Advances in Food Mycology
Advances in Food Mycology
Advances in Food Mycology
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<strong>Advances</strong> <strong>in</strong> <strong>Food</strong> <strong>Mycology</strong>
<strong>Advances</strong> <strong>in</strong> Experimental Medic<strong>in</strong>e and Biology<br />
Editorial Board:<br />
NATHAN BACK, State University of New York at Buffalo<br />
IRUN R. COHEN, The Weizmann Institute of Science<br />
DAVID KRITCHEVSKY, Wistar Institute<br />
ABEL LAJTHA, N.S. Kl<strong>in</strong>e Institute for Psychiatric Research<br />
RODOLFO PAOLETTI, University of Milan<br />
Recent Volumes <strong>in</strong> this Series<br />
Volume 563<br />
UPDATES IN PATHOLOGY<br />
Edited by David C. Chhieng and Gene P. Siegal<br />
Volume 564<br />
GLYCOBIOLOGY AND MEDICINE: PROCEEDINGS OF THE 7TH JENNER<br />
GLYCOBIOLOGY AND MEDICINE SYMPOSIUM<br />
Edited by John S. Axford<br />
Volume 565<br />
SLIDING FILAMENT MECHANISM IN MUSCLE CONTRACTION:<br />
FIFTY YEARS OF RESEARCH<br />
Edited by Haruo Sugi<br />
Volume 566<br />
OXYGEN TRANSPORT TO ISSUE XXVI<br />
Edited by Paul Okunieff, Jacquel<strong>in</strong>e Williams, and Yuhchyau Chen<br />
Volume 567<br />
THE GROWTH HORMONE-INSULIN-LIKE GROWTH FACTOR AXIS DURING<br />
DEVELOPMENT<br />
Edited by Isabel Varela-Nieto and Julie A. Chowen<br />
Volume 568<br />
HOT TOPICS IN INFECTION AND IMMUNITY IN CHILDREN II<br />
Edited by Andrew J. Pollard and Adam F<strong>in</strong>n<br />
Volume 569<br />
EARLY NUTRITION AND ITS LATER CONSEQUENCES: NEW OPPORTUNITIES<br />
Edited by Berthold Koletzko, Peter Dodds, Hans Akerbloom, and Margaret Ashwell<br />
Volume 570<br />
GENOME INSTABILITY IN CANCER DEVELOPMENT<br />
Edited by Erich A. Nigg<br />
Volume 571<br />
ADVANCES IN MYCOLOGY<br />
Edited by J.I. Pitts, A.D. Hock<strong>in</strong>g, and U. Thrane<br />
A Cont<strong>in</strong>uation Order Plan is available for this series. A cont<strong>in</strong>uation order will br<strong>in</strong>g delivery of<br />
each new volume immediately upon publication. Volumes are billed only upon actual shipment.<br />
For further <strong>in</strong>formation please contact the publisher.
<strong>Advances</strong> <strong>in</strong> <strong>Food</strong><br />
<strong>Mycology</strong><br />
Edited by<br />
A.D. Hock<strong>in</strong>g<br />
<strong>Food</strong> Science Australia<br />
North Ryde, Australia<br />
J.I. Pitt<br />
<strong>Food</strong> Science Australia<br />
North Ryde, Australia<br />
R.A. Samson<br />
Centraalbureau voor Schimmelcultures<br />
Utrecht, Netherlands<br />
and<br />
U. Thrane<br />
Technical University of Denmark<br />
Lyngby, Denmark
A.D. Hock<strong>in</strong>g<br />
<strong>Food</strong> Science Australia<br />
PO Box 52, North Ryde<br />
NSW 1670<br />
Australia<br />
Ailsa.Hock<strong>in</strong>g@csiro.au<br />
R.A. Samson<br />
Centraalbureau voor Schimmelcultures<br />
PO Box 85167<br />
3508 AD Utrecht<br />
Netherlands<br />
Samson@cbs.knaw.nl<br />
Library of Congress Control Number: 2005930810<br />
ISBN-10: 0-387-28385-4 e-ISBN: 0-387-28391-9<br />
ISBN-13: 978-0387-28385-2<br />
Pr<strong>in</strong>ted on acid-free paper.<br />
J.I. Pitt<br />
<strong>Food</strong> Science Australia<br />
PO Box 52, North Ryde<br />
NSW 1670<br />
Australia<br />
John.Pitt@csiro.au<br />
U. Thrane<br />
Biocentrum-DTU<br />
Technical University of Denmark<br />
Build<strong>in</strong>g 221<br />
DK-2800 Kgs. Lyngby<br />
Denmark<br />
ulf.thrane@biocentrum.dtu.dk<br />
© 2006 Spr<strong>in</strong>ger Science+Bus<strong>in</strong>ess Media, Inc.<br />
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FOREWORD<br />
This book represents the Proceed<strong>in</strong>gs of the Fifth International<br />
Workshop on <strong>Food</strong> <strong>Mycology</strong>, which was held on the Danish island of<br />
Samsø from 15-19 October, 2003. This series of Workshops commenced<br />
<strong>in</strong> Boston, USA, <strong>in</strong> July 1984, from which the proceed<strong>in</strong>gs<br />
were published as Methods for Mycological Exam<strong>in</strong>ation of <strong>Food</strong><br />
(edited by A. D. K<strong>in</strong>g et al., published by Plenum Press, New York,<br />
1986). The second Workshop was held <strong>in</strong> Baarn, the Netherlands, <strong>in</strong><br />
August 1990, and the proceed<strong>in</strong>gs were published as Modern Methods<br />
<strong>in</strong> <strong>Food</strong> <strong>Mycology</strong> (edited by R. A. Samson et al., and published by<br />
Elsevier, Amsterdam, 1992). The Third Workshop was held <strong>in</strong><br />
Copenhagen, Denmark, <strong>in</strong> 1994 and the Fourth near Uppsala,<br />
Sweden, <strong>in</strong> 1998. The proceed<strong>in</strong>gs of those two workshops were published<br />
as scientific papers <strong>in</strong> the International Journal of <strong>Food</strong><br />
Microbiology.<br />
International Workshops on <strong>Food</strong> <strong>Mycology</strong> are held under the<br />
auspices of the International Commission on <strong>Food</strong> <strong>Mycology</strong>, a<br />
Commission under the <strong>Mycology</strong> Division of the International Union<br />
of Microbiological Societies. Details of this Commission are given <strong>in</strong><br />
the f<strong>in</strong>al chapter of this book.<br />
This Fifth Workshop was organised by Ulf Thrane, Jens Frisvad,<br />
Per V. Nielsen and Birgitte Andersen from the Center for Microbial<br />
Biotechnology, Technical University of Denmark, Kgs. Lyngby,<br />
v
vi Foreword<br />
Denmark. This Center, through numerous publications and both<br />
undergraduate teach<strong>in</strong>g and graduate supervision has been highly<br />
<strong>in</strong>fluential <strong>in</strong> the world of food mycology for the past 20 years and<br />
more. Tr<strong>in</strong>e Bro and Lene Nordsmark from the Center also carried<br />
out the important tasks of provid<strong>in</strong>g secretarial help to the Organisers<br />
and solv<strong>in</strong>g the logistics of mov<strong>in</strong>g participants from Copenhagen to<br />
Samsø and back. Samsø provided an ideal sett<strong>in</strong>g for the Fifth<br />
Workshop, as the island is made up of rural agricultural communities,<br />
with old villages and rustic land and seascapes.<br />
The Fifth Workshop was attended by some 35 participants, drawn<br />
from among food mycology and related discipl<strong>in</strong>es around the world.<br />
The workshop was highly successful, with papers devoted to media<br />
and methods development <strong>in</strong> food mycology, as is usual with these<br />
workshops. Particular emphasis was placed on the fungi which produce<br />
mycotox<strong>in</strong>s, especially their ecology, and through ecology, potential<br />
control measures. Sessions were also devoted to yeasts, and the<br />
<strong>in</strong>activation of fungal spores by the use of heat and high pressure.<br />
Nearly 40 scientific papers were presented over three days of the workshop,<br />
and these papers are the major contributions <strong>in</strong> these<br />
Proceed<strong>in</strong>gs.<br />
The organisers especially wish to thank the sponsors of the Fifth<br />
Workshop: BCN Laboratories, Knoxville, Tennessee, USA; the<br />
Danish ECB5 Foundation, Copenhagen; Novozymes A/S, Bagsværd,<br />
Denmark; LMC Centre for Advanced <strong>Food</strong> Studies, Copenhagen; the<br />
Danish Research Agency STVF, Copenhagen, though Grant Number<br />
26-03-0188; Eurof<strong>in</strong>s Denmark A/S, Copenhagen and the <strong>Mycology</strong><br />
Division of the International Union of Microbiological Societies, for<br />
their support which made this workshop possible.<br />
A.D. Hock<strong>in</strong>g<br />
J.I. Pitt<br />
R.A. Samson<br />
U. Thrane
CONTENTS<br />
Foreword ..............................................v<br />
Contributors . .......................................... xi<br />
Section 1. Understand<strong>in</strong>g the fungi produc<strong>in</strong>g important<br />
mycotox<strong>in</strong>s . .................................... 1<br />
Important mycotox<strong>in</strong>s and the fungi which produce them ...... 3<br />
Jens C. Frisvad, Ulf Thrane, Robert A. Samson<br />
and John I. Pitt<br />
Recommendations concern<strong>in</strong>g the chronic problem of<br />
misidentification of mycotoxigenic fungi associated<br />
with foods and feeds ............................ 33<br />
Jens C. Frisvad, Kristian F. Nielsen and Robert A. Samson<br />
Section 2. Media and method development <strong>in</strong> food mycology . ... 47<br />
Comparison of hyphal length, ergosterol, mycelium dry<br />
weight, and colony diameter for quantify<strong>in</strong>g growth<br />
of fungi from foods ............................ 49<br />
Marta H. Taniwaki, John I. Pitt, Ailsa D. Hock<strong>in</strong>g<br />
and Graham H. Fleet<br />
vii
viii Contents<br />
Evaluation of molecular methods for the analysis of yeasts<br />
<strong>in</strong> foods and beverages .......................... 69<br />
Ai L<strong>in</strong> Beh, Graham H. Fleet, C. Prakitchaiwattana<br />
and Gillian M. Heard<br />
Standardization of methods for detect<strong>in</strong>g heat resistant fungi . . 107<br />
Jos Houbraken and Robert A. Samson<br />
Section 3. Physiology and ecology of mycotoxigenic fungi . ..... 113<br />
Ecophysiology of fumonis<strong>in</strong> producers <strong>in</strong> Fusarium<br />
section Liseola ............................ .... 115<br />
Vicente Sanchis, Sonia Marín, Naresh Magan and Antonio<br />
J. Ramos<br />
Ecophysiology of Fusarium culmorum and mycotox<strong>in</strong><br />
production ............................ ....... 123<br />
Naresh Magan, Russell Hope and David Aldred<br />
<strong>Food</strong>-borne fungi <strong>in</strong> fruit and cereals and their production<br />
of mycotox<strong>in</strong>s ............................ .... 137<br />
Birgitte Andersen and Ulf Thrane<br />
Black Aspergillus species <strong>in</strong> Australian v<strong>in</strong>eyards: from soil<br />
to ochratox<strong>in</strong> A <strong>in</strong> w<strong>in</strong>e ......................... 153<br />
Su-l<strong>in</strong> L. Leong, Ailsa D. Hock<strong>in</strong>g, John I. Pitt,<br />
Benozir A. Kazi, Robert W. Emmett and Eileen S. Scott<br />
Ochratox<strong>in</strong> A produc<strong>in</strong>g fungi from Spanish v<strong>in</strong>eyards ....... 173<br />
Marta Bau, M. Rosa Bragulat, M. Lourdes Abarca,<br />
Santiago M<strong>in</strong>guez and F. Javier Cabañes<br />
Fungi produc<strong>in</strong>g ochratox<strong>in</strong> <strong>in</strong> dried fruits ................ 181<br />
Beatriz T. Iamanaka, Marta H. Taniwaki, E. Vicente and<br />
Hilary C. Menezes<br />
An update on ochratoxigenic fungi and ochratox<strong>in</strong> A<br />
<strong>in</strong> coffee..................................... 189<br />
Marta H. Taniwaki
Contents ix<br />
Mycobiota, mycotoxigenic fungi, and citr<strong>in</strong><strong>in</strong> production <strong>in</strong><br />
black olives .................................. 203<br />
Dilek Heperkan, Burçak E. Meriç, Gülç<strong>in</strong> Sismanoglu,<br />
Gözde Dalkiliç and Funda K. Güler<br />
Byssochlamys: significance of heat resistance and mycotox<strong>in</strong><br />
production .................................. 211<br />
Jos Houbraken, Robert A. Samson and Jens C. Frisvad<br />
Effect of water activity and temperature on production<br />
of aflatox<strong>in</strong> and cyclopiazonic acid by Aspergillus<br />
flavus <strong>in</strong> peanuts ............................. 225<br />
Graciela Vaamonde, Andrea Patriarca and<br />
Virg<strong>in</strong>ia E. Fernández P<strong>in</strong>to<br />
Section 4. Control of fungi and mycotox<strong>in</strong>s <strong>in</strong> foods . ......... 237<br />
Inactivation of fruit spoilage yeasts and moulds us<strong>in</strong>g<br />
high pressure process<strong>in</strong>g ........................ 239<br />
Ailsa D. Hock<strong>in</strong>g, Mariam Begum and C<strong>in</strong>dy M. Stewart<br />
Activation of ascospores by novel food preservation<br />
techniques .................................. 247<br />
Jan Dijksterhuis and Robert A. Samson<br />
Mixtures of natural and synthetic antifungal agents ......... 261<br />
Aurelio López-Malo, Enrique Palou, Reyna León-Cruz<br />
and Stella M. Alzamora<br />
Probabilistic modell<strong>in</strong>g of Aspergillus growth.............. 287<br />
Enrique Palou and Aurelio López-Malo<br />
Antifungal activity of sourdough bread cultures ............ 307<br />
Lloyd B. Bullerman, Marketa Giesova, Yousef Hassan,<br />
Dwayne Deibert and Doj<strong>in</strong> Ryu<br />
Prevention of ochratox<strong>in</strong> A <strong>in</strong> cereals <strong>in</strong> Europe ............ 317<br />
Monica Olsen, Nils Jonsson, Naresh Magan, John Banks,<br />
Corrado Fanelli, Aldo Rizzo, Auli Haikara,<br />
Alan Dobson, Jens Frisvad, Stephen Holmes, Juhani Olkku,<br />
Sven-Johan Persson and Thomas Börjesson
x Contents<br />
Recommended methods for food mycology ............... 343<br />
Appendix 1 – Media ............................ .... 349<br />
Appendix 2 – International Commission on <strong>Food</strong> <strong>Mycology</strong> . . 358<br />
Index ............................ .... ............ 361
CONTRIBUTORS<br />
M. Lourdes Abarca, Departament de Sanitat i d’Anatomia Animals,<br />
Universitat Autónoma de Barcelona, 08193 Bellaterra, Barcelona,<br />
Spa<strong>in</strong><br />
David Aldred, Applied <strong>Mycology</strong> Group, Biotechnology Centre,<br />
Cranfield University, Silsoe, Bedford MK45 4DT, UK<br />
Stella M. Alzamora, Departament de Industrias, Facultad de Ciencias<br />
Exactas y Naturales, Universidad de Buenos Aires, Ciudad<br />
Universitaria 1428, Buenos Aires, Argent<strong>in</strong>a<br />
Birgitte Andersen, Center for Microbial Biotechnology, BioCentrum-<br />
DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby,<br />
Denmark<br />
Marta Bau, Departament de Sanitat i d’Anatomia Animals, Universitat<br />
Autónoma de Barcelona, 08193 Bellaterra, Barcelona, Spa<strong>in</strong><br />
John Banks, Central Science Laboratory, Sand Hutton, York YO41<br />
1LZ, UK<br />
Mariam Begum, <strong>Food</strong> Science Australia, CSIRO, P.O. Box 52, North<br />
Ryde, NSW 1670, Australia<br />
Ai L<strong>in</strong> Beh, <strong>Food</strong> Science and Technology, School of Chemical<br />
Eng<strong>in</strong>eer<strong>in</strong>g and Industrial Chemistry, University of New South<br />
Wales, Sydney, NSW 2052, Australia<br />
Thomas Börjesson, Svenska Lantmännen, Östra hamnen, SE-531 87<br />
Lidköp<strong>in</strong>g, Sweden<br />
xi
xii Contributors<br />
M. Rosa Bragulat, Departament de Sanitat i d’Anatomia Animals,<br />
Universitat Autónoma de Barcelona, 08193 Bellaterra, Barcelona,<br />
Spa<strong>in</strong><br />
Lloyd B. Bullerman, Department of <strong>Food</strong> Science and Technology,<br />
University of Nebraska, L<strong>in</strong>coln, NE 68583-0919, USA<br />
F. Javier Cabañes, Departament de Sanitat i d’Anatomia Animals,<br />
Universitat Autónoma de Barcelona, 08193 Bellaterra, Barcelona,<br />
Spa<strong>in</strong><br />
Gözde Dalkiliç, Department of <strong>Food</strong> Eng<strong>in</strong>eer<strong>in</strong>g, Istanbul Technical<br />
University, Istanbul, 34469 Maslak, Turkey<br />
Dwayne Deibert, Department of <strong>Food</strong> Science and Technology,<br />
University of Nebraska, L<strong>in</strong>coln, NE 68583-0919, USA<br />
Jan Dijksterhuis, Department of Applied Research and Services,<br />
Centraalbureau voor Schimmelcultures, Fungal Biodiversity<br />
Centre, Uppsalalaan 8, 3584 CT, Utrecht, Netherlands<br />
Alan Dobson, Microbiology Department, University College Cork,<br />
Cork, Ireland<br />
Robert W. Emmett, Department of Primary Industries, PO Box 905,<br />
Mildura, Vic. 3502, Australia<br />
Corrado Fanelli, Laboratorio di Micologia, Univerisità “La<br />
Sapienza”, Largo Crist<strong>in</strong>a di Svezia 24, I-00165 Roma, Italy<br />
Virg<strong>in</strong>ia E. Fernández P<strong>in</strong>to, Laboratorio de Microbiología de<br />
Alimentos, Departamento de Química Orgánica, Area<br />
Bromatología, Facultad de Ciencias Exactas y Naturales,<br />
Universidad de Buenos Aires, Ciudad Universitaria, Pabellón II, 3˚<br />
Piso, 1428, Buenos Aires, Argent<strong>in</strong>a<br />
Graham H. Fleet, <strong>Food</strong> Science and Technology, School of Chemical<br />
Eng<strong>in</strong>eer<strong>in</strong>g and Industrial Chemistry, University of New South<br />
Wales, Sydney, NSW 2052, Australia<br />
Jens C. Frisvad, BioCentrum-DTU, Build<strong>in</strong>g 221, Technical<br />
University of Denmark, 2800 Lyngby, Denmark<br />
Marketa Giesova, Department of Dairy and Fat Technology, Institute<br />
of Chemical Technology, Prague, Czech Republic<br />
Funda K. Güler Istanbul Technical University, Dept. of <strong>Food</strong><br />
Eng<strong>in</strong>eer<strong>in</strong>g Istanbul, Turkey, 34469 Maslak<br />
Auli Haikara, VTT Biotechnology, PO Box 1500, FIN-02044 Espoo,<br />
F<strong>in</strong>land<br />
Yousef Hassan, Department of <strong>Food</strong> Science and Technology,<br />
University of Nebraska, L<strong>in</strong>coln, NE 68583-0919, USA<br />
Gillian M. Heard, <strong>Food</strong> Science and Technology, School of Chemical<br />
Eng<strong>in</strong>eer<strong>in</strong>g and Industrial Chemistry, University of New South<br />
Wales, Sydney, NSW 2052, Australia
Contributors xiii<br />
Dilek Heperkan, Istanbul Technical University, Dept. of <strong>Food</strong><br />
Eng<strong>in</strong>eer<strong>in</strong>g Istanbul, 34469 Maslak, Turkey<br />
Ailsa D. Hock<strong>in</strong>g, <strong>Food</strong> Science Australia, CSIRO, PO Box 52, North<br />
Ryde, NSW 1670, Australia<br />
Stephen Holmes, ADGEN Ltd, Nellies Gate, Auch<strong>in</strong>cruive, Ayr KA6<br />
5HW, UK<br />
Russell Hope, Applied <strong>Mycology</strong> Group, Biotechnology Centre,<br />
Cranfield University, Silsoe, Bedford MK45 4DT, UK<br />
Jos Houbraken, Centraalbureau voor Schimmelcultures, PO Box<br />
85167, 3508 AD, Utrecht, The Netherlands<br />
Beatriz T. Iamanaka, <strong>Food</strong> Technology Institute, ITAL C.P 139<br />
CEP13.073-001 Camp<strong>in</strong>as-SP, Brazil<br />
Nils Jonsson, Swedish Institute of Agricultural and Environmental<br />
Eng<strong>in</strong>eer<strong>in</strong>g, PO Box 7033, SE-750 07 Uppsala, Sweden<br />
Benozir A. Kazi, Department of Primary Industries, PO Box 905,<br />
Mildura, Vic. 3502, Australia<br />
Reyna León-Cruz, Ingeniería Química y Alimentos, Universidad de las<br />
Américas, Puebla, Cholula 72820, Mexico<br />
Su-l<strong>in</strong> L. Leong, <strong>Food</strong> Science Australia, CSIRO, PO Box 52, North<br />
Ryde, NSW 1670, Australia<br />
Aurelio López-Malo, Ingeniería Química y Alimentos, Universidad de<br />
las Américas, Puebla, Cholula 72820, Mexico<br />
Naresh Magan, Applied <strong>Mycology</strong> Group, Biotechnology Centre,<br />
Cranfield University, Barton Road, Silsoe, Bedford MK45 4DT,<br />
UK<br />
Sonia Marín, <strong>Food</strong> Technology Department, Lleida University, 25198<br />
Lleida, Spa<strong>in</strong><br />
Hilary C. Menezes, <strong>Food</strong> Eng<strong>in</strong>eer<strong>in</strong>g Faculty (FEA), Unicamp,<br />
Camp<strong>in</strong>as-SP, Brazil<br />
Burçak E. Meriç, Istanbul Technical University, Dept. of <strong>Food</strong><br />
Eng<strong>in</strong>eer<strong>in</strong>g Istanbul, 34469 Maslak, Turkey<br />
Santiago M<strong>in</strong>ués, Institut Català de la V<strong>in</strong>ya i el Vi (INCAVI),<br />
Generalitat de Catalunya, Vilafranca del Penedés, Barcelona, Spa<strong>in</strong><br />
Kristian F. Nielsen, BioCentrum-DTU, Build<strong>in</strong>g 221, Technical<br />
University of Denmark, 2800 Lyngby, Denmark<br />
Juhani Olkku, Oy Panimolaboratorio-Bryggerilaboratorium AB, P.O.<br />
Box 16, FIN-02150 Espoo, F<strong>in</strong>land<br />
Monica Olsen, National <strong>Food</strong> Adm<strong>in</strong>istration, PO Box 622, SE-751<br />
26 Uppsala, Sweden<br />
Enrique Palou, Ingeniería Química y Alimentos, Universidad de las<br />
Américas, Puebla, Cholula 72820, Mexico
xiv Contributors<br />
Andrea Patriarca, Laboratorio de Microbiología de Alimentos,<br />
Departamento de Química Orgánica, Area Bromatología, Facultad<br />
de Ciencias Exactas y Naturales, Universidad de Buenos Aires,<br />
Ciudad Universitaria, Pabellón II, 3˚ Piso, 1428, Buenos Aires,<br />
Argent<strong>in</strong>a<br />
Sven-Johan Persson, Akron mask<strong>in</strong>er, SE-531 04 Järpås, Sweden<br />
John I. Pitt, <strong>Food</strong> Science Australia, CSIRO, PO Box 52, North Ryde,<br />
NSW 1670, Australia<br />
C. Prakitchaiwattana, <strong>Food</strong> Science and Technology, School of<br />
Chemical Eng<strong>in</strong>eer<strong>in</strong>g and Industrial Chemistry, University of<br />
New South Wales, Sydney, NSW 2052, Australia<br />
Antonio J. Ramos, <strong>Food</strong> Technology Department, Lleida University,<br />
25198 Lleida, Spa<strong>in</strong><br />
Aldo Rizzo, National Veter<strong>in</strong>ary and <strong>Food</strong> Res. Inst., PO Box 45,<br />
FIN-00581, Hels<strong>in</strong>ki, F<strong>in</strong>land<br />
Doj<strong>in</strong> Ryu, Department of <strong>Food</strong> Science and Technology, University<br />
of Nebraska, L<strong>in</strong>coln, NE 68583-0919, USA<br />
Robert A. Samson, Department of Applied Research and Services,<br />
Centraalbureau voor Schimmelcultures, Fungal Biodiversity<br />
Centre, Uppsalalaan 8, 3584 CT, Utrecht, Netherlands<br />
Vicente Sanchis, <strong>Food</strong> Technology Department, Lleida University,<br />
25198 Lleida, Spa<strong>in</strong><br />
Eileen S. Scott, School of Agriculture and W<strong>in</strong>e, University of<br />
Adelaide, PMB 1, Glen Osmond, SA 5064, Australia<br />
Gülç<strong>in</strong> S¸is¸manog˘lu, Department of <strong>Food</strong> Eng<strong>in</strong>eer<strong>in</strong>g, Istanbul<br />
Technical University, Istanbul, 34469 Maslak, Turkey<br />
C<strong>in</strong>dy Stewart, National Center for <strong>Food</strong> Safety and Technology, 6502<br />
S. Archer Rd, Summit-Argo, IL 60501, USA<br />
Marta H. Taniwaki, <strong>Food</strong> Technology Institute (ITAL), C.P 139<br />
CEP13.073-001 Camp<strong>in</strong>as-SP, Brazil<br />
Ulf Thrane, Center for Microbial Biotechnology, BioCentrum-DTU,<br />
Technical University of Denmark, DK-2800 Kgs. Lyngby,<br />
Denmark<br />
Graciela Vaamonde, Laboratorio de Microbiología de Alimentos,<br />
Departamento de Química Orgánica, Area Bromatología, Facultad<br />
de Ciencias Exactas y Naturales, Universidad de Buenos Aires,<br />
Ciudad Universitaria, Pabellón II, 3˚ Piso, 1428, Buenos Aires,<br />
Argent<strong>in</strong>a<br />
E. Vicente <strong>Food</strong> Technology Institute, ITAL C.P 139 CEP13.073-001<br />
Camp<strong>in</strong>as-SP, Brazil
Section 1.<br />
Understand<strong>in</strong>g the fungi produc<strong>in</strong>g<br />
important mycotox<strong>in</strong>s<br />
Important mycotox<strong>in</strong>s and the fungi which produce them<br />
Jens C. Frisvad, Ulf Thrane, Robert A. Samson and John I. Pitt<br />
Recommendations concern<strong>in</strong>g the chronic problem of misidentification of<br />
mycotoxigenic fungi associated with foods and feeds<br />
Jens C. Frisvad, Kristian F. Nielsen and Robert A. Samson
IMPORTANT MYCOTOXINS AND THE<br />
FUNGI WHICH PRODUCE THEM<br />
Jens C. Frisvad, Ulf Thrane, * Robert A. Samson † and John<br />
I. Pitt ‡<br />
1. INTRODUCTION<br />
The assessment of the relationship between species and mycotox<strong>in</strong>s<br />
production has proven to be very difficult. The modern literature<br />
is cluttered with examples of species purported to make particular<br />
mycotox<strong>in</strong>s, but where the association is <strong>in</strong>correct. In some cases,<br />
mycotox<strong>in</strong>s have even been named based on an erroneous association<br />
with a particular species: verruculogen, viridicatumtox<strong>in</strong> and rubratox<strong>in</strong><br />
come to m<strong>in</strong>d. As time has gone on, and more and more compounds<br />
have been described, lists of species-mycotox<strong>in</strong> associations<br />
have become so large, and the <strong>in</strong>accuracies <strong>in</strong> them so widespread <strong>in</strong><br />
acceptance, that determ<strong>in</strong><strong>in</strong>g true associations has become very difficult.<br />
It does not need to be emphasised how important it is that these<br />
associations be known accurately. The possible presence of mycotoxigenic<br />
fungi <strong>in</strong> foods, and rational decisions on the status of foods suspected<br />
to conta<strong>in</strong> mycotox<strong>in</strong>s, are ever present problems <strong>in</strong> the food<br />
<strong>in</strong>dustry around the world.<br />
In def<strong>in</strong><strong>in</strong>g mycotox<strong>in</strong>s, we exclude fungal metabolites which are<br />
active aga<strong>in</strong>st bacteria, protozoa, and lower animals <strong>in</strong>clud<strong>in</strong>g <strong>in</strong>sects.<br />
* J. C. Frisvad and U. Thrane, Center for Microbial Biotechnology, BioCentrum-<br />
DTU, Technical University of Denmark, Build<strong>in</strong>g 221, DK-2800 Kgs. Lyngby,<br />
Denmark. Correspondence to jcf@biocentrum.dtu.dk<br />
† R. A. Samson, Centraalbureau voor Schimmelcultures, PO Box 85167, 3508 AD<br />
Utrecht, Netherlands<br />
‡ J. I. Pitt, <strong>Food</strong> Science Australia, CSIRO, PO Box 52, North Ryde, NSW 1670,<br />
Australia<br />
3
4 Jens C. Frisvad et al.<br />
Furthermore we exclude Basidiomycete tox<strong>in</strong>s, because these are<br />
<strong>in</strong>gested by eat<strong>in</strong>g fruit<strong>in</strong>g bodies, a problem different from the <strong>in</strong>gestion<br />
of tox<strong>in</strong>s produced by microfungi. The def<strong>in</strong>ition of microfungi<br />
is not rigorous, but understood here to refer pr<strong>in</strong>cipally to<br />
Ascomycetous fungi, <strong>in</strong>clud<strong>in</strong>g those with no sexual stage. Lower<br />
fungi, from the subk<strong>in</strong>gdom Zygomycot<strong>in</strong>a, i.e. genera such as<br />
Rhizopus and Mucor, are not excluded, but compounds of sufficient<br />
toxicity to be termed mycotox<strong>in</strong>s have not been found <strong>in</strong> these genera,<br />
except perhaps for rhizon<strong>in</strong> A and B from Rhizopus microsporus<br />
(Jennessen et al., 2005).<br />
This paper sets out to provide an up to date authoritative list of<br />
mycotox<strong>in</strong>s which are known to have caused, or we believe have the<br />
potential to cause, disease <strong>in</strong> humans or vertebrate animals, and the<br />
fungal species which have been shown to produce them.<br />
We believe that all of the important and known mycotox<strong>in</strong>s produced<br />
by Aspergillus, Fusarium and Penicillium species have been<br />
<strong>in</strong>cluded <strong>in</strong> this list. However, it is possible that other species will be<br />
found which are capable of produc<strong>in</strong>g known tox<strong>in</strong>s, or other tox<strong>in</strong>s<br />
of consequence will arise. It is also important to note that there are<br />
many errors <strong>in</strong> the literature concern<strong>in</strong>g the mycotox<strong>in</strong>s and the fungi<br />
which produce them (Frisvad et al., 2006).<br />
Many other toxic chemicals, known to be produced by species from<br />
these genera, have been excluded from this list for one reason or<br />
another. The very toxic chemicals, the janthitrems, have been excluded<br />
from this list because the species which make them, <strong>in</strong>clud<strong>in</strong>g P. janth<strong>in</strong>ellum,<br />
normally do not grow to a significant extent <strong>in</strong> foods. On the<br />
other hand Penicillium tularense has recently been demonstrated to produce<br />
janthitrems <strong>in</strong> tomatoes (Andersen and Frisvad, 2004), so maybe<br />
these mycotox<strong>in</strong>s may occur sporadically. Other compounds which<br />
occur quite commonly <strong>in</strong> foods, <strong>in</strong>clud<strong>in</strong>g mycophenolic acid (Lafont<br />
et al., 1979, Lopez-Diaz et al., 1996; Overy and Frisvad, 2005), are of<br />
such low acute toxicity to vertebrate animals that their <strong>in</strong>volvement <strong>in</strong><br />
human or animal diseases appears unlikely. On the other hand<br />
mycophenolic acid has been reported to be strongly immunosuppressive<br />
(Bentley, 2000), so this fungal metabolite could pave the way for bacterial<br />
<strong>in</strong>fections. Toxic low molecular weight compounds that may not be<br />
considered mycotox<strong>in</strong>s <strong>in</strong> a strict sense <strong>in</strong>clude aflatrem, botryodiploid<strong>in</strong>,<br />
brefeld<strong>in</strong> A, chetom<strong>in</strong>, chetoc<strong>in</strong>s, emestr<strong>in</strong>, emod<strong>in</strong>,<br />
engleromyc<strong>in</strong>, fusar<strong>in</strong> C, lolitrems, paspalic<strong>in</strong>e, paspal<strong>in</strong>e, paspal<strong>in</strong><strong>in</strong>e,<br />
paspalitrems, paxill<strong>in</strong>e, territrems, tryptoquival<strong>in</strong>s, tryptoquivalons,<br />
verruculotox<strong>in</strong>, verticill<strong>in</strong>s, and viridicatumtox<strong>in</strong> which are among the<br />
fungal secondary metabolites listed as mycotox<strong>in</strong>s by Bet<strong>in</strong>a (1989).
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 5<br />
Future research may show that some of these are more important for<br />
human and domestic animals health than currently <strong>in</strong>dicated.<br />
For convenience the list below has been set out by genus, but it<br />
should be kept <strong>in</strong> m<strong>in</strong>d that some mycotox<strong>in</strong>s are common to both<br />
Aspergillus and Penicillium species (Samson, 2001). The list below sets<br />
out to be encyclopaedic, but at the same time we have <strong>in</strong>dicated, where<br />
possible, which species produc<strong>in</strong>g a particular tox<strong>in</strong> are more likely to<br />
occur <strong>in</strong> foods and which are probably of little consequence.<br />
2. ASPERGILLUS TOXINS<br />
2.1. Aflatox<strong>in</strong>s<br />
Aflatox<strong>in</strong>s are potent carc<strong>in</strong>ogens (Class 1; JECFA, 1997) affect<strong>in</strong>g<br />
man and all tested animal species <strong>in</strong>clud<strong>in</strong>g birds and fish. Four compounds<br />
are commonly produced <strong>in</strong> foods: aflatox<strong>in</strong>s B 1 ,B 2 ,G 1 and<br />
G 2 , named for the colour of their fluorescence under ultra violet light,<br />
and their relative position on TLC plates.<br />
Major sources. Aspergillus flavus is the most common species produc<strong>in</strong>g<br />
aflatox<strong>in</strong>s, occurr<strong>in</strong>g <strong>in</strong> most k<strong>in</strong>ds of foods <strong>in</strong> tropical countries.<br />
This species has a special aff<strong>in</strong>ity with three crops, maize,<br />
peanuts and cottonseed, and usually produces only B aflatox<strong>in</strong>s. Only<br />
about 40% of known isolates produce aflatox<strong>in</strong>.<br />
Aspergillus parasiticus occurs commonly <strong>in</strong> peanuts, but is quite rare<br />
<strong>in</strong> other foods. It is also restricted geographically, and is rare <strong>in</strong><br />
Southeast Asia (Pitt et al., 1993). A. parasiticus produces both B and<br />
G aflatox<strong>in</strong>s, and virtually all known isolates are toxigenic.<br />
M<strong>in</strong>or sources. Table 1 shows the species which are known to be capable<br />
of produc<strong>in</strong>g aflatox<strong>in</strong> <strong>in</strong> culture, and some details concern<strong>in</strong>g their<br />
appearance and their occurrence. Note that most of the m<strong>in</strong>or species<br />
are known from only a very few isolates, and their occurrence <strong>in</strong> foodstuffs<br />
or feedstuffs is at most rare. On the other hand A. nomius, A. toxicarius,<br />
and A. parvisclerotigenus may be more common than expected,<br />
because it is very difficult to dist<strong>in</strong>guish between those species and isolates<br />
may easily have been identified as A. flavus or A. parasiticus.<br />
2.2. Cyclopiazonic acid (see also Penicillium)<br />
Cyclopiazonic acid (CPA) (Holzapfel, 1968) is a potent mycotox<strong>in</strong><br />
that produces focal necrosis <strong>in</strong> most vertebrate <strong>in</strong>ner organs <strong>in</strong> high
6 Jens C. Frisvad et al.<br />
Table 1. Morphology and mycotox<strong>in</strong> production characteristic of species <strong>in</strong> Aspergillus that are known aflatox<strong>in</strong> producers a<br />
Species Heads Conidia Sclerotia Known occurrence Mycotox<strong>in</strong>s<br />
Aspergillus flavus Mostly biseriate Spherical to Large, spherical Ubiquitous <strong>in</strong> B aflatox<strong>in</strong>s<br />
ellipsoidal, smooth tropics and (40% of isolates);<br />
to f<strong>in</strong>ely rough walls subtropics CPAb , ca 50%<br />
A. parasiticus Rarely biseriate Spherical, rough walls Large, spherical USA, South B and G aflatox<strong>in</strong>s<br />
(uncommon) America, (nearly 100%)<br />
Australia<br />
A. nomius Mostly biseriate Spherical to Small, bullet USA, Thailand B and G aflatox<strong>in</strong>s<br />
ellipsoidal, smooth shaped (usually)<br />
to f<strong>in</strong>ely rough walls<br />
A. bombycis Mostly biseriate Spherical to Not reported Japan, Indonesia B and G aflatox<strong>in</strong>s<br />
subspheroidal, (silkworms only)<br />
roughened<br />
A. pseudotamarii Biseriate Spherical to Large, spherical Japan, Argent<strong>in</strong>a B aflatox<strong>in</strong>s, CPA<br />
subspheroidal,<br />
very rough walls<br />
A. toxicarius Rarely biseriate Sphaerical, rough Large, sphaerical USA, Uganda B and G aflatox<strong>in</strong>s<br />
walled<br />
A. parvisclerotigenus Mostly biseriate Spherical, rough Small, sphaerical USA, Argent<strong>in</strong>a, B and G aflatox<strong>in</strong>s,<br />
walled Japan, Nigeria CPA<br />
A. ochraceoroseus Biseriate Subspheroidal to Not reported Ivory Coast (soil) B aflatox<strong>in</strong>s,<br />
ellipsoidal, sterigmatocyst<strong>in</strong><br />
smooth walled<br />
A. rambellii Biseriate Ellipsoidal, smooth Not reported Ivory Coast (soil) B aflatox<strong>in</strong>s,<br />
walled sterigmatocyst<strong>in</strong><br />
Emericella astellata Biseriate Spheroidal, rugulose Ascomata and Ecuador (leaves B aflatox<strong>in</strong>s,<br />
walls hülle cells of Ilex) Sterigmatocyst<strong>in</strong><br />
Emericella Biseriate Sphaeroidal, Ascomata and Venezuela B aflatox<strong>in</strong>s,<br />
venezuelensis rugulose walls hülle cells (mangrove) Sterigmatocyst<strong>in</strong><br />
a From Kurtzman et al., 1987; Klich and Pitt, 1988; Pitt and Hock<strong>in</strong>g, 1997; Klich et al., 2000; Ito et al., 2001; Peterson et al., 2001; Frisvad<br />
et al., 2004a; Frisvad and Samson, 2004a; Frisvad et al., 2005a.<br />
b CPA, cyclopiazonic acid.
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 7<br />
concentrations and affects the ducts or organs orig<strong>in</strong>at<strong>in</strong>g from ducts.<br />
It was orig<strong>in</strong>ally believed that aflatox<strong>in</strong>s were responsible for all the<br />
toxic effects of Aspergillus flavus contam<strong>in</strong>ated peanuts to turkeys <strong>in</strong><br />
Turkey X disease, but it was later shown that cyclopiazonic acid had<br />
an additional severe effect on the muscles and bones of the turkeys<br />
(Jand et al., 2005).<br />
Major sources. Aspergillus flavus and the domesticated form<br />
A. oryzae often produce large amounts of CPA. A. flavus is common<br />
on oil seeds, nuts, peanuts and cereals, but may also produce aflatox<strong>in</strong><br />
on dried fruits (Pitt and Hock<strong>in</strong>g, 1997).<br />
M<strong>in</strong>or sources. Other producers of CPA <strong>in</strong> Aspergillus <strong>in</strong>clude<br />
A. tamarii, A. pseudotamarii, A. parvisclerotigenus, but the role of<br />
these fungi concern<strong>in</strong>g CPA production <strong>in</strong> foods or feeds is not clear.<br />
2.3. Cytochalas<strong>in</strong> E<br />
Cytochalas<strong>in</strong> E is a very toxic metabolite of Aspergillus clavatus.It<br />
may occur <strong>in</strong> malt<strong>in</strong>g barley (Lopez-Diaz and Flannigan, 1997)<br />
Major source. Aspergillus clavatus.<br />
M<strong>in</strong>or source. Rosell<strong>in</strong>ia necatrix is not found <strong>in</strong> foods.<br />
2.4. Gliotox<strong>in</strong><br />
Gliotox<strong>in</strong> is strongly immunosuppressive, but is probably only a<br />
potential problem <strong>in</strong> animal feeds (Bet<strong>in</strong>a, 1989).<br />
Major sources. Aspergillus fumigatus has been found <strong>in</strong> animal feeds.<br />
M<strong>in</strong>or sources. Gliocladium virens, P. lilac<strong>in</strong>oech<strong>in</strong>ulatum and few<br />
other soil-borne species also produce gliotox<strong>in</strong>.<br />
2.5. β-Nitropropionic Acid (BNP)<br />
β-nitropropionic acid has been reported to be <strong>in</strong>volved <strong>in</strong> sugar<br />
cane poison<strong>in</strong>g of children, but may potentially also cause other<br />
<strong>in</strong>toxications, as producers are widespread (Burdock et al., 2001).<br />
Furthermore BNP has been found <strong>in</strong> miso, shoyu and katsuobushi<br />
and it can be produced by A. oryzae when artificially <strong>in</strong>oculated on<br />
cheese, peanuts etc. Unfortunately A. flavus has not been tested for<br />
the production of BNP, but BNP production by A. oryzae on peanuts<br />
<strong>in</strong>dicates that A. flavus may be able to produce this mycotox<strong>in</strong> <strong>in</strong><br />
comb<strong>in</strong>ation with aflatox<strong>in</strong> B 1 , cyclopiazonic acid and kojic acid. The<br />
possible synergistic effect of these mycotox<strong>in</strong>s on mammals is<br />
unknown.
8 Jens C. Frisvad et al.<br />
Major sources. The BNP produc<strong>in</strong>g fungi from sugar cane are<br />
Arthr<strong>in</strong>ium phaeospermum and Art. sacchari, but other species such as<br />
Art. term<strong>in</strong>alis, Art. saccharicola, Art. aureum and Art. sereanis also<br />
produce BNP (Burdock et al., 2001).<br />
A. flavus may be an important producer of this mycotox<strong>in</strong> <strong>in</strong> foods,<br />
but there are no surveys that <strong>in</strong>clude analytical determ<strong>in</strong>ation of BNP<br />
alongside cyclopiazonic acid and aflatox<strong>in</strong> B 1 . A. oryzae and A. sojae<br />
can produce BNP <strong>in</strong> miso and shoyu, but it is probably more important<br />
that their wild-type forms, A. flavus and A. parasiticus respectively, may<br />
produce BNP <strong>in</strong> foods. More research is needed <strong>in</strong> this area.<br />
M<strong>in</strong>or sources. Penicillium atrovenetum is another authenticated<br />
producer of BNP, but this fungus is only found <strong>in</strong> soil.<br />
Incorrect sources. Penicillium cyclopium, P. chrysogenum, Aspergillus<br />
wentii, Eurotium spp., and A. candidus have been reported as producers<br />
of BNP (Burdock et al., 2001), but these identifications are<br />
doubtful.<br />
2.6. Ochratox<strong>in</strong> A (see also Penicillium)<br />
Ochratox<strong>in</strong> A (OA) is a nephrotox<strong>in</strong>, affect<strong>in</strong>g all tested animal<br />
species, though effects <strong>in</strong> man have been difficult to establish unequivocally.<br />
It is listed as a probable human carc<strong>in</strong>ogen (Class 2B) (JECFA,<br />
2001). L<strong>in</strong>ks between OA and Balkan Endemic Nephropathy have<br />
long been sought, but not established (JECFA, 2001).<br />
Major sources. Aspergillus ochraceus (van der Merwe et al., 1965),<br />
occurr<strong>in</strong>g <strong>in</strong> stored cereals (Pitt and Hock<strong>in</strong>g, 1997) and coffee<br />
(Taniwaki et al., 2003). A. ochraceus has been shown to consist of two<br />
species (Varga et al., 2000a, b; Frisvad et al., 2004b). The second and<br />
new species produc<strong>in</strong>g large amounts of ochratox<strong>in</strong> A consistently,<br />
has been described as A. westerdijkiae. Actually the orig<strong>in</strong>al producer<br />
of ochratox<strong>in</strong> A from Andropogon sorghum <strong>in</strong> South Africa, NRRL<br />
3174, has been designated as the type culture of A. westerdijkiae<br />
(Frisvad et al., 2004b). This is <strong>in</strong>terest<strong>in</strong>g as A. westerdijkiae is both a<br />
better and more consistent ochratox<strong>in</strong> producer than A. ochraceus,<br />
and it may be also more prevalent <strong>in</strong> coffee than A. ochraceus. The ex<br />
type culture of A. ochraceus CBS 108.08 only produces trace amounts<br />
of ochratox<strong>in</strong> A.<br />
Aspergillus carbonarius (Horie, 1995) is a major OA producer. It<br />
occurs <strong>in</strong> grapes, produc<strong>in</strong>g OA <strong>in</strong> grape products, <strong>in</strong>clud<strong>in</strong>g grape<br />
juice, w<strong>in</strong>es and dried v<strong>in</strong>e fruits (IARC, 2002; Leong et al., 2004) and<br />
sometimes on coffee beans (Taniwaki et al., 2003; Abarca et al., 2004).<br />
Aspergillus niger is an extremely common species, but only few stra<strong>in</strong>s
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 9<br />
appear to be producers of OA, so this species may be of much less<br />
importance than A. carbonarius <strong>in</strong> grapes, w<strong>in</strong>e and green coffee beans<br />
(Abarca et al., 1994; Taniwaki et al., 2003; Leong et al., 2004). It may<br />
be of major importance, however, as A. niger NRRL 337, referred to<br />
as the “food fungus”, produces large amounts of OA <strong>in</strong> pure culture.<br />
This fungus is used for fermentation of potato peel waste etc. and used<br />
for animal feed (Schuster et al., 2002).<br />
Petromyces alliaceus (Lai et al., 1970), produces large amounts of<br />
ochratox<strong>in</strong> A <strong>in</strong> pure culture, and OA produced by this fungus has<br />
been found <strong>in</strong> figs <strong>in</strong> California (Bayman et al., 2002). Aspergillus<br />
steynii, from the Aspergillus section Circumdati, is also a very efficient<br />
producer of OA, and has been found <strong>in</strong> green coffee beans, mouldy<br />
soy beans and rice (Frisvad et al., 2004b). As with A. westerdijkiae,<br />
A. steynii may have been identified as A. ochraceus earlier, so the relative<br />
abundance of these three species is difficult to evaluate at present.<br />
Penicillium verrucosum is the major producer of ochratox<strong>in</strong> A <strong>in</strong><br />
stored cereals (Frisvad, 1985; Pitt, 1987; Lund and Frisvad, 2003).<br />
Penicillium nordicum (Larsen et al., 2001) is the ma<strong>in</strong> OA producer<br />
found <strong>in</strong> meat products such as salami and ham. Both OA produc<strong>in</strong>g<br />
Penicillium species have been found on cheese also, but have only been<br />
reported to be of high occurrence on Swiss hard cheeses (as P. casei,<br />
Staub, 1911). The ex type culture of P. casei is a P. verrucosum (Larsen<br />
et al., 2001).<br />
M<strong>in</strong>or sources. Several Aspergilli can produce ochratox<strong>in</strong> A <strong>in</strong> large<br />
amounts, but they appear to be relatively rare. In Aspergillus section<br />
Circumdati (formerly the Aspergillus ochraceus group), the follow<strong>in</strong>g<br />
species can produce ochratox<strong>in</strong> A: Aspergillus cretensis, A. flocculosus,<br />
A. pseudoelegans, A. roseoglobulosus, A. sclerotiorum, A. sulphureus and<br />
Neopetromyces muricatus (Frisvad et al., 2004b). Accord<strong>in</strong>g to Ciegler<br />
(1972) and Hesselt<strong>in</strong>e et al. (1972) A. melleus, A. ostianus, A. persii and<br />
A. petrakii may produce trace amounts of OA, but this has not been<br />
confirmed s<strong>in</strong>ce publication of those papers. Stra<strong>in</strong>s of these species<br />
reported to produce large amounts of OA were reidentified by Frisvad<br />
et al. (2004b). In Aspergillus section Flavi, Petromyces albertensis produces<br />
ochratox<strong>in</strong> A. In Aspergillus section Nigri, A. lacticoffeatus and<br />
A. sclerotioniger produce ochratox<strong>in</strong> A (Samson et al., 2004).<br />
2.7. Sterigmatocyst<strong>in</strong><br />
Sterigmatocyst<strong>in</strong> is a possible carc<strong>in</strong>ogen. However, its low solubility<br />
<strong>in</strong> water or gastric juices limits its potential to cause human illness<br />
(Pitt and Hock<strong>in</strong>g, 1997).
10 Jens C. Frisvad et al.<br />
Major sources. The major source of sterigmatocyst<strong>in</strong> <strong>in</strong> foods is<br />
Aspergillus versicolor. This fungus is common on cheese, but may also<br />
occur on other substrates (Pitt and Hock<strong>in</strong>g, 1997).<br />
M<strong>in</strong>or sources. A large number of species are able to produce sterigmatocyst<strong>in</strong>,<br />
<strong>in</strong>clud<strong>in</strong>g Chaetomium spp., Emericella spp., Monocillium<br />
nord<strong>in</strong>ii and Humicola fuscoatra (Joshi et al., 2002). These species are<br />
unlikely to contam<strong>in</strong>ate foods.<br />
2.8. Verruculogen and Fumitremorg<strong>in</strong>s<br />
Verrucologen is an extremely toxic tremorgenic mycotox<strong>in</strong>, but it is<br />
unlikely to be found<strong>in</strong>g significant levels <strong>in</strong> foods. Neosartorya fisheri<br />
may be present <strong>in</strong> heat treated foods, but N. glabra and allied species<br />
are much more common <strong>in</strong> foods, and the latter species do not produce<br />
verrucologen.<br />
Major sources. Aspergillus fumigatus and Neosartorya fischeri are<br />
the major Aspergillus species produc<strong>in</strong>g verruculogen but these species<br />
are uncommon <strong>in</strong> foods. These species produce many other toxic compounds<br />
<strong>in</strong>clud<strong>in</strong>g gliotox<strong>in</strong>, fumigaclav<strong>in</strong>s, and tryptoquival<strong>in</strong>s (Cole<br />
et al., 1977; Cole and Cox, 1981; Panaccione and Coyle, 2005).<br />
M<strong>in</strong>or sources. Aspergillus caespitosus, Penicillium mononematosum<br />
and P. brasilianum are efficient producers of verrucologen and<br />
fumitremorg<strong>in</strong>s, but are very rare <strong>in</strong> foods and feeds.<br />
3. FUSARIUM TOXINS<br />
3.1. Antibiotic Y<br />
Antibiotic Y has significant antibiotic properties towards phytopathogenic<br />
bacteria but low cell toxicity (Gol<strong>in</strong>ski et al., 1986).<br />
However, this compound, which orig<strong>in</strong>ally was named lateropyrone<br />
(Bushnell et al., 1984), has not been studied <strong>in</strong> detail. Producers of<br />
antibiotic Y are widespread and common <strong>in</strong> agricultural products, so<br />
the natural occurrence of antibiotic Y may be of importance. Natural<br />
occurrence <strong>in</strong> cherries, apples and wheat gra<strong>in</strong>s has been reported<br />
(Andersen and Thrane, 2005).<br />
Major sources. The ma<strong>in</strong> producer is Fusarium avenaceum which<br />
occurs frequently <strong>in</strong> cereal gra<strong>in</strong>, fruit and vegetables. Another consistent<br />
producer is F. tric<strong>in</strong>ctum, which also is very frequently found on<br />
cereal gra<strong>in</strong>s <strong>in</strong> temperate climates.
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 11<br />
M<strong>in</strong>or sources. F. lateritium is known as a plant pathogen, but also<br />
causes spoilage <strong>in</strong> fruits and has been reported from apples and cherries<br />
<strong>in</strong> which antibiotic Y was detected (Andersen and Thrane, 2006).<br />
In warmer climates F. chlamydosporum is a potential producer of<br />
antibiotic Y <strong>in</strong> cereal gra<strong>in</strong> and other seeds.<br />
3.2. Butenolide<br />
Butenolide is a collective name for compounds with a given r<strong>in</strong>g<br />
structure; however <strong>in</strong> Fusarium mycotoxicology butenolide is a synonym<br />
for 4-acetamido-2-buten-4-olide, which has been associated<br />
with cattle diseases (fescue foot) s<strong>in</strong>ce the mid 1960s (Yates et al.,<br />
1969). The toxicology has been thoroughly discussed by Marasas et al.<br />
(1984). There have been no reports of butenolide <strong>in</strong> foods, but it may<br />
be an important tox<strong>in</strong> due to the reported synergistic effect with enniat<strong>in</strong><br />
B (Hershenhorn et al., 1992).<br />
Major sources. The orig<strong>in</strong>al reported producer of butenolide is<br />
F. sporotrichioides [reported as F. nivale, see Marasas et al. (1984) for<br />
details] and other frequent producers of butenolide <strong>in</strong> cereals are<br />
F. gram<strong>in</strong>earum and F. culmorum.<br />
M<strong>in</strong>or sources. Other potential producers of butenolide are F. avenaceum,<br />
F. poae and F. tric<strong>in</strong>ctum which are frequently found <strong>in</strong> cereal<br />
gra<strong>in</strong>s together with F. crookwellense, F. sambuc<strong>in</strong>um and F. venenatum.<br />
The latter three species also can be found <strong>in</strong> potatoes and other<br />
root vegetables.<br />
3.3. Culmor<strong>in</strong><br />
Culmor<strong>in</strong> has a low toxicity <strong>in</strong> several biological assays (Pedersen<br />
and Miller, 1999) but a synergistic effect with deoxynivalenol towards<br />
caterpillars has been demonstrated (Dowd et al., 1989). Culmor<strong>in</strong> and<br />
hydroxyculmor<strong>in</strong>s have been detected <strong>in</strong> cereals (Ghebremeskel and<br />
Langseth, 2000). These samples also conta<strong>in</strong>ed deoxynivalenol and<br />
acetyl-deoxynivalenol.<br />
Major sources. F. culmorum and F. gram<strong>in</strong>earum, found <strong>in</strong> cereals,<br />
are the major producers of culmor<strong>in</strong>. The less widely distributed<br />
species F. poae and F. langsethiae are also consistent producers of<br />
culmor<strong>in</strong> and derivatives (Thrane et al., 2004).<br />
M<strong>in</strong>or sources. Other species produc<strong>in</strong>g culmor<strong>in</strong> are F. crookwellense<br />
and F. sporotrichioides, also found <strong>in</strong> cereals.
12 Jens C. Frisvad et al.<br />
3.4. Cyclic Peptides<br />
The two groups of cyclic peptides, beauveric<strong>in</strong> and enniat<strong>in</strong>s, are<br />
structurally related and they show antibiotic and ionophoric activities<br />
(Kamyar et al., 2004). Both groups of cyclic peptides have been<br />
detected <strong>in</strong> agricultural products (Jestoi et al., 2004).<br />
3.4.1. Beauveric<strong>in</strong><br />
Beauveric<strong>in</strong> was orig<strong>in</strong>ally found <strong>in</strong> entomopathogenic fungi<br />
such as Beauveria bassiana and Isaria fumosorosea (formerly<br />
Paecilomyces fumosoroseus; Luangsa-Ard et al., 2005) but has also<br />
been detected <strong>in</strong> several Fusarium species occurr<strong>in</strong>g on food<br />
(Logrieco et al., 1998).<br />
Major sources. Fusarium subglut<strong>in</strong>ans, F. proliferatum and F. oxysporum<br />
are consistent producers of beauveric<strong>in</strong> and have often been<br />
found to produce high quantities under laboratory conditions. These<br />
species are often found on maize and fruits.<br />
M<strong>in</strong>or sources. Several species of the Gibberella fujikuroi complex<br />
have been reported to produce beauveric<strong>in</strong> <strong>in</strong> low amounts, <strong>in</strong>clud<strong>in</strong>g<br />
F. nygamai, F. dlam<strong>in</strong>ii and F. verticillioides from cereals and fruits.<br />
The systematics of these Fusaria has developed dramatically dur<strong>in</strong>g<br />
the last years, so a lot of species specific <strong>in</strong>formation of tox<strong>in</strong> production<br />
is not available.<br />
F. avenaceum, F. poae and F. sporotrichioides on cereal gra<strong>in</strong>, fruits<br />
and vegetables are known to produce beauveric<strong>in</strong> <strong>in</strong> low amounts<br />
(Morrison et al., 2002; Thrane et al., 2004). In addition, F. sambuc<strong>in</strong>um<br />
and a few stra<strong>in</strong>s of F. acum<strong>in</strong>atum, F.equiseti and F. longipes<br />
from agricultural products have also been reported low producers of<br />
beauveric<strong>in</strong> (Logrieco et al., 1998).<br />
3.4.2. Enniat<strong>in</strong>s<br />
Enniat<strong>in</strong>s are a group of more than 15 related compounds produced<br />
by several Fusarium species, but also from Halosarpeia sp. and<br />
Verticillium hemipterigenum; however these are not of food orig<strong>in</strong>.<br />
Major sources. Fusarium avenaceum is the most important enniat<strong>in</strong><br />
producer <strong>in</strong> cereals and other agricultural food plants, because this<br />
species is a very frequent and consistent producer of enniat<strong>in</strong> B<br />
(Morrison et al., 2002). Fusarium sambuc<strong>in</strong>um is a consistent producer<br />
of enniat<strong>in</strong> B and diacetoxyscirpenol and causes dry rot <strong>in</strong> potatoes;<br />
however the role of these tox<strong>in</strong>s has not been exam<strong>in</strong>ed.
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 13<br />
M<strong>in</strong>or sources. F. langsethiae, F. poae and F. sporotrichioides, ma<strong>in</strong>ly<br />
occur on cereal gra<strong>in</strong>, F. lateritium from fruits and F. acum<strong>in</strong>atum<br />
from herbs.<br />
3.5. Fumonis<strong>in</strong>s<br />
S<strong>in</strong>ce the discovery of fumonis<strong>in</strong>s <strong>in</strong> the late 1980s much attention<br />
has been paid to these highly toxic compounds. Several reviews on the<br />
chemistry, toxicology and mycology have been published (Marasas<br />
et al., 2001; Weidenbörner, 2001).<br />
Major sources. F. verticillioides (formerly known as F. moniliforme;<br />
Seifert et al., 2003) and F. proliferatum are the ma<strong>in</strong> sources of fumonis<strong>in</strong>s<br />
<strong>in</strong> maize. These species and fumonis<strong>in</strong>s <strong>in</strong> maize and to a lesser<br />
extent other cereal crops have been reported from all over the world <strong>in</strong><br />
numerous papers and book chapters.<br />
M<strong>in</strong>or sources. Other fumonis<strong>in</strong> produc<strong>in</strong>g species are Fusarium<br />
nygamai, F. napiforme, F. thaps<strong>in</strong>um, F. anthophilum and F. dlam<strong>in</strong>i<br />
from millet, sorghum and rice. Some stra<strong>in</strong>s of these species have also<br />
been isolated from soil debris.<br />
3.6. Fusaprolifer<strong>in</strong><br />
Fusaprolifer<strong>in</strong> is a recent discovered mycotox<strong>in</strong> which shows teratogenic<br />
and pathological effects <strong>in</strong> cell assays (Bryden et al., 2001).<br />
Fusaprolifer<strong>in</strong> has been detected <strong>in</strong> natural samples together with<br />
beauveric<strong>in</strong> and fumonis<strong>in</strong> (Munkvold et al., 1998). Noth<strong>in</strong>g is known<br />
about a possible synergistic effect <strong>in</strong> such tox<strong>in</strong> comb<strong>in</strong>ations.<br />
Major sources. Fusarium proliferatum and F. subglut<strong>in</strong>ans are the<br />
major sources <strong>in</strong> maize and other cereal gra<strong>in</strong>s. The fungi and fusaprolifer<strong>in</strong><br />
have been detected <strong>in</strong> Europe, North America and South Africa<br />
(Wu et al., 2003).<br />
M<strong>in</strong>or sources. A few stra<strong>in</strong>s of F. globosum, F. guttiforme,<br />
F. pseudocirc<strong>in</strong>atum, F. pseudonygamai and F. verticillioides have been<br />
found to produce fusaprolifer<strong>in</strong>, however the systematics <strong>in</strong> this section<br />
of Fusarium has developed dramatically with<strong>in</strong> recent years so<br />
specific <strong>in</strong>formation on the tox<strong>in</strong> production by recently described<br />
species is unknown.<br />
3.7. Moniliform<strong>in</strong><br />
Moniliform<strong>in</strong> is cytotoxic, <strong>in</strong>hibits prote<strong>in</strong> synthesis and enzymes,<br />
causes chromosome damages and <strong>in</strong>duces heart failure <strong>in</strong> mammals
14 Jens C. Frisvad et al.<br />
and poultry (Bryden et al., 2001). Moniliform<strong>in</strong> has been found world<br />
wide <strong>in</strong> cereal samples<br />
Major sources. In maize F. proliferatum and F. subglut<strong>in</strong>ans are the<br />
ma<strong>in</strong> producers of moniliform<strong>in</strong>, whereas F. avenaceum and F. tric<strong>in</strong>ctum<br />
are the key sources <strong>in</strong> cereals grown <strong>in</strong> temperate climates.<br />
M<strong>in</strong>or sources. In sorghum, millet and rice F. napiforme, F. nygamai,<br />
F. verticillioides and F. thaps<strong>in</strong>um may be responsible for moniliform<strong>in</strong><br />
production. Some stra<strong>in</strong>s of F. oxysporum produce a<br />
significant amount of moniliform<strong>in</strong> under laboratory condition; however<br />
there is no detailed <strong>in</strong>formation on a possible production <strong>in</strong> vegetables<br />
and fruits. An overview of other m<strong>in</strong>or sources has been<br />
published (Schütt et al., 1998).<br />
3.8. Trichothecenes<br />
More than 200 trichothecenes have been identified and the nonmacrocyclic<br />
trichothecenes are among the most important mycotox<strong>in</strong>s.<br />
Trichothecenes are haematotoxic and immunosuppressive. In<br />
animals, vomit<strong>in</strong>g, feed refusal and diarrhoea are typical symptoms.<br />
Sk<strong>in</strong> oedema <strong>in</strong> humans has also been observed. An EU work<strong>in</strong>g<br />
group on has reported on trichothecenes <strong>in</strong> food (Schothorst and van<br />
Egmond, 2004).<br />
3.8.1. Deoxynivalenol (DON) and Acetylated Derivatives<br />
(3ADON, 15ADON)<br />
Deoxynivalenol (DON) and its acetylated derivatives (3ADON,<br />
15ADON) are by far the most important trichothecenes. Numerous<br />
reports on world-wide occurrence have been published and several<br />
<strong>in</strong>ternational symposia and workshops have focussed on DON<br />
(Larsen et al., 2004).<br />
Major sources. Fusarium gram<strong>in</strong>earum and F. culmorum are consistent<br />
producers of DON, especially <strong>in</strong> cereals. With<strong>in</strong> both species<br />
stra<strong>in</strong>s have been grouped <strong>in</strong>to those that produce DON and its derivatives,<br />
and those that produce nivalenol and furarenon X as their<br />
major metabolites. Intermediates have also been found (Nielsen and<br />
Thrane, 2001). Recently, F. gram<strong>in</strong>earum has been divided <strong>in</strong>to n<strong>in</strong>e<br />
phylogenetic species (O’Donnell et al., 2004); however <strong>in</strong> the present<br />
context this species concept will not be used as a correlation to exist<strong>in</strong>g<br />
mycotoxicological literature is impossible at this stage.<br />
M<strong>in</strong>or sources. Production of DON by F. pseudogram<strong>in</strong>earum has<br />
been reported, but this species is restricted to warmer climates.
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 15<br />
3.8.2. Nivalenol (NIV) and Fusarenon X (FX, 4ANIV)<br />
Nivalenol (NIV) and fusarenon X (FX, 4ANIV) occur <strong>in</strong> the same<br />
commodities as DON and are <strong>in</strong> many cases covered by the same surveys<br />
due to the high degree of similiarity. NIV is often detected <strong>in</strong> much<br />
lower concentrations than DON, but is considered to be more toxic.<br />
Major sources. Fusarium gram<strong>in</strong>earum is a well known producer of<br />
NIV and FX <strong>in</strong> cereals. In temperate climates F. poae, which is a consistent<br />
producer of NIV (Thrane et al., 2004), may be responsible for<br />
NIV <strong>in</strong> cereals.<br />
M<strong>in</strong>or sources. Stra<strong>in</strong>s of F. culmorum that produce NIV are less<br />
commonly isolated than those that produce DON producers. F. equiseti<br />
and F. crookwellense found <strong>in</strong> some cereal samples and <strong>in</strong> vegetables<br />
may also produce NIV. In potatoes F. venenatum stra<strong>in</strong>s that<br />
produce NIV have been detected (Nielsen and Thrane, 2001).<br />
3.8.3. T-2 tox<strong>in</strong><br />
T-2 tox<strong>in</strong> is one of the most toxic trichothecenes, whereas the<br />
derivative HT-2 tox<strong>in</strong> is less toxic. Due to structural similarity these<br />
tox<strong>in</strong>s are often <strong>in</strong>cluded <strong>in</strong> the same analytical method.<br />
Major sources. Fusarium sporotrichioides and F. langsethiae, frequently<br />
isolated from cereals <strong>in</strong> Europe, are consistent producers of<br />
T-2 and HT-2 (Thrane et al., 2004).<br />
M<strong>in</strong>or sources. Only a few T-2 and HT-2 produc<strong>in</strong>g stra<strong>in</strong>s of<br />
F. poae and F. sambuc<strong>in</strong>um have been found (Nielsen and Thrane,<br />
2001; Thrane et al., 2004).<br />
3.8.4. Diacetoxyscirpenol (DAS)<br />
Diacetoxyscirpenol (DAS) and monoacetylated derivatives (MAS)<br />
are a fourth group of important trichothecenes <strong>in</strong> food.<br />
Major sources. Fusarium venenatum isolates often produce high levels<br />
of DAS and this species is frequently isolated from cereals and<br />
potatoes (Nielsen and Thrane, 2001). F. poae isolates also often produce<br />
high levels of DAS.<br />
M<strong>in</strong>or sources. Fusarium equiseti isolates can produce DAS and<br />
MAS <strong>in</strong> high amounts, but this species is <strong>in</strong>frequently isolated from<br />
cereals and vegetables. F. sporotrichioides and F. langsethiae also<br />
produce DAS and MAS; however at lower levels (Thrane et al., 2004).<br />
F. sambuc<strong>in</strong>um isolates produce DAS and MAS and are a probable<br />
cause of DAS <strong>in</strong> potatoes (Ellner, 2002).
16 Jens C. Frisvad et al.<br />
3.9. Zearalenone<br />
Zearalenone causes hyperoestrogenism <strong>in</strong> sw<strong>in</strong>e and possible<br />
effects <strong>in</strong> humans have also been reported. Derivatives of zearalenone<br />
have been used as growth promoters <strong>in</strong> livestock; however this<br />
is now banned <strong>in</strong> European Union (Launay et al., 2004). The toxicity<br />
of zearalenone and its derivatives have been reviewed recently<br />
(Hagler et al., 2001).<br />
Major sources. Fusarium gram<strong>in</strong>earum and F. culmorum are the most<br />
pronounced producers of zearalenone and several derivatives. They<br />
occur frequently <strong>in</strong> cereals all over the world. Recently, F. gram<strong>in</strong>earum<br />
has been divided <strong>in</strong>to n<strong>in</strong>e phylogenetic species (O’Donnell<br />
et al., 2004); however <strong>in</strong> the present context this species concept will<br />
not be used as a correlation to exist<strong>in</strong>g mycotoxicological literature is<br />
impossible at this stage.<br />
M<strong>in</strong>or sources. Under laboratory conditions Fusarium equiseti produces<br />
a number of zearalenone derivatives <strong>in</strong> high amounts, but little<br />
is known about production under natural conditions. F. crookwellense<br />
also produces zearalenone.<br />
4. PENICILLIUM TOXINS<br />
4.1. Chaetoglobos<strong>in</strong>s<br />
The chaetoglobos<strong>in</strong>s are toxic compounds that may be <strong>in</strong>volved<br />
<strong>in</strong> mycotoxicosis. They are produced by common food-borne<br />
Penicillia and have been found to occur naturally (Andersen et al.,<br />
2004).<br />
Major sources. Penicillium expansum and P. discolor are major<br />
sources of the chaetoglobos<strong>in</strong>s. Both species cause spoilage <strong>in</strong> fruits<br />
and vegetables, and the latter species also occurs on cheese (Frisvad<br />
and Samson, 2004b).<br />
M<strong>in</strong>or sources. Chaetomium globosum and P. mar<strong>in</strong>um are probably<br />
not of significance <strong>in</strong> foods.<br />
4.2. Citreovirid<strong>in</strong><br />
Citreovirid<strong>in</strong> was reported as a cause of acute cardiac beriberi<br />
(Ueno, 1974), but a more <strong>in</strong> depth toxicological evaluation of this<br />
metabolite is needed. It has been associated with yellow rice disease,
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 17<br />
but this disease has also been associated with P. islandicum and<br />
its toxic metabolites cyclic peptides cyclochlorot<strong>in</strong>e and islanditox<strong>in</strong>,<br />
and anthraqu<strong>in</strong>ones luteoskyr<strong>in</strong> and rugulos<strong>in</strong> (Enomoto and<br />
Ueno, 1974).<br />
Major sources. Eupenicillium c<strong>in</strong>namopurpureum has been found <strong>in</strong><br />
cereals <strong>in</strong> USA and <strong>in</strong> Slovakia (Labuda and Tanc<strong>in</strong>ova, 2003) and is<br />
an efficient producer of citreovirid<strong>in</strong>. P. citreonigrum may be of some<br />
importance <strong>in</strong> yellowed rice.<br />
M<strong>in</strong>or sources. P. smithii, P. miczynskii and P. mang<strong>in</strong>ii (Frisvad and<br />
Filtenborg, 1990) have most often been recovered from soil and only<br />
rarely from foods. Aspergillus terreus has occasionally been reported<br />
from foods, but is primarily a soil-borne fungus.<br />
4.3. Citr<strong>in</strong><strong>in</strong><br />
Citr<strong>in</strong><strong>in</strong> is a nephrotox<strong>in</strong>, but probably of less importance than<br />
ochratox<strong>in</strong> A (Reddy and Berndt, 1991), however, producers of citr<strong>in</strong><strong>in</strong><br />
are widespread and common <strong>in</strong> foods. Citr<strong>in</strong><strong>in</strong> has been found <strong>in</strong><br />
cereals, peanuts and meat products (Reddy and Berndt, 1991).<br />
Major sources. P. citr<strong>in</strong>um is an efficient and consistent producer of<br />
citr<strong>in</strong><strong>in</strong> and has been found <strong>in</strong> foods world-wide (Pitt and Hock<strong>in</strong>g,<br />
1997). P. verrucosum is predom<strong>in</strong>antly cereal-borne <strong>in</strong> Europe and<br />
often produces citr<strong>in</strong><strong>in</strong> as well as ochratox<strong>in</strong> A (Frisvad et al., 2005b).<br />
P. expansum, common <strong>in</strong> fruits and other foods, sometimes produces<br />
citr<strong>in</strong><strong>in</strong>. P. radicicola is commonly found <strong>in</strong> onions, carrots and potatoes<br />
(Overy and Frisvad, 2003).<br />
M<strong>in</strong>or sources. Aspergillus terreus, A. carneus, P. odoratum and<br />
P. westl<strong>in</strong>gii have been reported as producers of citr<strong>in</strong><strong>in</strong>, but are not<br />
likely to occur often <strong>in</strong> foods.<br />
4.4. Cyclopiazonic acid (see also Aspergillus)<br />
Major sources. Penicillium commune and its domesticated form<br />
P. camemberti, and the closely related species P. palitans, are common<br />
on cheese and meat products and may produce cyclopiazonic acid <strong>in</strong><br />
these products (Frisvad et al., 2004c). P. griseofulvum is also a major<br />
producer of cyclopiazonic acid, and may occur <strong>in</strong> long stored cereals<br />
and cereal products such as pasta (Pitt and Hock<strong>in</strong>g, 1997).<br />
M<strong>in</strong>or sources. P. dipodomyicola occurs <strong>in</strong> the environs of the<br />
kangaroo rat <strong>in</strong> the USA, but has also been reported from rice <strong>in</strong><br />
Australia and <strong>in</strong> a chicken feed mixture <strong>in</strong> Slovakia (Frisvad and<br />
Samson, 2004b).
18 Jens C. Frisvad et al.<br />
4.5. Mycophenolic acid<br />
Despite hav<strong>in</strong>g a low acute toxicity, mycophenolic acid may be a<br />
very important <strong>in</strong>direct mycotox<strong>in</strong> as it highly immunosuppressive,<br />
perhaps <strong>in</strong>fluenc<strong>in</strong>g the course of bacterial and fungal <strong>in</strong>fections<br />
(Bentley, 2000).<br />
Major sources. Penicillium brevicompactum is a ubiquitous species<br />
and may produce mycophenolic acid <strong>in</strong> foods, e.g. g<strong>in</strong>ger (Overy and<br />
Frisvad, 2005). Two other major species produc<strong>in</strong>g mycophenolic acid<br />
are P. roqueforti and P. carneum. Another important producer is<br />
Byssochlamys nivea (Puel et al., 2005). Mycophenolic acid has been<br />
found to occur naturally <strong>in</strong> blue cheeses (Lafont et al., 1979).<br />
M<strong>in</strong>or sources. The soil-borne species Penicillium fagi also produces<br />
mycophenolic acid (Frisvad and Filtenborg, 1990, as P. raciborskii).<br />
Septoria nodorum (Devys et al., 1980) is another source but is unimportant<br />
as a food contam<strong>in</strong>ant.<br />
4.6. Ochratox<strong>in</strong> A (see also Aspergillus)<br />
Major sources. Penicillium verrucosum (Frisvad, 1985; Pitt, 1987) is<br />
the major producer of ochratox<strong>in</strong> A <strong>in</strong> cool climate stored cereals<br />
(Lund and Frisvad, 2003).<br />
Penicillium nordicum (Larsen et al., 2001) is the ma<strong>in</strong> OA producer<br />
found <strong>in</strong> manufactured meat products such as salami and ham. Both<br />
OA produc<strong>in</strong>g Penicillium species have been found on cheese also, but<br />
have only been reported to be of high occurrence on Swiss hard<br />
cheeses (as P. casei Staub, 1911). The ex type culture of P. casei is a<br />
P. verrucosum (Larsen et al., 2001).<br />
4.7. Patul<strong>in</strong><br />
Patul<strong>in</strong> is generally very toxic for both prokaryotes and eukaryotes,<br />
but the toxicity for humans has not been conclusively demonstrated.<br />
Several countries <strong>in</strong> Europe and the USA have now set limits on the<br />
level of patul<strong>in</strong> <strong>in</strong> apple juice.<br />
Major sources. Penicillium expansum is by far the most important<br />
source of patul<strong>in</strong>. P. expansum is the major species caus<strong>in</strong>g spoilage of<br />
apples and pears, and is the major source of patul<strong>in</strong> <strong>in</strong> apple juice and<br />
other apple and pear products.<br />
Byssochlamys nivea may be present <strong>in</strong> pasteurised fruit juices and<br />
may produce patul<strong>in</strong> and mycophenolic acid (Puel et al., 2005).
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 19<br />
Penicillium griseofulvum is a very efficient producer of high levels of<br />
patul<strong>in</strong> <strong>in</strong> pure culture, and it may potentially produce patul<strong>in</strong> <strong>in</strong> cereals,<br />
pasta and similar products.<br />
P. carneum may produce patul<strong>in</strong> <strong>in</strong> beer, w<strong>in</strong>e, meat products and<br />
rye-bread as it has been found <strong>in</strong> those substrates (Frisvad and<br />
Samson, 2004b), but there are no reports yet on patul<strong>in</strong> production by<br />
this species <strong>in</strong> those foods. P. carneum also produces mycophenolic<br />
acid, roquefort<strong>in</strong>e C and penitrem A (Frisvad et al., 2004c). P. paneum<br />
occurs <strong>in</strong> rye-bread (Frisvad and Samson, 2004b), but aga<strong>in</strong> actual<br />
production of patul<strong>in</strong> <strong>in</strong> this product has not been reported.<br />
P. sclerotigenum is common <strong>in</strong> yams and has the ability to produce<br />
patul<strong>in</strong> <strong>in</strong> laboratory cultures.<br />
M<strong>in</strong>or sources. The coprophilous fungi P. concentricum, P. clavigerum,<br />
P. coprobium, P. formosanum, P. glandicola, P. vulp<strong>in</strong>um,<br />
Aspergillus clavatus, A. longivesica and A. giganteus are very efficient<br />
producers of patul<strong>in</strong> <strong>in</strong> the laboratory, but only A. clavatus may play<br />
any role <strong>in</strong> human health, as it may be present <strong>in</strong> beer malt (Lopez-<br />
Diaz and Flannigan, 1997). Aspergillus terreus, Penicillium novae-zeelandiae,<br />
P. mar<strong>in</strong>um, P. mel<strong>in</strong>ii and other soil-borne fungi may produce<br />
patul<strong>in</strong> <strong>in</strong> pure culture, but are less likely to occur <strong>in</strong> any foods.<br />
4.8. Penicillic acid<br />
Penicillic acid (Alsberg and Black, 1911) and dehydropenicillic acid<br />
(Obana et al., 1995) are small toxic polyketides, but their major role <strong>in</strong><br />
mycotoxicology may be <strong>in</strong> their possible synergistic toxic effect with<br />
OA (L<strong>in</strong>denfelser at al., 1973; Stoev et al., 2001) and possible additive<br />
or synergistic effect with the naphtoqu<strong>in</strong>ones hepatotox<strong>in</strong>s xanthomegn<strong>in</strong>,<br />
viomelle<strong>in</strong> and vioxanth<strong>in</strong>.<br />
Major sources. Penicillic acid is likely to co-occur with OA, xanthomegn<strong>in</strong>,<br />
viomelle<strong>in</strong> and vioxanth<strong>in</strong> produced by members of<br />
Aspergillus section Circumdati and Penicillium series Viridicata (which<br />
often co-occur with P. verrucosum). The Aspergillus species often occur<br />
<strong>in</strong> coffee and the Penicillia are common <strong>in</strong> cereals. The major sources of<br />
penicillic acid are P. aurantiogriseum, P. cyclopium, P. melanoconidium<br />
and P. polonicum (Frisvad and Samson, 2004b) and all members of<br />
Aspergillus section Circumdati (Frisvad and Samson, 2000). Penicillic<br />
acid is produced by P. tulipae and P. radicicola, which are occasionally<br />
found on onions, carrots and potatoes (Overy and Frisvad, 2003).<br />
M<strong>in</strong>or sources. Penicillic acid has been found <strong>in</strong> one stra<strong>in</strong> of<br />
P. carneum (Frisvad and Samson, 2004b).
20 Jens C. Frisvad et al.<br />
4.9. Penitrem A<br />
Penitrem A is a highly toxic tremorgenic <strong>in</strong>dol-terpene. It has primarily<br />
been implicated <strong>in</strong> animal mycotoxicoses (Rundberget and<br />
Wilk<strong>in</strong>s, 2002), but has also been suspected to cause tremors <strong>in</strong><br />
humans (Cole et al., 1983; Lewis et al., 2005).<br />
Major sources. Penicillium crustosum is the most important producer<br />
of penitrem A (Pitt, 1979). This species is of world-wide distribution<br />
and often found <strong>in</strong> foods. This mycotox<strong>in</strong>s is produced by all<br />
isolates of P. crustosum exam<strong>in</strong>ed (Pitt, 1979; Sonjak et al., 2005).<br />
P. melanoconidium is common <strong>in</strong> cereals (Frisvad and Samson, 2004b),<br />
but it is not known whether this species can produce penitrem A <strong>in</strong><br />
<strong>in</strong>fected cereals.<br />
M<strong>in</strong>or sources. P. glandicola, P. clavigerum, and P. janczewskii are<br />
further producers of penitrem A (Ciegler and Pitt, 1970; Frisvad and<br />
Samson, 2004b; Frisvad and Filtenborg, 1990), but have been recovered<br />
from foods only sporadically.<br />
4.10. PR tox<strong>in</strong><br />
PR tox<strong>in</strong> is a mycotox<strong>in</strong> that is acutely toxic and can damage DNA<br />
and prote<strong>in</strong>s (Moule et al., 1980; Arnold et al., 1987). It is unstable <strong>in</strong><br />
cheese (Teuber and Engel, 1983), but it may be produced <strong>in</strong> silage and<br />
other substrates.<br />
Major sources. Penicillium roqueforti is the major source of PR<br />
tox<strong>in</strong>. It has been reported also from P. chrysogenum (Frisvad and<br />
Samson, 2004b).<br />
4.11. Roquefort<strong>in</strong>e C<br />
The status of roquefort<strong>in</strong>e C as a mycotox<strong>in</strong> has been questioned,<br />
but it is a very widespread fungal metabolite, and is produced by a<br />
large number of species. The acute toxicity of roquefort<strong>in</strong>e C is not<br />
very high (Cole and Cox, 1981), but it has been reported as a neurotox<strong>in</strong>.<br />
Major sources. Penicillium albocoremium, P. atramentosum, P. allii,<br />
P. carneum, P. chrysogenum, P. crustosum, P. expansum, P. griseofulvum,<br />
P. hirsutum, P. hordei, P. melanoconidium, P. paneum, P. radicicola,<br />
P. roqueforti, P. sclerotigenum, P. tulipae and P. venetum are all<br />
producers that have been found <strong>in</strong> foods, but the natural occurrence of<br />
roquefort<strong>in</strong>e C has been reported only rarely.
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 21<br />
M<strong>in</strong>or sources. P. concentricum, P. confertum, P. coprobium,<br />
P. coprophilum, P. flavigenum, P. glandicola, P. mar<strong>in</strong>um, P. persic<strong>in</strong>um<br />
and P. vulp<strong>in</strong>um are less likely food contam<strong>in</strong>ants.<br />
4.12. Rubratox<strong>in</strong><br />
Rubratox<strong>in</strong> is a potent hepatotox<strong>in</strong> (Engelhardt and Carlton,<br />
1991) and is of particular <strong>in</strong>terest as it has been implicated <strong>in</strong> severe<br />
liver damage <strong>in</strong> three Canadian boys, who drank rhubarb w<strong>in</strong>e contam<strong>in</strong>ated<br />
with Penicillium crateriforme. One of the boys needed to<br />
have the liver transplanted (Richer et al., 1997).<br />
Major producers. P. crateriforme is the only known major producer<br />
of rubratox<strong>in</strong> A and B (Frisvad, 1989).<br />
4.13. Secalonic Acid D<br />
The toxicological data on secalonic acid D and F are somewhat<br />
equivocal (Reddy and Reddy, 1991), so the significance of this<br />
metabolite <strong>in</strong> human and animal health is somewhat uncerta<strong>in</strong>.<br />
Major sources. Claviceps purpurea, Penicillium oxalicum, Phoma<br />
terrestris and Aspergillus aculeatus produce large amounts of<br />
secalonic acid D and F <strong>in</strong> pure culture. Secalonic acid D has been<br />
found to occur <strong>in</strong> gra<strong>in</strong> dust <strong>in</strong> USA (Palmgren, 1985; Reddy and<br />
Reddy, 1991).<br />
4.14. Verrucosid<strong>in</strong><br />
Verrucosid<strong>in</strong> is a of the mycotox<strong>in</strong> from species <strong>in</strong> Penicillium<br />
series Viridicata that has been claimed to cause mycotoxicosis <strong>in</strong> animals<br />
(Burka et al., 1983).<br />
Major sources. Penicillium polonicum, P. aurantiogriseum and<br />
P. melanoconidium are the major known sources of verrucosid<strong>in</strong><br />
(Frisvad and Samson, 2004b).<br />
4.15. Xanthomegn<strong>in</strong>, Viomelle<strong>in</strong> and Vioxanth<strong>in</strong><br />
These tox<strong>in</strong>s have been reported to cause experimental mycotoxicosis<br />
<strong>in</strong> pigs and they apparently are more toxic to the liver than to<br />
kidneys <strong>in</strong> mammals (Zimmerman et al., 1979). They have been found<br />
to be naturally occurr<strong>in</strong>g <strong>in</strong> cereals (Hald et al., 1983; Scudamore<br />
et al., 1986).
22 Jens C. Frisvad et al.<br />
Major sources. P. cyclopium, P. freii, P. melanoconidium, P. tricolor<br />
and P. viridicatum are common <strong>in</strong> cereals. A. ochraceus, A. westerdijkiae<br />
and possibly A. steynii are common <strong>in</strong> green coffee beans and<br />
are occasionally found <strong>in</strong> grapes and on rice.<br />
M<strong>in</strong>or sources. P. janth<strong>in</strong>ellum and P. mariaecrucis are soil-borne<br />
species produc<strong>in</strong>g these hepatotox<strong>in</strong>s (Frisvad and Filtenborg, 1990).<br />
5. TOXINS FROM OTHER GENERA<br />
5.1. Claviceps Tox<strong>in</strong>s<br />
Ergot alkaloids are common <strong>in</strong> sclerotia of Claviceps, which are<br />
produced on cereals, especially <strong>in</strong> whole rye. These sclerotia are often<br />
removed before mill<strong>in</strong>g of the rye, and outbreaks of ergotism rarely<br />
occur now.<br />
Major sources. Claviceps purpurea and C. paspali are the major<br />
sources of ergot alkaloids (Blum, 1995). Several Penicillia and<br />
Aspergilli can produce clav<strong>in</strong>et type alkaloids also, but their possible<br />
role <strong>in</strong> mycotoxicology is unknown.<br />
5.2. Alternaria Tox<strong>in</strong>s<br />
Tenuazonic acid is regarded as the most toxic of the secondary<br />
metabolites from Alternaria (Blaney, 1991). It is also produced by a<br />
Phoma species.<br />
Major sources. Phoma sorgh<strong>in</strong>a appears to be the most important<br />
producer of tenuazonic acid. It has been associated with onyalai, a<br />
haematological disease (Steyn and Rabie, 1976). Species <strong>in</strong> the<br />
Alternaria tenuissima complex often produce tenuazonic acid, but it<br />
has not been found <strong>in</strong> isolates of A. alternata sensu stricto. A. citri,<br />
A. japonica, A. kikuchiana, A. longipes, A. mali, A. oryzae, and A. solani<br />
have also been reported to produce tenuazonic acid (Sivanesan, 1991).<br />
Many other metabolites have been found <strong>in</strong> Alternaria, and some<br />
can occur naturally <strong>in</strong> tomatoes, apples and other fruits (Sivanesan,<br />
1991; Andersen and Frisvad, 2004). The toxicity of such compounds,<br />
<strong>in</strong>clud<strong>in</strong>g alternariols, is not well exam<strong>in</strong>ed.<br />
5.3. Phoma and Phomopsis Tox<strong>in</strong>s<br />
Lup<strong>in</strong>osis tox<strong>in</strong> (phomops<strong>in</strong>) is produced when Phomopsis leptostromiformis<br />
grows on lup<strong>in</strong> plants (Lup<strong>in</strong>us species) and lup<strong>in</strong> seeds
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 23<br />
(Culvenor et al., 1977). It is a hepatotox<strong>in</strong> which has caused widespread<br />
disease <strong>in</strong> sheep graz<strong>in</strong>g lup<strong>in</strong>s <strong>in</strong> Australia, South Africa and<br />
parts of Europe (Marasas, 1974; Culvenor et al., 1977). As lup<strong>in</strong> seed<br />
is used for human food <strong>in</strong> South Asia, quality control of phomops<strong>in</strong><br />
is important.<br />
5.4. Pithomyces Tox<strong>in</strong>s<br />
Sporidesm<strong>in</strong> is produced by Pithomyces chartarum and causes<br />
facial eczema <strong>in</strong> sheep (Atherton et al., 1974). However, this is a disease<br />
of pasture only.<br />
5.5. Stachybotrys Tox<strong>in</strong>s<br />
Stachybotrys and Memnoniella spp. are primarily of importance<br />
for <strong>in</strong>door air, but stachybotrytoxicosis was one of the first equ<strong>in</strong>e<br />
mycotoxicosis to be reported (Rodrick and Eppley, 1974).<br />
Stachybotrys chartarum and S. chlorohalonata are the two important<br />
fungi produc<strong>in</strong>g cyclic trichothecenes (satratox<strong>in</strong>s) and toxic atranones<br />
(Andersen et al., 2003; Jarvis, 2003).<br />
5.6. Monascus Tox<strong>in</strong>s<br />
Monascus ruber is used <strong>in</strong> the production of red rice <strong>in</strong> the Orient,<br />
and is a source of red food colour<strong>in</strong>g. However, it has been repeatedly<br />
reported to produce citr<strong>in</strong><strong>in</strong> (Blanc et al., 1995).<br />
6. DISCUSSION<br />
A large number of filamentous fungi are able to produce secondary<br />
metabolites that are toxic to vertebrate animals, i.e. mycotox<strong>in</strong>s.<br />
Only a fraction of these fungi can produce mycotox<strong>in</strong>s <strong>in</strong> food or<br />
feeds, and among those, pathogenic field fungi and deteriorat<strong>in</strong>g storage<br />
fungi are the most significant. When misidentified fungi are<br />
excluded, only a few fungal species are highly toxigenic, and produc<strong>in</strong>g<br />
their tox<strong>in</strong>s <strong>in</strong> sufficiently large amounts to cause public alarm.<br />
The most important among these are trichothecenes, fumonis<strong>in</strong>s, aflatox<strong>in</strong>,<br />
ochratox<strong>in</strong> A and zearalenone (Miller, 1995), because their fungal<br />
producers are widespread and can grow and produce their tox<strong>in</strong>s<br />
on many k<strong>in</strong>ds of foods. Other mycotox<strong>in</strong>s are important, but may
24 Jens C. Frisvad et al.<br />
only occur on a s<strong>in</strong>gle type of crop and cause mycotoxicosis <strong>in</strong> one<br />
k<strong>in</strong>d of animal. Phomops<strong>in</strong> is an example of this.<br />
Correct identification and knowledge of the associated mycobiota<br />
of the different foods and feeds will assist <strong>in</strong> determ<strong>in</strong><strong>in</strong>g which mycotox<strong>in</strong>s<br />
to look for. There have been examples of mycotox<strong>in</strong>s analysis<br />
for aflatox<strong>in</strong>, trichothecenes, zearalenone, fumonis<strong>in</strong> and ochratox<strong>in</strong><br />
A <strong>in</strong> silage, where the dom<strong>in</strong>ant mycobiota is P. roqueforti, P. paneum,<br />
Monascus ruber and Byssochlamys nivea. In that particular case patul<strong>in</strong>,<br />
mycophenolic acid, PR tox<strong>in</strong>, and citr<strong>in</strong><strong>in</strong> would be more relevant<br />
mycotox<strong>in</strong>s to analyse for. Rapid methods may are effective to<br />
secure healthy foods and feeds, but such methods should be based on<br />
mycological and ecological knowledge. We hope that our compilation<br />
of mycotox<strong>in</strong> producers will help <strong>in</strong> decid<strong>in</strong>g the most appropriate<br />
mycotox<strong>in</strong> analyses of foods and feeds. New mycotox<strong>in</strong>s and new<br />
mycotox<strong>in</strong> producers will no doubt appear, but we believe that the<br />
most important ones are listed here.<br />
7. ACKNOWLEDGEMENTS<br />
Jens Frisvad and Ulf Thrane thank LMC and the Technical<br />
Research Council for f<strong>in</strong>ancial support.<br />
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and their mycotox<strong>in</strong>s, <strong>in</strong>: Fungi and mycotox<strong>in</strong>s <strong>in</strong> stored products, B. R. Champ,<br />
E. Highley, A. D. Hock<strong>in</strong>g and J. I. Pitt, eds, ACIAR Proceed<strong>in</strong>gs No. 36,<br />
Australian Centre for International Agricultural Research, Canberra, pp. 47-64.
Important Mycotox<strong>in</strong>s and the Fungi Which Produce Them 31<br />
Sonjak, S., Frisvad, J. C. and Gunde-Cimerman, N., 2005, Comparison of secondary<br />
metabolite production by Penicillium crustosum stra<strong>in</strong>s, isolated from Arctic and<br />
other various ecological niches, FEMS Microbiol. Ecol. 53: 51-60.<br />
Staub, W., 1911, Penicillium casei n. sp. als Ursache die rotbraunen R<strong>in</strong>derfarbung bei<br />
Emmenthaler Käsen, Centrabl. f. Bakt. (II) 31:454.<br />
Steyn, P. S., and Rabie, C. J., 1976, Characterisation of magnesium and calcium tenuazonate<br />
from Phoma sorgh<strong>in</strong>a, Phytochemistry 15:1977-1979.<br />
Stoev, S. D., Vitanov, S., Anguelov, G., Petkova-Bocharova, T., and Creppy, E. E.,<br />
2001, Experimental mycotoxic nephropathy <strong>in</strong> pigs provoked by a diet conta<strong>in</strong><strong>in</strong>g<br />
ochratox<strong>in</strong> A and penicillic acid, Vet. Res. Commun. 25:205-223.<br />
Taniwaki, M. H., Pitt, J. I., Teixeira, A. A., and Iamanaka, B. T., 2003, The source of<br />
ochratox<strong>in</strong> A <strong>in</strong> Brazilian coffee and its formation <strong>in</strong> relation to process<strong>in</strong>g methods,<br />
Int. J. <strong>Food</strong> Microbiol. 82:173-179.<br />
Teuber, M., and Engel, G., 1983, Low risk of mycotox<strong>in</strong> production <strong>in</strong> cheese,<br />
Microbiol. Alim. Nutr. 1:193-197.<br />
Thrane, U., Adler, A., Clasen, P.-E., Galvano, F., Langseth, W., Lew, H., Logrieco, A.,<br />
Nielsen, K. F., and Ritieni, A., 2004, Diversity <strong>in</strong> metabolite production by<br />
Fusarium langsethiae, Fusarium poae, and Fusarium sporotrichioides, Int. J. <strong>Food</strong><br />
Microbiol. 95:257-266.<br />
Ueno, Y., 1974, Citreovirid<strong>in</strong> from Penicillium citreo-viride Biourge, <strong>in</strong>: Mycotox<strong>in</strong>s I.<br />
F. H. Purchase, ed., Elsevier, Amsterdam, pp. 283-302.<br />
Van der Merwe, K. J., Steyn, P. S., Fourie, L., Scott., D. B., and Theron, J. J., 1965,<br />
Ochratox<strong>in</strong> A, a toxic metabolite produced by Aspergillus ochraceus Wilh., Nature<br />
205:1112-1113.<br />
Varga, J., Tóth, B., Rigó, K, Téren, J, Hoekstra, R. F., and Kozakiewics, Z., 2000a,<br />
Phylogenetic analysis of Aspergillus section Circumdati based on sequences of the<br />
<strong>in</strong>ternal transcribed spacer regions of the 5.8 S rRNA gene, Fungal Gen. Biol.<br />
30:71-80.<br />
Varga, J., Kevei, É., Tóth, B., Kozakiewicz, Z., and Hoekstra, R. F., 2000b, Molecular<br />
analysis of variability with<strong>in</strong> the toxigenic Aspergillus ochraceus species, Can.<br />
J. Microbiol. 46:593-599.<br />
Weidenbörner, M., 2001, <strong>Food</strong> and fumonis<strong>in</strong>s, Eur. <strong>Food</strong> Res. Technol. 212:262-273.<br />
Wu, X., Leslie, J. F., Thakur, R. A., and Smith, J. S., 2003, Purifiction of fusaprolifer<strong>in</strong><br />
form cultures of Fusarium subglut<strong>in</strong>ans by preparative high-performance<br />
liquid chromatography, J. Agric. <strong>Food</strong> Chem. 51: 383-388.<br />
Yates, S. G., Tookey, H. L., Ellis, J. J., Tallent, W. H., and Wolff, I. A., 1969,<br />
Mycotox<strong>in</strong>s as a possible cause of fescue toxicity, J. Agric. <strong>Food</strong> Chem. 17:437-442.<br />
Zimmerman, J. L., Carlton, W. W., and Tuite, J., 1979, Mycotoxicosis produced by<br />
cultural products of an isolate of Aspergillus ochraceus. 1. Cl<strong>in</strong>ical observations<br />
and pathology, Vet. Pathol. 16:583-592.
RECOMMENDATIONS CONCERNING THE<br />
CHRONIC PROBLEM OF<br />
MISIDENTIFICATION OF<br />
MYCOTOXIGENIC FUNGI ASSOCIATED<br />
WITH FOODS AND FEEDS<br />
Jens C. Frisvad, Kristian F. Nielsen and Robert A. Samson *<br />
1. INTRODUCTION<br />
S<strong>in</strong>ce the aflatox<strong>in</strong>s were first reported <strong>in</strong> 1961 from Aspergillus<br />
flavus, mycotox<strong>in</strong>s have often been named after the fungus which was<br />
first found to produce them. A long list of connections between fungal<br />
species and mycotox<strong>in</strong>s and antibiotics has been reported, but<br />
unfortunately many of the identifications, and hence the connection<br />
between mycotox<strong>in</strong> name and the source of the tox<strong>in</strong>, are <strong>in</strong>correct<br />
(Frisvad, 1989). The most famous example of such <strong>in</strong>correct connections<br />
was Alexander Flem<strong>in</strong>g’s identification of the orig<strong>in</strong>al penicill<strong>in</strong><br />
producer as Penicillium rubrum. Fortunately, <strong>in</strong> this example, the substance<br />
was named after the genus Penicillium, rather than the species,<br />
as K. B. Raper re-identified the stra<strong>in</strong> as P. notatum, which was subsequently<br />
determ<strong>in</strong>ed to be a synonym of P. chrysogenum (Pitt, 1979b).<br />
Later, penicill<strong>in</strong> was found <strong>in</strong> other stra<strong>in</strong>s of P. chrysogenum (Raper<br />
and Thom, 1949).<br />
The early aflatox<strong>in</strong> literature is plagued with wrong reports of aflatox<strong>in</strong><br />
production by Penicillium puberulum (Hodges et al., 1964),<br />
* J. C. Frisvad and K. F. Nielsen: BioCentrum-DTU, Build<strong>in</strong>g 221, Technical<br />
University of Denmark, 2800 Lyngby, Denmark; R. A. Samson: Centraalbureau voor<br />
Schimmelcultures, PO Box 85167, 3508 AD, Utrecht, Netherlands. Correspondence<br />
to: jcf@biocentrum.dtu.dk<br />
33
34 Jens C. Frisvad et al.<br />
Aspergillus ostianus (Scott et al., 1967), Rhizopus sp. (Kulik and<br />
Holaday, 1966), the bacterium Streptomyces (Mishra and Murthy,<br />
1968) and several other taxa. The most famous of these reports was<br />
the paper of El-Hag and Morse (1976). They reported that Aspergillus<br />
oryzae, the domesticated species used <strong>in</strong> the manufacture of soy sauce<br />
and other Oriental fermented foods, produced aflatox<strong>in</strong>. However, the<br />
culture of A. oryzae they used was quickly shown to be contam<strong>in</strong>ated<br />
by an aflatox<strong>in</strong> produc<strong>in</strong>g A. parasiticus (Fennell, 1976). Immediate<br />
correction of this error did not prevent Adebajo et al. (1992), El-Kady<br />
et al. (1994), Atalla et al. (2003) or Drusch and Ragab (2003) report<strong>in</strong>g<br />
that A. oryzae produces aflatox<strong>in</strong>.<br />
Often, publications report<strong>in</strong>g mycotox<strong>in</strong> production are reviewed<br />
by people who have little or no understand<strong>in</strong>g of mycological<br />
taxonomy. For example, “P. patul<strong>in</strong>um” and “P. clavatus” are<br />
mentioned <strong>in</strong> Drusch and Ragab (2003). In Bhatnagar et al. (2002),<br />
“P. niger” is mentioned as produc<strong>in</strong>g ochratox<strong>in</strong> A. Each of these<br />
names is an <strong>in</strong>correct comb<strong>in</strong>ation of genus and species. Bhatnagar<br />
et al. (2002) give P. viridicatum as produc<strong>in</strong>g ochratox<strong>in</strong> A <strong>in</strong> a<br />
table, while us<strong>in</strong>g P. verruculosum as the species name <strong>in</strong> the text,<br />
confus<strong>in</strong>g it with P. verrucosum, the correct name for the producer<br />
of this tox<strong>in</strong>.<br />
Such mistakes could have been avoided. This paper provides a set of<br />
recommendations to be followed to ensure correct reports of connections<br />
between mycotox<strong>in</strong> production and fungal species.<br />
2. EXAMPLES OF INCORRECT CITATIONS<br />
OF SOME FUNGI PRODUCING WELL<br />
KNOWN MYCOTOXINS<br />
2.1. Aflatox<strong>in</strong><br />
The known producers of aflatox<strong>in</strong> are given <strong>in</strong> a separate paper <strong>in</strong><br />
these Proceed<strong>in</strong>gs (Frisvad et al., 2006). The list of other species that have<br />
been (<strong>in</strong>correctly) reported to produce aflatox<strong>in</strong>s <strong>in</strong>cludes Aspergillus<br />
flavo-fuscus, A. glaucus, A. niger, A. oryzae, A. ostianus, A. sulphureus,<br />
A. tamarii, A. terreus, A. terricola, A. wentii, Emericella nidulans (as<br />
A. nidulans), Emer. rugulosa (as A. rugulosus), Eurotium chevalieri, Eur.<br />
repens, Eur. rubrum, Mucor mucedo, Penicillium citr<strong>in</strong>um, P. citromyces,<br />
P. digitatum, P. frequentans, P. expansum, P. glaucum, P. puberulum,<br />
P. variabile, Rhizopus sp. and the bacterium Streptomyces sp. None of
Recommendations Concern<strong>in</strong>g the Chronic Problem 35<br />
these species produce aflatox<strong>in</strong>s, and many of these names are not<br />
accepted as valid species <strong>in</strong> any case.<br />
2.2. Sterigmatocyst<strong>in</strong><br />
Fungi known to produce sterigmatocyst<strong>in</strong> <strong>in</strong>clude Aspergillus versicolor,<br />
Emericella nidulans, several other Emericella species and some<br />
Chaetomium species. Although sterigmatocyst<strong>in</strong> is a precursor of aflatox<strong>in</strong>s<br />
(Frisvad, 1989), only Aspergillus ochraceoroseus (Frisvad et al.,<br />
1999; Klich et al., 2000), and some Emericella species accumulate both<br />
sterigmatocyst<strong>in</strong> and aflatox<strong>in</strong> (Frisvad et al., 2004a; Frisvad and<br />
Samson, 2004a). Species <strong>in</strong> Aspergillus section Flavi, which <strong>in</strong>cludes the<br />
major aflatox<strong>in</strong> producers, efficiently convert sterigmatocyst<strong>in</strong> <strong>in</strong>to<br />
3-methoxysterigmatocyst<strong>in</strong> and then <strong>in</strong>to aflatox<strong>in</strong>s (Frisvad et al., 1999).<br />
Many Aspergillus species have been reported to produce sterigmatocyst<strong>in</strong>,<br />
<strong>in</strong>correctly except for those cited above. Sterigmatocyst<strong>in</strong> production<br />
by Penicillium species has not been reported, apart from an<br />
obscure reference to Penicillium luteum (Dean, 1963). However,<br />
Wilson et al. (2002) claimed that P. camemberti, P. commune and<br />
P. griseofulvum produce sterigmatocyst<strong>in</strong>. Perhaps they mistook<br />
sterigmatocyst<strong>in</strong> for cyclopiazonic acid. Three Eurotium species have<br />
been claimed to produce sterigmatocyst<strong>in</strong> (Schroeder and Kelton,<br />
1975), but this was based only on unconfirmed TLC assays.<br />
Unfortunately the stra<strong>in</strong>s used were not placed <strong>in</strong> a culture collection.<br />
2.3. Ochratox<strong>in</strong> A<br />
Ochratox<strong>in</strong> A is produced by four ma<strong>in</strong> species, Aspergillus carbonarius,<br />
A. ochraceus, Petromyces alliaceus, Penicillium verrucosum,<br />
and a few other related species as detailed elsewhere (Frisvad and<br />
Samson, 2004b; Samson and Frisvad, 2004; Frisvad et al., 2006). A very<br />
large number of species have been claimed to produce ochratox<strong>in</strong> A, but<br />
not all will be detailed here. However, some of the names frequently<br />
cited <strong>in</strong> reviews will be mentioned. Of the Penicillia, P. viridicatum was<br />
the name cited for many years as the major ochratox<strong>in</strong> A producer, but<br />
it was shown that P. verrucosum was the correct name for this fungus,<br />
the only species that produces ochratox<strong>in</strong> A <strong>in</strong> cereals <strong>in</strong> Europe<br />
(Frisvad and Filtenborg,1983; Frisvad, 1985; Pitt. 1987). The closely<br />
related P. nordicum, which occurs on dried meat <strong>in</strong> Europe, was mentioned<br />
as produc<strong>in</strong>g ochratox<strong>in</strong> A by Frisvad and Filtenborg (1983) and<br />
Land and Hult (1987), but not accepted as a separate species until the<br />
publication of Larsen et al. (2001).
36 Jens C. Frisvad et al.<br />
P. verrucosum has been correctly cited as the ma<strong>in</strong> Penicillium<br />
species produc<strong>in</strong>g ochratox<strong>in</strong> A for a number of years now, but <strong>in</strong> a<br />
series of recent reviews and papers P. viridicatum and P. verruculosum<br />
(no doubt mistaken for P. verrucosum) have been mentioned aga<strong>in</strong><br />
(Mantle and McHugh, 1993; Bhatnagar et al., 2002; Czerwiecki et al.,<br />
2002a, b). In the latter two papers P. chrysogenum, P. cyclopium,<br />
P. griseofulvum, P. solitum, Aspergillus flavus, A. versicolor and Eurotium<br />
glaucum were listed as ochratox<strong>in</strong> A producers. The stra<strong>in</strong> of P. solitum<br />
reported by Mantle and McHugh (1993) to produce ochratox<strong>in</strong> A<br />
were assigned more recently to P. polonicum, but neither species produces<br />
ochratox<strong>in</strong> A (Lund and Frisvad, 1994; 2003). These isolates<br />
were contam<strong>in</strong>ated by P. verrucosum. The reports by Czerwiecki et al.<br />
(2002 a, b) are more problematic <strong>in</strong> that the fungi have been discarded,<br />
so it will never be possible to check the results.<br />
The follow<strong>in</strong>g species were listed as ochratox<strong>in</strong> A producers by<br />
Varga et al. (2001): Aspergillus auricomus, A. fumigatus, A. glaucus,<br />
A. melleus, A. ostianus, A. petrakii, A. repens, A. sydowii, A. terreus,<br />
A. ustus, A. versicolor, A. wentii, Penicillium aurantiogriseum,<br />
P. canescens, P. chrysogenum, P. commune, P. corylophilum, P. cyaneum,<br />
P. expansum, P. fuscum, P. hirayamae, P. implicatum, P. janczewskii,<br />
P. mel<strong>in</strong>ii, P. miczynskii, P. montanense, P. purpurescens, P. purpurogenum,<br />
P. raistrickii, P. sclerotiorum, P. sp<strong>in</strong>ulosum,, P. simplicissimum,<br />
P. variabile and P. verruculosum. None of these species<br />
produces ochratox<strong>in</strong> A, and it seems clear that the authors have<br />
uncritically accepted lists from earlier reviews. In the recent<br />
Handbook of Fungal Secondary Metabolites (Cole and Schweikert,<br />
2003a, b; Cole et al., 2003), only two of the species cited as produc<strong>in</strong>g<br />
ochratox<strong>in</strong> A are correct: A. ochraceus and A. sulphureus. The<br />
others mentioned are not.<br />
2.4. Citr<strong>in</strong><strong>in</strong><br />
Citr<strong>in</strong><strong>in</strong> is produced by a number of species <strong>in</strong> Penicillium and<br />
Aspergillus, notably P. citr<strong>in</strong>um, P. expansum, P. verrucosum, A. carneus,<br />
A. niveus and an Aspergillus species resembl<strong>in</strong>g A. terreus (Frisvad,<br />
1989; Frisvad et al., 2004b), but not by Aspergillus oryzae or<br />
P. camemberti, as claimed by Bennett and Klich (2003). Critical<br />
check<strong>in</strong>g of the orig<strong>in</strong>al reports clearly did not occur. Many other<br />
species have been claimed to produce citr<strong>in</strong><strong>in</strong>, <strong>in</strong>clud<strong>in</strong>g A. ochraceus<br />
(Mantle and McHugh, 1993), A. wentii (Abu-Seidah, 2002) and<br />
Eurotium pseudoglaucum (El-Kady et al., 1994), but either fungus or<br />
mycotox<strong>in</strong> may have been misidentified <strong>in</strong> these cases.
Recommendations Concern<strong>in</strong>g the Chronic Problem 37<br />
2.5. Patul<strong>in</strong><br />
A number of species <strong>in</strong> different genera, notably Penicillium,<br />
Aspergillus and Byssochlamys, produce patul<strong>in</strong>. Among the most efficient<br />
producers of patul<strong>in</strong> are Aspergillus clavatus, A. giganteus, A. terreus,<br />
Byssochlamys nivea, P. carneum, P. dipodomyicola, Penicillium<br />
expansum, P. griseofulvum, P. mar<strong>in</strong>um, P. paneum and several dung<br />
associated Penicillia (Frisvad, 1989; Frisvad et al., 2004b). It is not,<br />
however, produced by species <strong>in</strong> all of the 42 genera listed by Steiman<br />
et al. (1989) and Okele et al. (1993). These papers <strong>in</strong>clude erroneous<br />
statements that Alternaria alternata, Fusarium culmorum, Mucor<br />
hiemalis, Trichothecium roseum and many others produce patul<strong>in</strong>. The<br />
production of patul<strong>in</strong> by Alternaria alternata was later reported by<br />
Laidou et al. (2001), and mentioned <strong>in</strong> a review by Drusch and Ragab<br />
(2003). However patul<strong>in</strong> was not found <strong>in</strong> hundreds of analyses of<br />
Alternaria extracts (Montemurro and Visconti, 1992), or <strong>in</strong> extracts<br />
from more than 200 Alternaria cultures tested by us at the Technical<br />
University of Denmark (B. Andersen, personal communication).<br />
2.6. Penitrem A<br />
Many species have been claimed to produce penitrem A, but most<br />
have been misidentifications of Penicillium crustosum (Pitt, 1979;<br />
Frisvad, 1989). Names given to isolates that were <strong>in</strong> fact P. crustosum<br />
<strong>in</strong>clude P. cyclopium, P. verrucosum var. cyclopium, P. verrucosum var.<br />
melanochlorum, P. viridicatum, P. commune, P. lanosum, P. lanosocoeruleum,<br />
P. granulatum, P. griseum, P. martensii, P. palitans and<br />
P. piceum (Frisvad, 1989). Other species which do produce penitrem<br />
A <strong>in</strong>clude P. carneum, P. melanoconidium, P. tulipae, P. janczewskii,<br />
P. glandicola and P. clavigerum (Frisvad et al., 2004b). Only the first<br />
three of these species are likely to occur <strong>in</strong> foods.<br />
2.7. Cyclopiazonic Acid<br />
Cyclopiazonic acid is produced by Aspergillus flavus, A. oryzae,<br />
A. tamarii, A. pseudotamarii, Penicillium camemberti, P. commune,<br />
P. dipodomyicola, P. griseofulvum and P. palitans (Goto et al., 1996;<br />
Huang et al., 1994; Pitt et al., 1986; Polonelli et al., 1987; Frisvad<br />
et al., 2004b). Cyclopiazonic acid was orig<strong>in</strong>ally isolated from and<br />
named after P. cyclopium CSIR 1082, but this fungus was reidentified<br />
as P. griseofulvum (Hermansen et al., 1984; Frisvad, 1989). Despite<br />
this, most reviews still cite P. cyclopium or P. aurantiogriseum [of which
38 Jens C. Frisvad et al.<br />
Pitt (1979) considered P. cyclopium to be a synonym] as producers<br />
(Scott, 1994; Bhatnagar, 2002; Bennett and Klich, 2003). Scott (1994)<br />
drew an <strong>in</strong>correct conclusion<br />
“α-cyclopiazonic acid is a metabolite of several Penicillium and Aspergillus<br />
species and is of Canadian <strong>in</strong>terest from two viewpo<strong>in</strong>ts. First, one of the<br />
important producers (P. aurantiogriseum, formerly P. cyclopium, Pitt et al.,<br />
1986), commonly occurs <strong>in</strong> stored Canadian gra<strong>in</strong>s...”<br />
Although P. aurantiogriseum no doubt occurs <strong>in</strong> cereal gra<strong>in</strong>s, it is<br />
not a producer of cyclopiazonic acid.<br />
Another example of an error be<strong>in</strong>g cited repeatedly is the claimed<br />
production of cyclopiazonic acid by Aspergillus versicolor (Ohmomo<br />
et al., 1973; cited by Bhatnagar et al., 2002) even though Domsch et al.<br />
(1980) and Frisvad (1989) had stated that the isolate described by<br />
Ohmomo et al. (1973) was correctly identified as A. oryzae, a wellknown<br />
producer of cyclopiazonic acid (Orth, 1977). Penicillium hirsutum,<br />
P. viridicatum, P. chrysogenum, P. nalgiovense, Aspergillus<br />
nidulans and A. wentii have also wrongly been claimed to produce<br />
cyclopiazonic acid (Cole et al., 2003; Abu-Seidah, 2003).<br />
2.8. Xanthomegn<strong>in</strong>, Viomelle<strong>in</strong> and Vioxanth<strong>in</strong><br />
Xanthomegn<strong>in</strong>, viomelle<strong>in</strong> and vioxanth<strong>in</strong> are nephrotox<strong>in</strong>s produced<br />
by all members of Aspergillus section Circumdati (Frisvad and<br />
Samson, 2000), Penicillium cyclopium, P. freii, P. melanoconidium,<br />
P. tricolor and P. viridicatum (Lund and Frisvad, 1994), and by P. janth<strong>in</strong>ellum<br />
and some other genera and species which do not occur <strong>in</strong><br />
foods. Some of these Penicillium species occur <strong>in</strong> cereals, so these tox<strong>in</strong>s<br />
have been found occurr<strong>in</strong>g naturally (Scudamore et al., 1986).<br />
These tox<strong>in</strong>s are not produced, however, by P. crustosum as reported<br />
by Hald et al. (1983), by P. oxalicum as reported by Lee and Skau<br />
(1981) or by A. nidulans, A. flavus, A. oryzae or A. terreus as reported<br />
by Abu-Seidah (2003).<br />
2.9. Penicillic Acid<br />
Penicillic acid is associated with Penicillium series Viridicata and<br />
Aspergillus section Circumdati (Lund and Frisvad, 1994; Frisvad<br />
and Samson, 2000; Frisvad et al., 2004). Production reported by<br />
P. roqueforti (Moubasher et al., 1978; Olivigni and Bullman, 1978)<br />
is now considered to be due to the similar species P. carneum<br />
(Boysen et al., 1996).
Recommendations Concern<strong>in</strong>g the Chronic Problem 39<br />
2.10. Rubratox<strong>in</strong>s<br />
Rubratox<strong>in</strong>s are hepatoxic mycotox<strong>in</strong>s known to be produced only<br />
by the rare species Penicillium crateriforme (Frisvad, 1989).<br />
Rubratox<strong>in</strong>s are not produced by P. rubrum, P. purpurogenum or<br />
Aspergillus ochraceus as reported by Moss et al. (1968), Natori et al.<br />
(1970) and Abu-Seibah (2003).<br />
2.11. Trichothecenes<br />
Trichothecenes are especially troublesome as it is only after the<br />
<strong>in</strong>troduction of capillary gas chromatography coupled to mass spectrometry<br />
(MS) and more recently the <strong>in</strong>troduction of liquid chromatography<br />
comb<strong>in</strong>ed with atmospheric ionization MS that reliable<br />
methods have been available for these mycotox<strong>in</strong>s. Because immunochemical<br />
methods have been improved <strong>in</strong> recent years they also can<br />
now be considered valid. However results from TLC and HPLC based<br />
methods are dubious, unless comb<strong>in</strong>ed with immunoaff<strong>in</strong>ity cleanup,<br />
as many authors have neglected very time consum<strong>in</strong>g but crucial<br />
clean-up steps.<br />
Trichothecene have been reported to be produced by several<br />
Fusarium species as detailed elsewhere <strong>in</strong> these proceed<strong>in</strong>gs (Frisvad<br />
et al., 2006). Marasas et al. (1984) showed that Fusarium nivale, which<br />
gave nivalenol its name, does not produce trichothecenes. However,<br />
under its newer, correct name, Microdochium nivale was still <strong>in</strong>correctly<br />
cited as a trichothecene producer <strong>in</strong> a recent review (Bhatnagar<br />
et al., 2002). It has even been claimed recently that Aspergillus species<br />
(A. oryzae, A. terreus, A. parasiticus and A. versicolor) produce<br />
nivalenol, deoxynivalenol and T-2 tox<strong>in</strong> (Atilla et al., 2003). A. parasiticus<br />
was claimed to produce very high amounts of deoxynivalenol<br />
and T-2 tox<strong>in</strong> after growth on wheat held at 80% relative humidity for<br />
1-2 months. These data are totally implausible. Possibly the wheat was<br />
already contam<strong>in</strong>ated with trichothecenes before use, but the high levels<br />
<strong>in</strong>dicate that there may have been false positives as well.<br />
3. RECOMMENDATIONS<br />
To avoid <strong>in</strong>correct report<strong>in</strong>g of fungal species produc<strong>in</strong>g particular<br />
mycotox<strong>in</strong>s, we recommend the follow<strong>in</strong>g rules when work<strong>in</strong>g with<br />
mycotox<strong>in</strong> produc<strong>in</strong>g fungi and the report<strong>in</strong>g of the results:
40 Jens C. Frisvad et al.<br />
3.1. Ensure correct identification and purity of fungal<br />
isolates<br />
● Fungal isolates from the particular substrate should be checked with<br />
the literature on the mycobiota of foods, e.g. Filtenborg et al. (1996),<br />
Pitt and Hock<strong>in</strong>g (1997) or Samson et al. (2004), which correlate<br />
particular fungal species with particular food types or substrates.<br />
Unusual f<strong>in</strong>d<strong>in</strong>gs especially should be carefully checked. For example,<br />
Aspergillus oryzae is the domesticated form of A. flavus and A.<br />
sojae is the domesticated form of A. parasiticus, and these fungi are<br />
not expected to be isolated other than from production plants used<br />
for mak<strong>in</strong>g Oriental foods or enzymes.<br />
● Use typical cultures as reference for comparison, both for identification<br />
and mycotox<strong>in</strong> production. Frisvad et al. (2000), lists typical<br />
cultures for each species of common foodborne Penicillium subgenus<br />
Penicillium species. Some effective mycotox<strong>in</strong> produc<strong>in</strong>g cultures<br />
are listed <strong>in</strong> Table 1.<br />
● Check the purity of cultures, as contam<strong>in</strong>ated cultures are a very<br />
common problem. Check for contam<strong>in</strong>ants by grow<strong>in</strong>g cultures on<br />
standard media such as CYA (Pitt and Hock<strong>in</strong>g, 1997). Especially<br />
when fungi are grown on cereals or liquid cultures it is very difficult<br />
to assess if the culture is pure, and it necessary to streak them out on<br />
agar substrates where it is much easier to see if the culture is pure.<br />
Table 1. Reference cultures for the production of the more common Aspergillus and<br />
Penicillium mycotox<strong>in</strong>s<br />
Mycotox<strong>in</strong> Produc<strong>in</strong>g species and reference culture<br />
Aflatox<strong>in</strong>s B and B Aspergillus parasiticus CBS 1 2<br />
a 100926<br />
Aspergillus flavus CBS 573.65<br />
Aflatox<strong>in</strong>s G 1 and G 2<br />
Aspergillus parasiticus CBS 100926<br />
Sterigmatocyst<strong>in</strong> Aspergillus versicolor CBS 563.90<br />
Ochratox<strong>in</strong> A Petromvces alliaceus CBS 110.26<br />
Penicillium verrucosum CBS 223.71<br />
Patul<strong>in</strong> Aspergillus clavatus CBS 104.45<br />
Penicillium griseofulvum CBS 295.97<br />
Cyclopiazonic acid Penicillium griseofulvum CBS 295.97<br />
Roquefort<strong>in</strong>e C Penicillium griseofulvum CBS 295.97<br />
Citr<strong>in</strong><strong>in</strong> Penicillium citr<strong>in</strong>um CBS 252.55<br />
Penicillium verrucosum CBS 223.71<br />
Penicillic acid Penicillium cyclopium CBS 144.45<br />
Penitrem A Penicillium crustosum CBS 181.89<br />
Verrucosid<strong>in</strong> Penicillium polonicum CBS 101479<br />
Xanthomegn<strong>in</strong> Penicillium cyclopium CBS 144.45<br />
Rubratox<strong>in</strong> B Penicillium crateriforme CBS 113161<br />
aCBS = Culture collection of the Centraalbureau voor Schimmelcultures, Utrecht,<br />
Netherlands
Recommendations Concern<strong>in</strong>g the Chronic Problem 41<br />
● If unusual producers are found, check them carefully for purity and<br />
correct identity us<strong>in</strong>g the references cited above. A specialist taxonomist<br />
may be consulted.<br />
3.2. Ensure that cultures are deposited <strong>in</strong> a recognised<br />
culture collection<br />
● Deposit all <strong>in</strong>terest<strong>in</strong>g stra<strong>in</strong>s produc<strong>in</strong>g mycotox<strong>in</strong>s <strong>in</strong> <strong>in</strong>ternational<br />
culture collections, and cite the culture collection numbers<br />
<strong>in</strong> any publications regard<strong>in</strong>g the stra<strong>in</strong>s. This procedure<br />
should be mandatory for all microbial, biochemical and chemical<br />
journals.<br />
3.3. Ensure substrate is sterile and not already<br />
contam<strong>in</strong>ated with mycotox<strong>in</strong>s<br />
● If natural substrates, such as cereals, are used for mycotox<strong>in</strong> production,<br />
they should be sterilised before use (e.g. by autoclav<strong>in</strong>g or<br />
by gamma-irradiation). Control assays should be carried out for all<br />
mycotox<strong>in</strong>s be<strong>in</strong>g studied on the material <strong>in</strong>tended for use as the<br />
substrate. This will ensure false positives are not reported.<br />
● Also check for <strong>in</strong>terfer<strong>in</strong>g peaks. Natural substrates such as gra<strong>in</strong>s<br />
may conta<strong>in</strong> <strong>in</strong>terfer<strong>in</strong>g compounds, and the chemical composition<br />
of these matrices may change dur<strong>in</strong>g fungal growth. In such matrices,<br />
highly selective cleanup procedures should be used and comb<strong>in</strong>ed<br />
with highly selective analytical methods.<br />
3.4. Ensure optimal conditions for mycotox<strong>in</strong> production<br />
are used<br />
● Use several media and growth conditions to ensure that the fungus<br />
can actually produce the mycotox<strong>in</strong>s. Four good media for mycotox<strong>in</strong><br />
production are listed <strong>in</strong> Table 2.<br />
3.5. Ensure appropriate analytical and confirmatory<br />
procedures for mycotox<strong>in</strong> extraction and<br />
identification<br />
● Sample preparation methods are important and should be validated.<br />
Sample preparation is specific for the food matrix it is<br />
designed for. Use only validated analytical methods.
42 Jens C. Frisvad et al.<br />
Table 2. Efficient media for mycotox<strong>in</strong> production<br />
Czapek Yeast Autolysate agar (CYA) Yeast Extract Sucrose agar (YES)<br />
(Pitt, 1979; Pitt and Hock<strong>in</strong>g, 1997) (Frisvad and Filtenborg, 1983)<br />
NaNO 3 3 g Yeast extract (Difco) 20 g<br />
K 2 HPO 4 1 g Sucrose 150 g<br />
KCl 0.5 g MgSO 4 • 7H 2 O 0.5 g<br />
MgSO 4 • 7H 2 O 0.5 g ZnSO 4 • 7H 2 O 0.01 g<br />
FeSO 4 • 7H 2 O 0.01 g CuSO 4 • 5H 2 O 0.005 g<br />
ZnSO 4 • 7H 2 O 0.01 g Agar 20 g<br />
CuSO 4 • 5H 2 O 0.005 g Distilled water l litre<br />
Yeast extract (Difco) 5 g<br />
Sucrose 30 g<br />
Agar 20 g<br />
Distilled water l litre<br />
Rice powder Corn steep agar Mercks Malt Extract (MME) agar<br />
(RC) (Bullerman, 1974) (El-Banna and Leistner, 1988)<br />
Rice powder 50 g Malt extract 30 g<br />
Corn steep liquid 40 g Soy peptone 3 g<br />
ZnSO 4 • 7H 2 O 0.01 g ZnSO 4 • 7H 2 O 0.01 g<br />
CuSO 4 • 5H 2 O 0.005 g CuSO 4 • 5H 2 O 0.005 g<br />
Agar 20 g Agar 20 g<br />
Distilled water l litre Distilled water l litre<br />
pH 5.6<br />
● Use efficient extraction techniques, for example, fumonis<strong>in</strong>s are very<br />
polar and penitrem A is very apolar. Extractions should be validated<br />
by recovery experiments.<br />
● Use authenticated standards of the mycotox<strong>in</strong>s for comparison, ideally<br />
as <strong>in</strong>ternal and external standards.<br />
● More than one separation technique should be use, comb<strong>in</strong>ed with<br />
selective detection pr<strong>in</strong>ciples. S<strong>in</strong>gle UV, refractive <strong>in</strong>dex, evaporative<br />
light scatter<strong>in</strong>g, or flame ionisation detection are non-specific.<br />
Fluorescence and full UV spectra are specific to some compounds,<br />
while mass spectrometry and especially tandem mass spectrometry is<br />
very selective for most compounds when monitor<strong>in</strong>g several ions.<br />
Generally four identification po<strong>in</strong>ts should give a very specific detection,<br />
e.g. obta<strong>in</strong>ed by LC-MS/MS monitor<strong>in</strong>g two fragmentation<br />
reactions.<br />
● Use more than one discretionary test to secure correct identification<br />
of the mycotox<strong>in</strong>, Comb<strong>in</strong>ed these with derivatization or alternative<br />
clean-up procedures when f<strong>in</strong>d<strong>in</strong>g unexpected results.
Recommendations Concern<strong>in</strong>g the Chronic Problem 43<br />
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Pitt, J. I., Cruickshank, R. H., and Leistner, L., 1986, Penicillium commune, P. camembertii,<br />
the orig<strong>in</strong> of white cheese moulds, and the production of cyclopiazonic acid,<br />
<strong>Food</strong> Microbiol. 3:363-371.<br />
Polonelli, L., Morace, G., Rosa, R., Castagnola, M., and Frisvad, J. C., 1987,<br />
Antigenic characterization of Penicillium camemberti and related common cheese<br />
contam<strong>in</strong>ants, Appl. Environ. Microbiol. 53:872-878.<br />
Raper, K. B. and Thom, C., 1949, A Manual of the Penicillia, Williams and Wilk<strong>in</strong>s,<br />
Baltimore.<br />
Samson, R. A., and Frisvad, J. C., 2004, New ochratox<strong>in</strong> or sclerotium produc<strong>in</strong>g<br />
species <strong>in</strong> Aspergillus section Nigri, Stud. Mycol. 50:45-61.<br />
Samson, R. A., Hoekstra, E. S., and Frisvad, J. C., eds, 2004, Introduction to <strong>Food</strong>and<br />
Airborne Fungi, 7th edition, Centraalbureau voor Schimmelcultures, Utrecht,<br />
Netherlands, 389 pp.<br />
Schroeder, H. W., and Kelton, W. H., 1975, Production of sterigmatocyst<strong>in</strong> by some<br />
species of the genus Aspergillus and its toxicity to chicken embryos, Appl.<br />
Microbiol. 30:589-591.<br />
Scott, P. M., 1994, Penicillium and Aspergillus tox<strong>in</strong>s, <strong>in</strong>: Mycotox<strong>in</strong>s <strong>in</strong> Gra<strong>in</strong>.<br />
Compounds other than Aflatox<strong>in</strong>. J. D. Miller and H. L. Trenholm, H. L., eds,<br />
Eagan Press, St. Paul, M<strong>in</strong>nesota, pp. 261-285.<br />
Scott, P. M., van Walbeek, W., and Forgacs, J., 1967, Formation of aflatox<strong>in</strong>s by<br />
Aspergillus ostianus Wehmer, Appl. Microbiol. 15:945.<br />
Scudamore, K. A., Atk<strong>in</strong>, P., and Buckle, A. E., 1986, Natural occurrence of the<br />
naphthoqu<strong>in</strong>one mycotox<strong>in</strong>s, xanthomegn<strong>in</strong>, viomelle<strong>in</strong> and vioxanth<strong>in</strong> <strong>in</strong> cereals<br />
and animal foodstuffs, J. Stored Prod. Res. 22:81-84.<br />
Steiman R., Seigle-Murandi, F., Sage, L., and Krivobok S., 1989, Production of patul<strong>in</strong><br />
by micromycetes, Mycopathologia 105:129-133.<br />
Varga, J., Rigó, K., Réren, J., and Mesterházy, Á., 2001, Recent advances <strong>in</strong> ochratox<strong>in</strong><br />
research. I. Production, detection and occurrence of ochratox<strong>in</strong>s, Cereal Res.<br />
Commun. 29:85-92.<br />
Wilson, D. M., Mutabanhema, W., and Jurjevic, Z., 2002, Biology and ecology of<br />
mycotoxigenic Aspergillus species as related to economy and health concerns, <strong>in</strong>:<br />
Mycotox<strong>in</strong>s and <strong>Food</strong> Safety, J. W. DeVries, M. W. Trucksess and L. S. Jackson, eds,<br />
Kluwer Academic Publishers, Dordrech, Netherlands. pp. 3-17.
Section 2.<br />
Media and method development<br />
<strong>in</strong> food mycology<br />
Comparison of hyphal length, ergosterol, mycelium dry weight and colony<br />
diameter for quantify<strong>in</strong>g growth of fungi from foods<br />
Marta H. Taniwaki, John I. Pitt, Ailsa D. Hock<strong>in</strong>g and Graham H. Fleet<br />
Evaluation of molecular methods for the analysis of yeasts <strong>in</strong> foods and beverages<br />
Ai L<strong>in</strong> Beh, Graham H. Fleet, C. Prakitchaiwattana and Gillian M. Heard<br />
Standardization of methods for detect<strong>in</strong>g heat resistant fungi<br />
Jos Houbraken and Robert A. Samson
COMPARISON OF HYPHAL LENGTH,<br />
ERGOSTEROL, MYCELIUM DRY WEIGHT,<br />
AND COLONY DIAMETER FOR<br />
QUANTIFYING GROWTH OF FUNGI FROM<br />
FOODS<br />
M. H. Taniwaki, J. I. Pitt, A. D. Hock<strong>in</strong>g and G. H. Fleet *<br />
1. INTRODUCTION<br />
Fungi are significant environmental microorganisms, as they are<br />
responsible for spoilage of foods, production of mycotox<strong>in</strong>s and <strong>in</strong><br />
some cases desirable bioconversions. It is important therefore to have<br />
reliable, convenient methods for measur<strong>in</strong>g fungal growth. However,<br />
the growth of fungi is not easy to quantify because, unlike bacteria<br />
and yeasts, fungi do not grow as s<strong>in</strong>gle cells, but as hyphal filaments<br />
that cannot be quantified by the usual enumeration techniques.<br />
Fungal hyphae can penetrate solid substrates, such as foods, mak<strong>in</strong>g<br />
their extraction difficult. In addition, fungi differentiate to produce<br />
spores, result<strong>in</strong>g <strong>in</strong> large <strong>in</strong>creases <strong>in</strong> viable counts often with little<br />
relationship to biomass (Pitt, 1984).<br />
A number of methods have been developed for quantify<strong>in</strong>g fungal<br />
growth and their pr<strong>in</strong>ciples and applications comprehensively<br />
reviewed (Matcham et al.,1984; Hartog and Notermans, 1988;<br />
Williams, 1989; Newell, 1992; Samson et al., 1992; de Ruiter et al.,<br />
1993; Pitt and Hock<strong>in</strong>g, 1997). The most frequently used method is<br />
* M. H. Taniwaki, Instituto de Tecnologia de Alimentos, Camp<strong>in</strong>as-Sp, Brazil; J. I.<br />
Pitt, A. D. Hock<strong>in</strong>g, <strong>Food</strong> Science Australia, PO Box 52, North Ryde, NSW 2113,<br />
Australia; G. H. Fleet, <strong>Food</strong> Science and Technology, University of New South Wales,<br />
Sydney, NSW 2052, Australia. Correspondence to: mtaniwak@ital.sp.gov.br<br />
49
50 M. H. Taniwaki et al.<br />
the count<strong>in</strong>g of viable propagules, i.e. colony form<strong>in</strong>g units (CFU), a<br />
technique derived from food bacteriology. However, this method suffers<br />
from serious drawbacks. Viable counts usually reflect spore numbers<br />
rather than biomass (Pitt, 1984). When fungal growth consists<br />
predom<strong>in</strong>antly of hyphae, i.e. <strong>in</strong> young colonies or <strong>in</strong>side food particles,<br />
viable counts will be low, but when sporulation occurs, counts<br />
often <strong>in</strong>crease rapidly without any great <strong>in</strong>crease <strong>in</strong> biomass. Some<br />
fungal genera, e.g. Alternaria and Fusarium, produce low numbers of<br />
spores <strong>in</strong> relation to hyphal growth, whereas others, e.g. Penicillium,<br />
produce very high numbers of spores. Consequently, viable counts are<br />
a poor <strong>in</strong>dicator of the extent of fungal growth and appear to correlate<br />
poorly with other measures such as ergosterol (Saxena et al.,<br />
2001).<br />
A second commonly used method is measurement of colony diameter<br />
(Brancato and Gold<strong>in</strong>g, 1953). When measured over several time<br />
<strong>in</strong>tervals, colony diameters can be translated <strong>in</strong>to growth rates, which<br />
are frequently l<strong>in</strong>ear over quite long periods (Pitt and Hock<strong>in</strong>g, 1977)<br />
and have been widely used <strong>in</strong> water activity studies (e.g. Pitt and<br />
Hock<strong>in</strong>g, 1977; Pitt and Miscamble, 1995) and to model growth<br />
(Gibson et al., 1994). However colony diameter as a measure of fungal<br />
biomass takes no account of colony density (Wells and Uota,<br />
1970).<br />
Estimation of mycelium dry weight is a third commonly used<br />
method to assess fungal growth or biomass. This is the method of<br />
choice for growth <strong>in</strong> liquid systems, such as fermentors, however,<br />
mycelium dry weight measurements lack sensitivity and are destructive<br />
(Deploey and Fergus, 1975). A fourth approach, measurement<br />
of hyphal length, was used by Schnürer (1993) to estimate the biomass<br />
of three fungi grown <strong>in</strong> pure culture. This technique has the<br />
advantage of actually measur<strong>in</strong>g growth, but is particularly laborious.<br />
None of these methods can be used to estimate fungal biomass<br />
<strong>in</strong> foods.<br />
Chemical assays have also been used to measure fungal growth. The<br />
two substances commonly assayed are chit<strong>in</strong> and ergosterol. The chit<strong>in</strong><br />
assay is well documented (Ride and Drysdale, 1972), but has major<br />
disadvantages: it lacks sensitivity, is time consum<strong>in</strong>g, and is subject to<br />
<strong>in</strong>terference from <strong>in</strong>sect fragments (Pitt and Hock<strong>in</strong>g, 1997).<br />
Ergosterol is the dom<strong>in</strong>ant sterol <strong>in</strong> most fungi (Weete, 1974), and is<br />
not found to any significant extent <strong>in</strong> plants, animals or bacteria<br />
(Schwardorf and Muller, 1989). Thus, its measurement <strong>in</strong> environmental<br />
samples can be taken as an <strong>in</strong>dex of the presence of fungi<br />
(Seitz et al., 1977, 1979; Nylund and Wallander, 1992; Miller and
Comparison of Hyphal Length, Ergosterol, Mycelium Dry Weight 51<br />
Young, 1997). The advantages of this method are high sensitivity,<br />
specificity and relatively short analysis time (Seitz et al., 1977;<br />
Schwardorf and Muller, 1989). However, the ergosterol assay has<br />
never been validated aga<strong>in</strong>st more traditional methods, an essential<br />
step before it can be accepted as a reliable method for quantify<strong>in</strong>g fungal<br />
growth <strong>in</strong> foods. In addition, the <strong>in</strong>fluences of such factors as<br />
medium composition, water activity and age of colony on the ergosterol<br />
content of mycelium have not been evaluated adequately. One<br />
study has attempted to compare ergosterol content with mycelial dry<br />
weight over a range of species: for n<strong>in</strong>e aquatic fungi, only three<br />
showed correlations between these parameters (Berm<strong>in</strong>gham et al.,<br />
1995). Studies compar<strong>in</strong>g ergosterol content with mould viable counts<br />
have reported mixed results <strong>in</strong> gra<strong>in</strong>s (Schnürer and Jonsson, 1992)<br />
and pure cultures (Saxena et al., 2001).<br />
This paper reports a comparison of the ergosterol, colony diameter,<br />
dry weight and hyphal length methods for quantify<strong>in</strong>g the growth of<br />
several fungal species significant <strong>in</strong> foods. Studies were carried out <strong>in</strong><br />
pure culture under a range of conditions.<br />
2. MATERIALS AND METHODS<br />
2.1. Fungi<br />
S<strong>in</strong>gle isolates of n<strong>in</strong>e food spoilage fungi, represent<strong>in</strong>g examples<br />
of heat resistant, xerophilic and toxigenic species commonly found <strong>in</strong><br />
foods, were obta<strong>in</strong>ed from the FRR culture collection at <strong>Food</strong> Science<br />
Australia North Ryde, NSW, Australia (Table 1). These species were<br />
Aspergillus flavus, Byssochlamys fulva, Byssochlamys nivea, Eurotium<br />
chevalieri, Fusarium oxysporum, Mucor plumbeus, Penicillium commune,<br />
Penicillium roqueforti and Xeromyces bisporus.<br />
2.2. Media<br />
The follow<strong>in</strong>g media were used: Czapek Yeast Extract Agar<br />
(CYA), Malt Extract Agar (MEA) and Potato Dextrose Agar (PDA)<br />
representative of high water activity (a w ) media, (all of ca 0.997 a w );<br />
and Czapek Yeast Extract 20% Sucrose Agar (CY20S), 0.98 a w and<br />
Malt Extract Yeast Extract 50% Glucose Agar (MY50G), 0.89 a w ,as<br />
reduced a w media. PDA was from Oxoid Ltd, Bas<strong>in</strong>gstoke, UK, and<br />
the formulae for the others are given by Pitt and Hock<strong>in</strong>g (1997).
52 M. H. Taniwaki et al.<br />
Table 1. Orig<strong>in</strong>s of cultures useda Species Stra<strong>in</strong> number Source<br />
Aspergillus flavus FRR 2757 Peanut, Queensland, Australia, 1984<br />
Byssochlamys fulva FRR 3792 Strawberry puree, NSW, Australia, 1990<br />
Byssochlamys nivea FRR 4421 Strawberry, Brazil, 1993<br />
Eurotium chevalieri FRR 547 Animal feed, Queensland, Australia,<br />
1970<br />
Fusarium oxysporum FRR 3414 Orange juice, NSW, Australia, 1987<br />
Mucor plumbeus FRR 2412 Apple juice, NSW, Australia, 1981<br />
Penicillium commune FRR 3932 Cheddar cheese, NSW, Australia, 1991<br />
Penicillium roqueforti FRR 2162 Cheddar cheese, USA, 1978<br />
Xeromyces bisporus FRR 2351 Dates, NSW, 1981<br />
a FRR denotes the culture collection of <strong>Food</strong> Science Australia, North Ryde, NSW,<br />
Australia<br />
2.3. Cultivation<br />
Inocula were prepared from 5 to 7 day cultures grown on CYA,<br />
except for B. nivea, E. chevalieri and X. bisporus which were grown on<br />
MEA for 7 to 10 days, CY20S for 10 to 15 days and MY50G for 15 to<br />
20 days, respectively. Cultures for growth estimates and assays were<br />
grown <strong>in</strong> 90 mm plastic Petri dishes, <strong>in</strong>oculated at a s<strong>in</strong>gle central<br />
po<strong>in</strong>t. Each fungus was grown on several plates of each medium.<br />
Plates were <strong>in</strong>cubated upright at 25°C.<br />
2.4. Growth Measurement<br />
Fungal growth was measured by the methods described below<br />
throughout the growth period, but at <strong>in</strong>tervals which varied widely<br />
with species and medium. Measurements and assays were carried out<br />
<strong>in</strong> duplicate.<br />
2.4.1. Colony Diameters<br />
Colonies were measured from the reverse side <strong>in</strong> millimetres with a<br />
ruler. Only well formed, circular colonies were chosen for measurement.<br />
2.4.2. Mycelium Dry Weight<br />
A colony and surround<strong>in</strong>g agar were cut from a Petri dish, transferred<br />
to a beaker conta<strong>in</strong><strong>in</strong>g distilled water (100 ml), then heated <strong>in</strong> a
Comparison of Hyphal Length, Ergosterol, Mycelium Dry Weight 53<br />
steamer for 30 m<strong>in</strong> to melt the agar. The mycelium, which rema<strong>in</strong>ed<br />
<strong>in</strong>tact, was r<strong>in</strong>sed once <strong>in</strong> distilled water and then transferred to a<br />
dried, weighed filter paper which was placed <strong>in</strong> an alum<strong>in</strong>ium dish and<br />
dried <strong>in</strong> an oven at 80°C for 18 h. After cool<strong>in</strong>g to room temperature<br />
<strong>in</strong> a desiccator, the filter papers and mycelium were weighed and the<br />
dry weight calculated by difference. The method was based on those<br />
of Paster et al. (1983) and Zill et al. (1988).<br />
2.4.3. Hyphal Length<br />
Hyphal lengths were estimated by direct microscopy us<strong>in</strong>g a<br />
haemocytometer and a modification of the method of Schnürer<br />
(1993). Colonies and associated agar were cut <strong>in</strong>to pieces and homogenized<br />
with distilled water (3-100 ml, depend<strong>in</strong>g on the size of the<br />
colony), for about 30 s us<strong>in</strong>g an Ultra-Turrax homogeniser (Ystral<br />
GmbH, Dott<strong>in</strong>gen, Germany). The suspension was then treated <strong>in</strong> a<br />
sonicator (Branson Sonic Power Company, Danbury, CT) at 100 watts<br />
for about 20 s to break up hyphal clumps. After dilution <strong>in</strong> distilled<br />
water, drops (0.5 ml) were placed <strong>in</strong> a haemocytometer and hyphal<br />
fragments counted. Hyphal lengths were measured us<strong>in</strong>g the <strong>in</strong>tersection<br />
technique (Olson, 1950) at a magnification of 400 X. Colonies<br />
from two plates were measured separately and for each plate 10 fields<br />
were counted. Results were calculated from the means of the two<br />
plates.<br />
2.4.4. Ergosterol Assay<br />
A colony was excised from a Petri dish culture, transferred to a<br />
beaker of distilled water (100 ml) conta<strong>in</strong><strong>in</strong>g Tween 80 (0.05%) and<br />
steamed for 30 m<strong>in</strong> to melt the agar. The <strong>in</strong>tact mycelium was collected,<br />
r<strong>in</strong>sed with water and transferred to a round bottomed flask.<br />
Ergosterol was extracted from the mycelium by reflux<strong>in</strong>g with 95%<br />
ethanol: water (100 ml, 50: 50 v/v) and potassium hydroxide (5 g) for<br />
30 m<strong>in</strong> (Zill et al., 1988). This crude extract was partitioned three<br />
times with n-hexane <strong>in</strong> a separat<strong>in</strong>g funnel. The comb<strong>in</strong>ed hexane<br />
extracts were concentrated under vacuum to near dryness. The residue<br />
was redissolved <strong>in</strong> n-hexane (2 ml), then filtered through a polypropylene<br />
membrane, 0.45 µm pore size, 13 mm diameter (Activon, Sydney,<br />
NSW). The filtrate was dried under N 2 and redissolved <strong>in</strong> n-hexane for<br />
ergosterol quantification.<br />
Ergosterol was assayed by high pressure liquid chromatography<br />
(HPLC) us<strong>in</strong>g a Millipore Waters system fitted with a LiChrosorb SI
54 M. H. Taniwaki et al.<br />
60 column (Gold Pak, Activon). The column was eluted with<br />
nhexane: isopropanol (97: 3, v/v) at 1 ml/m<strong>in</strong> and ergosterol was<br />
detected by absorption at 280 nm about 8-10 m<strong>in</strong> after <strong>in</strong>jection of<br />
the sample. Ergosterol was quantified by reference to an ergosterol<br />
standard calibration curve, prepared from a standard solution<br />
(2 mg/ml, Sigma Chemicals, St. Louis, MO). For five of the fungal<br />
Figure 1. HPLC traces of ergosterol from six fungi: (a) Penicillium commune, (b)<br />
Penicillium roqueforti, (c) Byssochlamys nivea, (d) Fusarium oxysporum, (e) Aspergillus<br />
flavus, (f) Eurotium chevalieri. The ergosterol peak is <strong>in</strong>dicated by an arrow.
Comparison of Hyphal Length, Ergosterol, Mycelium Dry Weight 55<br />
species, well separated s<strong>in</strong>gle peaks for ergosterol were obta<strong>in</strong>ed <strong>in</strong><br />
HPLC traces of mycelial extracts (Figure 1). However, <strong>in</strong> extracts<br />
from E. chevalieri and X. bisporus the peak eluted close to <strong>in</strong>terfer<strong>in</strong>g<br />
substances. In these cases a second filtration of the extract or<br />
addition of ergosterol standard was necessary to conclusively identify<br />
the ergosterol peaks.<br />
3. RESULTS<br />
3.1. Validation of ergosterol assay<br />
A l<strong>in</strong>ear relationship was observed between peak heights measured<br />
on HPLC chromatograms and ergosterol concentration. The lower<br />
limit of detection was 0.01 µg of ergosterol. The coefficient of variation<br />
for ergosterol peaks detected by HPLC <strong>in</strong> extracts ranged<br />
between 1 and 14% provided that the ergosterol content was greater<br />
than 20 µg. Variation was greatest when the ergosterol content was<br />
less than 50 µg and least for high amounts (e.g. 500 µg) (Table 2).<br />
Ergosterol recoveries from spiked samples of F. oxysporum and B.<br />
fulva mycelium were 80-100% us<strong>in</strong>g the described method.<br />
For most species, the ergosterol peak <strong>in</strong> the HPLC traces was clear<br />
and well separated from other peaks (Figure 1a-d). However, the<br />
ergosterol peaks for Aspergillus flavus (Figure 1e) and more especially<br />
for Eurotium chevalieri (Figure 1f) and Xeromyces bisporus (not<br />
shown) were close to peaks likely to be other sterols. Levels of ergosterol<br />
observed <strong>in</strong> these species were lower than expected.<br />
Table 2. Reproducibility of ergosterol analysis (n=3) <strong>in</strong> colonies of Fusarium oxysporum<br />
and Byssochlamys fulva grown on Czapek yeast extract agar for various <strong>in</strong>cubation<br />
periods<br />
Incubation Coefficient of<br />
Species time (d) Ergosterol (µg) variation (%)<br />
Fusarium<br />
oxysporum 2 5.5, 7.5,11.1 34.9<br />
5 620.5, 654.8, 696.2 5.8<br />
6 884.2, 767.4, 835.0 7.0<br />
Byssochlamys<br />
fulva 2 0.76, 0.42, 0.76 28.4<br />
4 23.5, 31.1, 27.7 13.9<br />
7 694.7, 682.0, 686.2 0.9
56 M. H. Taniwaki et al.<br />
3.2. Growth of Fungi as Assessed by Colony Diameters<br />
Most of the fungi grew on all of the media used, though with vary<strong>in</strong>g<br />
vigour, reflect<strong>in</strong>g their water relations (Table 3). Best growth of<br />
most species occurred on CY20S, 0.98 a w except for B. fulva and F.<br />
oxysporum which grew faster at 25°C on CYA, 0.997 a w . Along with<br />
M. plumbeus, these species produced little or no growth on MY50G at<br />
0.89 a w , an a w near their lower limit for growth (Pitt and Hock<strong>in</strong>g,<br />
1997). A. flavus and P. commune grew strongly at all a w tested. Growth<br />
of E. chevalieri, a xerophilic species, was slow on CYA, and faster on<br />
CY20S and MY50G. X. bisporus, an extreme xerophile, grew only on<br />
MY50G.<br />
3.3. Influence of Colony Age on Growth Parameters<br />
The <strong>in</strong>fluence of colony age on some of the data obta<strong>in</strong>ed by the<br />
four methods used for measur<strong>in</strong>g fungal growth is shown <strong>in</strong> Table 4,<br />
for four representative species. Ratios were calculated for colony<br />
diameter over hyphal length, mycelium dry weight and ergosterol content<br />
over hyphal length, and ergosterol over mycelium dry weight. The<br />
ratio of colony diameter over hyphal length showed a general downward<br />
trend, <strong>in</strong>dicat<strong>in</strong>g a greater rate of hyphal extension than colony<br />
diameter <strong>in</strong>crease as colonies aged. Colony diameters are therefore not<br />
a good measure of fungal biomass production <strong>in</strong> ag<strong>in</strong>g colonies. The<br />
other ratios were reasonably constant, <strong>in</strong>dicat<strong>in</strong>g a general correspondence<br />
between mycelial dry weight, ergosterol content and hyphal<br />
length.<br />
Table 3. Colony diameters of fungi grown on media of various water activities at<br />
25°C<br />
Species Colony diameter (mm) at 7 days on<br />
CYA 0.997 a w CY20S 0.98 a w MY50G 0.89 a w<br />
Aspergillus flavus 68 84 16<br />
Byssochlamys fulva 78 58 0<br />
Byssochlamys nivea 43 - a -<br />
Eurotium chevalieri 19 50 36<br />
Fusarium oxysporum 90 78 6<br />
Mucor plumbeus 63 85 4<br />
Penicillium commune 32 44 18<br />
Penicillium roqueforti 52 - -<br />
Xeromyces bisporus 0 0 12<br />
a not tested
Comparison of Hyphal Length, Ergosterol, Mycelium Dry Weight 57<br />
Table 4. Growth of four species of fungi on Czapek yeast extract agar as measured by four techniques, and ratios derived from those<br />
measurementsa Hyphal Colony Mycelium Ergolength<br />
diam Ratio dry Ratio sterol Ratio Ratio<br />
Species Time (d) (m ×1000) (mm) CD/HL wt (mg) MDW/HL (µg) E/HL E/MDW<br />
Mucor 3 2.04 51 25 16.5 8.1 11.2 5.5 0.68<br />
plumbeus 6 2.62 73 27.9 30.9 11.8 62.0 23.7 2.00<br />
16 4.34 83 19.2 36.0 8.3 81.2 18.7 2.26<br />
Fusarium 4 10.45 44 4.2 40.4 3.9 128.6 12.3 3.18<br />
oxysporum 6 32.38 70 2.1 133.8 4.1 444.3 13.7 3.32<br />
9 83.32 86 1.0 308.6 3.7 598.3 7.1 1.94<br />
Byssochlamys 5 2.51 33 13.1 14.7 5.9 77.5 30.9 5.27<br />
fulva 8 19.83 71 3.6 119.6 6.0 379.6 19.1 3.17<br />
9 30.75 86 2.8 185.4 6.0 977.7 31.8 5.27<br />
Penicillium 4 3.23 29 9.0 19.6 6.1 43.6 13.5 2.22<br />
roqueforti 7 8.36 57 6.8 103.4 12.3 168.6 20.2 1.63<br />
14 19.22 86 4.5 238.4 12.4 280.5 14.6 1.18<br />
a Ratio CD/HL, ratio of colony diameter (mm) / hyphal length (m ×1000); ratio MDW/HL, ratio of mycelial dry weight (mg) / hyphal length<br />
(m ×1000), ratio E/HL, ergosterol (µg) / hyphal length (m ×1000).
58 M. H. Taniwaki et al.<br />
3.4. Estimates of Fungal Growth by Mycelial Dry<br />
Weight and Ergosterol Compared with Hyphal<br />
Length<br />
To provide a common reference po<strong>in</strong>t, colonies of diameter 83-86<br />
mm, i.e. virtually full plate growth from a s<strong>in</strong>gle <strong>in</strong>oculum po<strong>in</strong>t, were<br />
selected where possible. To take account of type of medium, two data<br />
sets were developed, for colonies on CYA and PDA. Species other<br />
than Penicillium commune, E. chevalieri and X. bisporus produced full<br />
plate colonies on both media, although after varied <strong>in</strong>cubation periods.<br />
P. commune reached 86 mm on PDA after 21 days, but a maximum<br />
of only 39 mm on CYA, after 17 days. E. chevalieri colonies<br />
reached a maximum of only 46 mm diameter on PDA, after 16 days,<br />
and were smaller on CYA. For comparisons with other species on<br />
CYA, therefore, E. chevalieri colonies on CY20S were used. As<br />
expected, X. bisporus did not grow on either CYA or CY20S, so data<br />
from MY50G were used, where this species reached a maximum of 64<br />
mm after 42 days <strong>in</strong>cubation. The overall results from analyses of<br />
hyphal length, mycelium dry weight and ergosterol under these conditions<br />
are given <strong>in</strong> Table 5.<br />
3.5. Hyphal Length<br />
Hyphal lengths, estimated for colonies of similar diameters as set<br />
out <strong>in</strong> Table 5, varied widely between species. On CYA, hyphal<br />
length varied almost 20 fold between F. oxysporum (83,000 m) and<br />
B. nivea (4200 m). Results were more uniform on PDA, with less<br />
than 6 fold variation among the species. F. oxysporum produced only<br />
23,000 m of hyphae on PDA. B. nivea produced the greatest hyphal<br />
length on PDA (28,000 m) but the lowest on CYA (4200 m). Despite<br />
profuse growth, M. plumbeus produced less than 6000 m of hyphae<br />
under any of the varied conditions used. Mycelial dry weights were<br />
also lower for M. plumbeus than for many of the other species<br />
studied.<br />
3.6. Ergosterol Content<br />
Ergosterol content per colony varied widely between genus,<br />
between medium and even with<strong>in</strong> genus, reflect<strong>in</strong>g differences <strong>in</strong><br />
growth density and membrane composition. When grown on CYA,<br />
B. fulva produced the highest ergosterol content and M. plumbeus the
Comparison of Hyphal Length, Ergosterol, Mycelium Dry Weight 59<br />
Table 5. Comparison of measurements of mature growth of various fungi on CYAa Colony diameter Hyphal Mycelium<br />
mm (<strong>in</strong>cubation length dry Ratio Ergosterol Ratio Ratio<br />
Species time, d) (m ×1000) weight (mg) MDW/HL (µg) E/HL E/MDW<br />
Medium: CYA<br />
Aspergillus flavus 86 (11) 6.42 297 46.3 298 46.4 1.00<br />
Byssochlamys fulva 86 (9) 30.8 185 6.01 977 31.7 5.28<br />
B. nivea 86 (17) 4.17 23.5 5.63 64.5 15.5 2.74<br />
Fusarium oxysporum 86 (9) 83.3 309 3.71 598 7.17 1.93<br />
Mucor plumbeus 83 (13) 4.34 36 8.29 81.2 18.7 2.25<br />
Penicillium commune 39 (17) 7.67 160 20.9 440 57.4 2.75<br />
P. roqueforti 86 (14) 19.2 238 12.4 281 14.6 1.18<br />
Average 14.7 21.4 2.44<br />
Medium: PDA<br />
Aspergillus flavus 86 (11) 4.78 186 38.9 84.3 17.6 0.45<br />
Byssochlamys fulva 86 (7) 22.2 156 7.02 1463 65.9 9.37<br />
B. nivea 86 (9) 28.4 180 6.33 183 6.4 1.02<br />
Eurotium chevalieri 46 (16) 8.6 86 10.0 669 7.8 7.78<br />
Fusarium oxysporum 86 (7) 23.3 171 7.33 1884 80.9 11.0<br />
Mucor plumbeus 86 (4) 5.2 109 21.0 259 49.8 2.36<br />
Penicillium commune 86 (2 1) 7.02 108 15.4 818 116.5 7.57<br />
P. roqueforti 86 (11) 7.48 222 29.7 509 68 2.29<br />
Average 17.0 51.6 5.23<br />
Medium: CY20S<br />
Eurotium chevalieri 86 (14) 6.59 354 53.7 25.1 3.8 0.07<br />
Medium: MY50G<br />
Xeromyces bisporus 64 (42) 4 28.9 7.22 42.7 10.7 1.47<br />
Overall average 17.6 36.4 4.1<br />
a Ratio CD/HL, ratio of colony diameter (mm)/ hyphal length (m ×1000); ratio MDW/HL, ratio of mycelial dry weight (mg)/ hyphal length<br />
(m ×1000), ratio E/HL, ergosterol (µg) /hyphal length (m × 1000).
60 M. H. Taniwaki et al.<br />
lowest; about a 12 fold difference. On PDA, F. oxysporum produced<br />
the highest ergosterol content, with A. flavus the lowest, about a 22<br />
fold difference.<br />
For some species, medium composition greatly affected ergosterol<br />
content. This was most evident for E. chevalieri, which produced 25<br />
times as much ergosterol on PDA (670 µg/colony) from colonies less<br />
than 50 mm <strong>in</strong> diameter, than from 86 mm diameter colonies on<br />
CY20S (25 µg), despite comparable hyphal lengths on the two media.<br />
In contrast, ergosterol production by A. flavus was much higher on<br />
CYA than on PDA, aga<strong>in</strong> with comparable hyphal lengths.<br />
3.7. Mycelium Dry Weights<br />
Mycelium dry weights of 83-86 mm diameter colonies varied<br />
between species. On CYA, A. flavus and F. oxysporum produced<br />
colonies with a high mycelium dry weight (approximately 300<br />
mg/plate). On CY20S, E. chevalieri colonies were equally heavy. The<br />
weights of M. plumbeus colonies, however were only 12% of these values<br />
(Table 5). For the vigorously grow<strong>in</strong>g Aspergillus, Penicillium and<br />
Fusarium species, mycelium dry weights on PDA were lower than values<br />
obta<strong>in</strong>ed on CYA. However, mycelium weights for M. plumbeus<br />
and B. nivea were much higher on PDA than on CYA. In the case of<br />
B. nivea, this difference was more than 8 fold.<br />
3.8. Relationship Between Hyphal Length and<br />
Mycelium Dry Weight<br />
The relationship between hyphal length and mycelium dry weight<br />
over time of growth was reasonably constant with<strong>in</strong> species (Table 4)<br />
except for very small colonies of Penicillium roqueforti, and varied<br />
only four fold between the four species shown <strong>in</strong> Table 4. When all<br />
n<strong>in</strong>e species were compared, on more than one medium, much<br />
greater variability was seen. This appears to be due mostly to variation<br />
<strong>in</strong> hyphal length measurements, which is not so precise as the<br />
other techniques used here. Increases <strong>in</strong> hyphal length sometimes<br />
occurred with little <strong>in</strong>crease <strong>in</strong> mycelial dry weight, e.g. for<br />
M. plumbeus when grown on CYA and E. chevalieri grown on PDA.<br />
These observations were reproducible (data not shown). As discussed<br />
by Schnürer (1993), vacuole formation and autolysis of cell<br />
contents may occur <strong>in</strong> ag<strong>in</strong>g cultures which would lead to a reduction<br />
<strong>in</strong> weight per unit length.
Comparison of Hyphal Length, Ergosterol, Mycelium Dry Weight 61<br />
On the other hand, some species sporulated heavily <strong>in</strong> age, e.g.<br />
P. roqueforti grown on PDA, A. flavus on CYA, and E. chevalieri on<br />
CY20S. Here large <strong>in</strong>creases <strong>in</strong> mycelial dry weight were accompanied<br />
by little or no hyphal growth. The Penicillium, Aspergillus, and<br />
Eurotium species showed ratios above 12 mg/1000 m, while for the<br />
Mucor, Fusarium and Byssochlamys species ratios were 8 mg/1000 m<br />
or below. On PDA, ratios ranged from 6.3 mg/1000 m (B. nivea) to<br />
38.9 mg/1000 m (A. flavus).<br />
3.9. Relationship Between Hyphal Length and<br />
Ergosterol Content<br />
The ratios of ergosterol production (µg) to hyphal length (m<br />
×1000) for colonies of various ages were found to be more variable<br />
with<strong>in</strong> species than those for mycelial dry weight over hyphal length<br />
(Table 4). No pattern with age of cultures was apparent. On PDA,<br />
values varied from 6.4 (B. nivea) to 116.5 (P. commune), an 18 fold difference<br />
(Table 5). Low ergosterol production on PDA by A. flavus and<br />
on CY20S by E. chevalieri (see above) was reflected <strong>in</strong> very low ratios<br />
of ergosterol to hyphal length.<br />
3.10. Relationship Between Ergosterol Content and<br />
Mycelial Dry Weight<br />
Reasonable agreement was seen between the ratios of ergosterol<br />
(µg) to mycelium dry weight (mg) both for cultures of different age <strong>in</strong><br />
each species and between species (Table 4). If the very low value for<br />
small colonies of M. plumbeus (0.68) is omitted, ratios varied between<br />
1.18 and 5.27, less than 5 fold. When the effect of medium is considered<br />
with the full range of species (Table 5), ratios were aga<strong>in</strong> reasonably<br />
constant for colonies grown on CYA and PDA. With omission<br />
aga<strong>in</strong> of a very low figure (0.45 for A. flavus on PDA), values varied<br />
from 1.0 to 11.0. The average for all species was 4.1 (Table 5). Most<br />
species produced higher amounts of ergosterol and mycelium dry<br />
weight on PDA than on CYA except for A. flavus, which produced<br />
higher ergosterol and mycelium dry weight on CYA than on PDA.<br />
However, the very low ratio (0.07) observed from growth of<br />
E. chevalieri on CY20S is anomalous. No doubt this is due to the very<br />
low level of ergosterol produced on this medium by this species. The<br />
HPLC spectrum showed several peaks elut<strong>in</strong>g close to ergosterol, so<br />
other sterols may have been present.
62 M. H. Taniwaki et al.<br />
4. DISCUSSION<br />
This study attempted to validate ergosterol measurements as an<br />
<strong>in</strong>dex of fungal growth for important food spoilage fungi, chosen<br />
because of the great differences <strong>in</strong> their growth patterns, us<strong>in</strong>g hyphal<br />
length and mycelium dry weight as standards.<br />
Colony diameter, hyphal length, mycelium dry weight and ergosterol<br />
content all gave useful <strong>in</strong>formation about the growth of the<br />
species exam<strong>in</strong>ed. However, each technique exhibited advantages and<br />
limitations. Colony diameter is a sensitive technique, <strong>in</strong> that a colony<br />
as small as 2 mm is easily measured. It was not possible to determ<strong>in</strong>e<br />
accurately dry weight, hyphal length or ergosterol concentration on<br />
such small amounts of material. However, colony diameter did not<br />
show a consistent correlation with the other parameters, especially as<br />
colony diameters became larger and more mature. In particular,<br />
sporulation caused a reduced correlation between colony diameters<br />
and the other parameters. If colony diameters are measured from relatively<br />
early growth, e.g. us<strong>in</strong>g the Petrislide technique of Pitt and<br />
Hock<strong>in</strong>g (1977), then correlations could be higher.<br />
Values for hyphal length obta<strong>in</strong>ed from s<strong>in</strong>gle colonies on standard<br />
Petri dishes by Schnürer (1993) ranged from 10,000 m for Rhizopus<br />
stolonifer to 54,000 m for Fusarium culmorum. In this study, comparable<br />
fungi produced more hyphae: 43,000 m for Mucor plumbeus and<br />
83,000 m for Fusarium oxysporum. These differences may be due to the<br />
lower nutritional value of the medium used by Schnürer (1993).<br />
In this study, the average ratio of mycelium dry weight over hyphal<br />
length was 17.6 mg dry weight/1000 m of mycelium. Schnürer (1993)<br />
found values of 4.2 to 6.7 mg/1000 m, calculated from the mycelium<br />
and hyphal volume, respectively. Values obta<strong>in</strong>ed by us for Fusarium<br />
oxysporum, Byssochlamys spp. grown on CYA and PDA, and Mucor<br />
plumbeus when grown on CYA were comparable with those of<br />
Schnürer (1993). Much larger differences were seen with the rapidly<br />
grow<strong>in</strong>g and/or highly sporulat<strong>in</strong>g Aspergillus and Penicillium species<br />
studied here. Given the differences <strong>in</strong> fungi studied and media used,<br />
the data of Schnürer (1993) and our data are comparable. Work<strong>in</strong>g<br />
with aquatic fungi, ratios of ergosterol to mycelium dry weight of 2.3<br />
to 11.5 were given by Gessner and Chauvet (1993), similar to those of<br />
Schnürer (1993).<br />
Reports have suggested that ergosterol content <strong>in</strong>creases as colonies<br />
age (Nout et al., 1987; Torres et al., 1992). However, as discussed by<br />
Schnürer (1993), vacuole formation and autolysis of cell contents may<br />
occur <strong>in</strong> ag<strong>in</strong>g cultures which would lead to a reduction <strong>in</strong> weight per
Comparison of Hyphal Length, Ergosterol, Mycelium Dry Weight 63<br />
unit of length and, consequently, to an <strong>in</strong>creased ergosterol to dry<br />
weight ratio. Differences <strong>in</strong> degree and type of sporulation by the<br />
species studied here also clearly play a major part <strong>in</strong> the variations <strong>in</strong><br />
the ratios of mycelial dry weight to hyphal length and ergosterol content<br />
to hyphal length observed here. For example, Aspergillus flavus<br />
and the Penicillium species produce relatively little vegetative<br />
mycelium relative to conidiophores and conidia, <strong>in</strong>creas<strong>in</strong>g mycelial<br />
dry weight and probably decreas<strong>in</strong>g ergosterol <strong>in</strong> relation to hyphal<br />
length.<br />
Mycelium dry weight is often considered to be a basic measure of<br />
fungal growth but fundamental questions rema<strong>in</strong> unanswered. In this<br />
study, mycelium was separated from agar medium us<strong>in</strong>g a heat treatment.<br />
Cochrane (1958) criticised the separation of agar medium from<br />
fungal biomass us<strong>in</strong>g hot water because the water may extract soluble<br />
fungal components, result<strong>in</strong>g <strong>in</strong> a loss of dry weight. However, it is<br />
also important to note that dry weights measured without prior<br />
extraction are greatly affected by the variation <strong>in</strong> <strong>in</strong>ternal solutes<br />
caused by different medium formulations, especially <strong>in</strong> media of<br />
reduced a w (Hock<strong>in</strong>g and Norton, 1983).<br />
An alternative approach is to scrape or peel the fungal growth from<br />
the medium surface. This may lead to <strong>in</strong>complete removal and underestimation<br />
of dry weight. The technique of Hock<strong>in</strong>g (1986) where<br />
fungi were grown on dialysis membrane on the surface of agar media<br />
enables ready separation of fungus from medium, and is a notable<br />
improvement. However, <strong>in</strong> the current study, where some species<br />
sporulated profusely, a wet extraction technique was considered<br />
preferable for safety reasons.<br />
In this study, extracted mycelium dry weight showed a reasonably<br />
good correlation with hyphal length (Tables 4, 5), <strong>in</strong>dicat<strong>in</strong>g the value<br />
of this parameter as a measure of fungal growth <strong>in</strong> media. However,<br />
the measurement of mycelium dry weight is not readily applicable to<br />
the estimation of growth of fungi <strong>in</strong> foods.<br />
Schnürer (1993) noted differences <strong>in</strong> growth patterns between different<br />
fungal species. For a nonsporulat<strong>in</strong>g Fusarium culmorum, good<br />
agreement was found between hyphal length, colony counts and ergosterol<br />
content. For Penicillium rugulosum and Rhizopus stolonifer,<br />
changes <strong>in</strong> ergosterol level were related more closely to changes <strong>in</strong><br />
hyphal length rather than to production of spores or colony counts. In<br />
the present study, hyphal length correlated rather poorly with ergosterol<br />
content (Table 5). Schnürer and Jonsson (1992) found reasonable<br />
correlation between ergosterol and mould viable counts <strong>in</strong> Swedish<br />
gra<strong>in</strong>s under certa<strong>in</strong> conditions, but Saxena et al. (2001) found that
64 M. H. Taniwaki et al.<br />
viable counts and ergosterol did not correlate well for pure cultures of<br />
Aspergillus ochraceus and Penicillium verrucosum.<br />
The accuracy of the hyphal length technique is affected by factors<br />
<strong>in</strong>clud<strong>in</strong>g variation <strong>in</strong> hyphal width for different species, degree of<br />
sporulation, formation of reproductive structures (e.g. cleistothecia),<br />
fragmentation dur<strong>in</strong>g homogenization, and clump<strong>in</strong>g of hyphae. The<br />
<strong>in</strong>tersection technique of Olsen (1950) is probably only statistically<br />
sound when large numbers of microscopic fields are counted. Despite<br />
the laboriousness of the procedure used here for hyphal length estimations<br />
(ten fields from two colonies), this rigour is lack<strong>in</strong>g. All of<br />
these factors lead to variation <strong>in</strong> estimates of hyphae length.<br />
Ergosterol content was a sensitive <strong>in</strong>dication of fungal biomass. As<br />
little as 0.01 µg of ergosterol could be detected from mycelium <strong>in</strong> a<br />
colony of 4 mm diameter. However, the amount of ergosterol found <strong>in</strong><br />
the fungi varied with the growth medium, species and culture <strong>in</strong>cubation<br />
time. This variation was reflected <strong>in</strong> the data shown <strong>in</strong> Tables 4<br />
and 5. The ratio of ergosterol over mycelium dry weight ranged from<br />
0.07 µg/mg for E. chevalieri on CY20S to 11.0 µg/mg for F. oxysporum<br />
on PDA, a 150 fold variation. Even when these two extreme values are<br />
omitted, variation of about 20 fold rema<strong>in</strong>ed (i.e. 0.45 µg/mg for<br />
A. flavus to 9.37 µg/mg for B. fulva, both on PDA).<br />
Weete (1974) noted that <strong>in</strong> general sterol levels <strong>in</strong> fungi varied with<br />
medium composition and culture conditions. Four to 10 fold variations<br />
have been reported <strong>in</strong> the ergosterol content of the same fungus<br />
under different growth conditions (Newell et al., 1987; Nout et al.,<br />
1987). Increased nutritional complexity of the medium, the presence<br />
of free fatty acid precursors of the ergosterol biosynthetic pathway<br />
and <strong>in</strong>creased availability of oxygen all gave mycelium with <strong>in</strong>creased<br />
ergosterol contents.<br />
The measurement of ergosterol alone does not give the absolute<br />
amount of fungus present. For this, it is necessary to convert ergosterol<br />
values <strong>in</strong>to biomass <strong>in</strong> terms of mycelium dry weight. After<br />
study<strong>in</strong>g 14 aquatic hyphomycetes, Gessner and Chauvet (1993) gave<br />
the range of ratios of ergosterol to mycelium dry weight as 2.3 -11.5<br />
µg/g, figures similar to those derived <strong>in</strong> this work (0.45 -11.0 µg/g).<br />
This ratio varies between fungal species and with growth condition,<br />
limit<strong>in</strong>g the direct use of ergosterol as a means of calculat<strong>in</strong>g<br />
mycelium dry weight.<br />
A further explanation for variation <strong>in</strong> the ergosterol content of<br />
fungi is that sterols other than ergosterol can be produced by some<br />
species. For example, ergosterol and 22-dihydroergosterol have been<br />
reported as the predom<strong>in</strong>ant sterol <strong>in</strong> A. flavus (Vacheron and Michel,
Comparison of Hyphal Length, Ergosterol, Mycelium Dry Weight 65<br />
1968; Weete, 1973). Other sterols identified as products of<br />
deuteromycetous fungi <strong>in</strong>clude cerevisterol, ergosterol peroxide, lanosterol,<br />
24-methylenelophenol and 14-dehydroergosterol (Weete, 1973).<br />
In this study, A. flavus produced a low level of ergosterol despite its<br />
high production of biomass, and showed extra peaks <strong>in</strong> its extract.<br />
Similarly, colonies of E. chevalieri were found to conta<strong>in</strong> only low<br />
amounts of ergosterol, despite high amounts of biomass. Additional<br />
peaks seen <strong>in</strong> the HPLC profile of E. chevalieri extracts also <strong>in</strong>dicate<br />
that it may produce sterols other than ergosterol. The ergosterol content<br />
of X. bisporus mycelium was also low, and several additional<br />
peaks were observed <strong>in</strong> the HPLC trace from its extract. Further studies<br />
are needed to determ<strong>in</strong>e if the additional peaks found <strong>in</strong> the HPLC<br />
profiles are sterols.<br />
The low level of ergosterol produced by E. chevalieri has important<br />
consequences. Eurotium species are very common <strong>in</strong> stored gra<strong>in</strong>s, <strong>in</strong><br />
which ergosterol has been used to estimate fungal growth. The overall<br />
average ratio of ergosterol to mycelium dry weight was 4.1, about 60<br />
times higher than that obta<strong>in</strong>ed for E. chevalieri on CY20S.<br />
Hypothetically, a sample of gra<strong>in</strong> <strong>in</strong>fected by E. chevalieri, estimated to<br />
conta<strong>in</strong> a particular ergosterol content, could conta<strong>in</strong> 150 times as much<br />
fungal biomass as one <strong>in</strong>fected by Fusarium oxysporum, which gave an<br />
ergosterol to mycelium dry weight ratio of 11 µg/mg <strong>in</strong> this study.<br />
Estimation of ergosterol content, colony diameter, mycelium dry<br />
weight and hyphal length were shown to be good <strong>in</strong>dices for measur<strong>in</strong>g<br />
fungal growth, but it is important to keep <strong>in</strong> m<strong>in</strong>d the limitations of<br />
each techniques. The most reliable <strong>in</strong>formation about fungal growth will<br />
be obta<strong>in</strong>ed by us<strong>in</strong>g two or more techniques for quantification.<br />
5. ACKNOWLEDGMENTS<br />
The authors wish to thank to Mr N. Tob<strong>in</strong> and Ms S. L. Leong of<br />
<strong>Food</strong> Science Australia, North Ryde, for helpful advice on chemical<br />
analyses and hyphal length measurement, respectively, and to<br />
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br />
for fund<strong>in</strong>g the PhD program for M.H.T.<br />
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Newell, S. Y., Miller, J. D., and Fallon, R. D., 1987, Ergosterol content of salt-marsh<br />
fungi: effect of growth conditions and mycelial age, Mycologia 79:688-695.<br />
Nout, M. J. R., Bonants-van Laarhoven, T. M. G., de Jongh, P., and Koster, P. G.,<br />
1987, Ergosterol content of Rhizopus oligosporus NRRL 5905 grown <strong>in</strong> liquid and<br />
solid substrates, Appl. Microbiol. Biotechnol. 26:456-461.<br />
Nylund, J. E., and Wallander, H., 1992, Ergosterol analysis as a means of quantify<strong>in</strong>g<br />
mycorrhizal biomass, <strong>in</strong>: Methods <strong>in</strong> Microbiology, Vol 24, Techniques for the Study<br />
of Mycorrhiza. J. R. Norris, D. J. Read, and A. K. Varma, eds, Academic Press,<br />
London, pp.77-88.<br />
Olson, F. C. W., 1950, Quantitative estimates of filamentous algae, Trans. Am.<br />
Microsc. Soc. 69:272-279.<br />
Paster, N., Lisker, N., and Chet, I., 1983, Ochratox<strong>in</strong> A production by Aspergillus<br />
ochraceus Wilhelm grown under controlled atmospheres, Appl. Environ. Microbiol.<br />
45:1136-1139.<br />
Pitt, J. I., 1984, The significance of potentially toxigenic fungi <strong>in</strong> foods, <strong>Food</strong> Technol.<br />
Aust. 36:218-219.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1977, Influence of solute and hydrogen ion concentration<br />
on the water relations of some xerophilic fungi, J. Gen. Microbiol. 101:35-40.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, 2nd edition, Blackie<br />
Academic and Professional, London.
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Pitt, J. I., and Miscamble, B. F., 1995, Water relations of Aspergillus flavus and closely<br />
related species, J. <strong>Food</strong> Prot. 58:86-90.<br />
Ride, J. P., and Drysdale, R. B., 1972, A rapid method for the chemical estimation of<br />
filamentous fungi <strong>in</strong> plant tissue, Physiol. Plant Pathol. 2:7-15.<br />
Samson, R. A., Hock<strong>in</strong>g, A. D., Pitt J. I., and K<strong>in</strong>g, A. D., 1992, Modern Methods <strong>in</strong><br />
<strong>Food</strong> <strong>Mycology</strong>, Elsevier Publishers, Amsterdam.<br />
Saxena, J., Munimbazi, C., and Bullerman, L. B., 2001, Relationship of mould count,<br />
ergosterol and ochratox<strong>in</strong> A production, Int. J. <strong>Food</strong> Microbiol. 71: 29-34.<br />
Schnürer, J., 1993, Comparison of methods for estimat<strong>in</strong>g the biomass of three foodborne<br />
fungi with different growth patterns, Appl. Environ. Microbiol. 59:552-555.<br />
Schnürer, J., and Jonsson, A., 1992, Ergosterol levels and mould colony form<strong>in</strong>g units<br />
<strong>in</strong> Swedish gra<strong>in</strong>s of food and feed grade, Acta Agric. Scand. Sect B. 42:240-245.<br />
Schwardorf, K., and Muller, H. M., 1989, Determ<strong>in</strong>ation of ergosterol <strong>in</strong> cereals,<br />
mixed feed components, and mixed feeds by liquid chromatography, J. Assoc. Off.<br />
Anal. Chem. 72:457-462.<br />
Seitz, L. M., Mohr, H. E., Burroughs, R., and Sauer D. B., 1977, Ergosterol as an <strong>in</strong>dicator<br />
of fungal <strong>in</strong>vasion <strong>in</strong> gra<strong>in</strong>, Cereal Chem. 54:1207-1217.<br />
Seitz, L. M., Sauer, D. B., Burroughs, R., Mohr, H. E., and Hubbard J. D., 1979,<br />
Ergosterol as a measure of fungal growth, Phytopathology 69:1202-1203.<br />
Torres, M., Viladrich, R., Sanchis, V., and Canela, R., 1992, Influence of age on ergosterol<br />
content <strong>in</strong> mycelium of Aspergillus ochraceus, Lett. Appl. Microbiol. 15:20-22.<br />
Vacheron, M. J., and Michel, G., 1968, Composition en sterols et en acides gras de<br />
deux souches d’Aspergillus flavus, Phytochemistry 7:1645-1651.<br />
Weete, J. D., 1973, Sterols of fungi: distribution and biosynthesis, Phytochemistry<br />
12:1843-1864.<br />
Weete, J. D., 1974, Distribution of sterols <strong>in</strong> the fungi. 1. Fungal spores, Lipids 9:<br />
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and high-carbon dioxide atmospheres, Phytopathologia 60:50-53.<br />
Williams, A. P., 1989, Methodological developments <strong>in</strong> food mycology, J. Appl.<br />
Bacteriol. 67: Symp. Suppl. 61S-67S.<br />
Zill, G., Engelhardt, G., and Wallnofer, P. R., 1988. Determ<strong>in</strong>ation of ergosterol as<br />
a measure of fungal growth us<strong>in</strong>g Si 60 HPLC, Z. Lebensm. Unters. Forsch. 187:<br />
246-249.
EVALUATION OF MOLECULAR METHODS<br />
FOR THE ANALYSIS OF YEASTS IN FOODS<br />
AND BEVERAGES<br />
Ai L<strong>in</strong> Beh, Graham H. Fleet, C. Prakitchaiwattana and<br />
Gillian M. Heard *<br />
1. INTRODUCTION<br />
The analysis of yeasts <strong>in</strong> foods and beverages <strong>in</strong>volves the sequential<br />
operations of isolation, enumeration, taxonomic identification to<br />
genus and species, and stra<strong>in</strong> differentiation. Although well established<br />
cultural methods are available to perform these operations,<br />
many molecular methods have now been developed as alternatives.<br />
These newer methods offer various advantages, <strong>in</strong>clud<strong>in</strong>g faster<br />
results, <strong>in</strong>creased specificity of analysis, decreased workload, computer<br />
process<strong>in</strong>g of data and possibilities for automation. Molecular<br />
methods for yeast analysis are now at a stage of development where<br />
they can move from the research laboratory <strong>in</strong>to the quality assurance<br />
laboratories of the food and beverage <strong>in</strong>dustries. However, many practical<br />
questions need to be considered for this transition to progress.<br />
A diversity of molecular methods with similar analytical objectives<br />
are available. Which methods should the food analyst choose and what<br />
pr<strong>in</strong>ciples should be used to guide this choice? <strong>Food</strong> analysts are<br />
required to make judgements and decisions about the microbiological<br />
quality and safety of consignments of products often worth many<br />
millions of dollars <strong>in</strong> national and <strong>in</strong>ternational trade. Moreover,<br />
* <strong>Food</strong> Science and Technology, School of Chemical Eng<strong>in</strong>eer<strong>in</strong>g and Industrial<br />
Chemistry, University of New South Wales, Sydney, New South Wales, Australia,<br />
2052. Correspondence to: g.fleet@unsw.edu.au<br />
69
70 Ai L<strong>in</strong> Beh et al.<br />
these decisions need to conform to the requirements of supplier and<br />
customer contracts, and government legislation. Consequently, they<br />
have the potential to encounter <strong>in</strong>tense legal scrut<strong>in</strong>y (Fleet 2001). For<br />
these reasons, the food analyst requires basic <strong>in</strong>formation about<br />
method standardisation, accuracy, reproducibility, precision, specificity<br />
and detection sensitivity (Cox and Fleet, 2003). While these criteria<br />
have guided the selection and choice of currently accepted<br />
cultural methods, they have not been critically applied to the newer<br />
molecular techniques.<br />
This Chapter has the follow<strong>in</strong>g goals: (1) to provide an overview of<br />
the diversity of molecular methods that are f<strong>in</strong>d<strong>in</strong>g rout<strong>in</strong>e application<br />
to the analysis of yeasts <strong>in</strong> foods and beverages, (ii) to outl<strong>in</strong>e the<br />
variables that affect the performance and reliability of these methods,<br />
and (iii) to suggest strategies for the <strong>in</strong>ternational standardisation and<br />
validation of these methods. Molecular methods have found most<br />
application to the identification of yeast species and to stra<strong>in</strong> differentiation,<br />
but there is <strong>in</strong>creas<strong>in</strong>g use of culture-<strong>in</strong>dependent methods<br />
to detect and monitor yeasts <strong>in</strong> food and beverage ecosystems.<br />
2. MOLECULAR METHODS FOR YEAST<br />
IDENTIFICATION<br />
The traditional, standard approach to yeast identification has been<br />
based on cultural, phenotypic analyses. The yeast isolate is exam<strong>in</strong>ed<br />
for a vast range of morphological, biochemical and physiological<br />
properties which are systematically compared with standard descriptions<br />
to give a genus and species identity. Generally, it is necessary to<br />
conduct approximately 100 <strong>in</strong>dividual tests to obta<strong>in</strong> a reasonably reliable<br />
identification (Kurtzman and Fell, 1998; Barnett et al., 2000).<br />
Consequently, the entire process is very labour-<strong>in</strong>tensive, lengthy and<br />
costly. Although various technical and diagnostic <strong>in</strong>novations have<br />
been developed to facilitate this process, they are not universal <strong>in</strong> their<br />
application and the data generated are not always equivalent (Deak<br />
and Beuchat, 1996; Deak 2003; Robert, 2003; Kurtzman et al., 2003).<br />
Molecular methods based on DNA analysis are now be<strong>in</strong>g used to<br />
quickly identify yeasts to genus and species level. The workload is m<strong>in</strong>imal<br />
and, usually, reliable data can be obta<strong>in</strong>ed with<strong>in</strong> 1-2 days. Several<br />
approaches are be<strong>in</strong>g used. The most def<strong>in</strong>itive and universal assay<br />
determ<strong>in</strong>es the sequence of bases <strong>in</strong> segments of the ribosomal DNA.<br />
Other approaches are based on determ<strong>in</strong>ation of restriction fragment
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 71<br />
length polymorphisms (RFLP) of segments of ribosomal DNA,<br />
hybridisation with specific nucleic acid probes, and polymerase cha<strong>in</strong><br />
reaction (PCR) assays with species-specific primers. Aspects of these<br />
methods and their application to food and beverage yeasts have been<br />
reviewed by Loureiro and Querol (1999), Giudici and Pulvirenti (2002),<br />
Loureiro and Malfeito-Ferreira (2003), and van der Vossen et al. (2003).<br />
2.1. Sequenc<strong>in</strong>g of Ribosomal DNA<br />
The discovery that ribosomal RNA is highly conserved throughout<br />
nature but has certa<strong>in</strong> segments which are species variable, has lead to<br />
the widespread use of ribosomal DNA sequenc<strong>in</strong>g <strong>in</strong> develop<strong>in</strong>g<br />
microbial phylogeny and taxonomy. As a consequence, ribosomal<br />
DNA sequences are known for most microorganisms, <strong>in</strong>clud<strong>in</strong>g<br />
yeasts, and are now rout<strong>in</strong>ely used for diagnostic and identification<br />
objectives (Valente et al., 1999). The ribosomal DNA repeat unit<br />
found <strong>in</strong> yeasts is schematically shown <strong>in</strong> Figure 1. It consists of conserved<br />
and variable regions which are arranged <strong>in</strong> tandem repeats of<br />
several hundred copies per genome. The conserved sequences are<br />
found <strong>in</strong> genes encod<strong>in</strong>g for small (18S), 5.8S, 5S, and large (25-28S)<br />
subunits of ribosomal RNA. With<strong>in</strong> each cluster, variable spacer<br />
regions occur between the subunits, called <strong>in</strong>ternal transcribed spacer<br />
(ITS) regions, and between gene clusters, called the <strong>in</strong>tergenic spacer<br />
regions (IGS) or the non-transcribed spacer region (NTS). All of these<br />
regions have some potential for differentiat<strong>in</strong>g yeast genera and<br />
species, but most focus has been on the 18S, 26S and ITS regions<br />
(Valente et al., 1999; Kurtzman, 2003).<br />
The D1/D2 doma<strong>in</strong> of the large subunit (26S) ribosomal DNA consists<br />
of about 600 nucleotides and has been sequenced for virtually all<br />
known yeast species. Databases of these sequences can be accessed<br />
through GenBank (http://www.ncbi.nlm.nih.gov/), DataBank of Japan<br />
(http://www.ddbj.nig.ac.jp/) or the European Molecular Biology<br />
Laboratory (http://www.ebi.ac.uk/embl/). There is sufficient variation<br />
<strong>in</strong> these sequences to allow differentiation of most ascomycetous<br />
(Kurtzman and Robnett, 1998, 2003) and basidiomycetous (Fell et al.<br />
ss rDNA ITS ls rDNA SS rDNA<br />
ITS NTS NTS<br />
18S 26S 18S<br />
5.8SS 5S<br />
Figure 1. The ribosomal DNA repeat unit
72 Ai L<strong>in</strong> Beh et al.<br />
2000; Scorzetti et al., 2002) yeast species. Sequenc<strong>in</strong>g of the D1/D2<br />
doma<strong>in</strong> of the 26S ribosomal DNA is now widely used for the rout<strong>in</strong>e<br />
identification of yeasts and the construction of phylogenetic taxonomy.<br />
Sequence comparisons have also been done for the small subunit,<br />
18S ribosomal DNA but, so far, the databases are not extensive and<br />
sequence differences may not be sufficient to allow the discrim<strong>in</strong>ation<br />
of closely related species (James et al., 1997; Naumov et al., 2000;<br />
Daniel and Meyer, 2003). The ribosomal spacer regions (ITS) show<br />
higher rates of sequence divergence than the D1/D2 doma<strong>in</strong> of the<br />
26S subunit and have proven useful for species differentiation (James<br />
et al., 1996; Naumov et al., 2000; Cadez et al., 2003). For example, the<br />
Hanseniaspora uvaurm-guillermondii cluster is poorly resolved, and<br />
species of Saccharomyces pastorianus/Saccharomyces bayanus, and<br />
Cryptococcus magnus/Filobasidium floriforme/Filobasidium elegans are<br />
<strong>in</strong>dist<strong>in</strong>guishable us<strong>in</strong>g D1/D2 sequences. Sequenc<strong>in</strong>g of the ITS<br />
region can provide the required level of differentiation.<br />
Sequenc<strong>in</strong>g of mitochondrial and prote<strong>in</strong> encod<strong>in</strong>g genes are also<br />
be<strong>in</strong>g used to determ<strong>in</strong>e phylogenetic relationships among yeasts.<br />
These genes <strong>in</strong>clude the translation elongation factor 1α, act<strong>in</strong>-1,<br />
RNA polymerase II, pyruvate decarboxylase, beta tubul<strong>in</strong> gene, small<br />
subunit rDNA and cytochrome oxidase II (Daniel et al., 2001;<br />
Kurtzman and Robnett 2003; Daniel and Meyer 2003).<br />
The basic protocol for sequenc<strong>in</strong>g ribosomal DNA segments is: (i)<br />
prepare a pure culture of the yeast isolate, (ii) extract and purify the<br />
DNA, (iii) perform PCR amplification of the region to be sequenced,<br />
(iv) verify the amplified product by gel eletrophoresis, and (v)<br />
sequence the product us<strong>in</strong>g <strong>in</strong>ternal or external primers. Procedures<br />
for conduct<strong>in</strong>g these operations are well established but are not standardised,<br />
and may vary from one laboratory to another. Primer<br />
sequences used to amplify the different segments of the rDNA have<br />
been tabulated <strong>in</strong> White et al. (1990), Valente et al. (1999), Sipiczki<br />
(2002) and Kurtzman and Robnett (2003). Table 1 lists some key publications<br />
on the identification of yeasts by ribosomal DNA sequenc<strong>in</strong>g.<br />
Some yeasts are not reliably identified by sequenc<strong>in</strong>g s<strong>in</strong>gle gene<br />
segments and it is suggested that sequences be obta<strong>in</strong>ed for multiple<br />
genes or gene segments for more reliable data (Kurtzman, 2003).<br />
2.2. Restriction Fragment Length Polymorphism<br />
(RFLP)<br />
RFLP analysis of the ribosomal DNA segments is emerg<strong>in</strong>g as one<br />
of the most useful methods for rapidly identify<strong>in</strong>g food and beverage
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 73<br />
Table 1. Application of gene sequenc<strong>in</strong>g technology to the identification of species<br />
of food and beverage yeasts<br />
Region Application References<br />
18S Phylogenetic relationships;<br />
Zygosaccharomyces and<br />
Torulaspora species James et al. (1994)<br />
Brettanomyces, Dekkera, Cai et al. (1996)<br />
Debaryomyces, Kluyveromyces<br />
species<br />
Candida, Pichia, Citeromyces species Suzuki and Nakase (1999)<br />
Saccharomyces genus; new species James et al. (1997)<br />
S. kunashirensis, S. mart<strong>in</strong>iae<br />
Saccharomyces sensu lato group; Mikata et al. (2001)<br />
new species S. naganishii,<br />
S. humaticus, S. yukushimaensis<br />
18S, ITS Phylogenetic relationships of Naumov et al. (2000)<br />
Saccharomyces sensu stricto<br />
complex; new species<br />
S. cariocanus, S. kudriavzevii,<br />
S. mikatae<br />
18S; 834- Identification of yeast from Cappa and Cocconcelli (2001)<br />
1415 dairy products<br />
D1/D2 Systematics of ascomycetous yeasts Kurtzman and Robnett (1998)<br />
of 26S Systematics of basdidiomycetous Fell et al. (2000)<br />
yeasts<br />
D1/D2 of Systematics of basdidiomycetous Scorzetti et al. (2002)<br />
26S, ITS yeasts<br />
D1/D2 of Candida davenportii sp. nov. from Stratford et al. (2002)<br />
26S a wasp <strong>in</strong> a soft-dr<strong>in</strong>k production<br />
facility<br />
D1/D2 of Tetrapisispora fleetii sp. nov. from Kurtzman et al. (2004)<br />
26S, ITS a food process<strong>in</strong>g plant<br />
D1/D2 Identification of yeast species;<br />
of 26S <strong>in</strong> Sicilian sourdough Pulvirenti et al. (2001)<br />
<strong>in</strong> orange juice Arias et al. (2002)<br />
<strong>in</strong> spontaneous w<strong>in</strong>e fermentation van Keulen et al. (2003)<br />
from bark of cork oak Villa-Carvajal et al. (2004)<br />
from Malbec grape berries Comb<strong>in</strong>a et al. (2005)<br />
from fermentation of West Jespersen et al. (2005)<br />
African cocoa beans<br />
contam<strong>in</strong>ant <strong>in</strong> carbonated orange P<strong>in</strong>a et al. (2005)<br />
juice production cha<strong>in</strong><br />
ITS Phylogenetic relationships of Belloch et al. (2002)<br />
Kluyveromyces marxianus group<br />
ITS Phylogenetic relationships of James et al. (1996)<br />
Zygosaccharomyces and<br />
Torulaspora species
74 Ai L<strong>in</strong> Beh et al.<br />
Table 1. Application of gene sequenc<strong>in</strong>g technology to the identification of species<br />
of food and beverage yeasts—cont’d<br />
Region Application References<br />
ITS Phylogenetic analysis of the Oda et al. (1997)<br />
Saccharomyces species<br />
Phylogenetic analysis of the Montrocher et al. (1998)<br />
Saccharomyces sensu stricto<br />
complex<br />
ITS Identification of yeast species;<br />
<strong>in</strong> orange fruit and orange juice Heras-Vazques et al. (2002)<br />
from Italian sourdough baked Fosch<strong>in</strong>o et al. 2004<br />
products<br />
ITS1 Separation of S. cerevisiae stra<strong>in</strong>s Naumova et al. 2003<br />
<strong>in</strong> African sorghum beer<br />
IGS Separation of Clavispora opuntiae Lachance et al. 2000<br />
varieties<br />
ITS, IGS Intraspecies diversity of Mrakia Diaz and Fell 2000<br />
and Phaffia species<br />
Act<strong>in</strong> Phylogenetic relationships of Daniel et al. 2001<br />
anamorphic Candida and<br />
related teleomorphic genera<br />
mt COX II Phylogeny of the genus Belloch et al. 2000<br />
Kluyveromyces<br />
Multigenes Ascomycete phylogeny Kurtzman and Robnett<br />
Ascomycete species separation (2003), Kurtzman (2003),<br />
Daniel and Meyer (2003)<br />
Multigenes Taxonomic position of the van der Aa Kühle and<br />
biotherapeutic agent Jespersen (2003)<br />
Saccharomyces boulardii<br />
yeasts. The preferred region for analysis represents the ITS1-5.8S-<br />
ITS2 segment (Figure 1). Us<strong>in</strong>g appropriate primers, this segment is<br />
specifically amplified by PCR. The PCR product is then cleaved with<br />
specific restriction endonucleases, and the result<strong>in</strong>g fragments are separated<br />
by gel electrophoresis. The size (number of base pairs) of the<br />
ITS amplicon itself can be useful <strong>in</strong> discrim<strong>in</strong>at<strong>in</strong>g between yeast<br />
species. The number (usually 1-4) and size (base pairs) of the fragments<br />
as determ<strong>in</strong>ed by band<strong>in</strong>g patterns on the gel are the ma<strong>in</strong> pr<strong>in</strong>ciples<br />
used to discrim<strong>in</strong>ate between yeast species. Generally, more than<br />
one restriction enzyme needs to be used <strong>in</strong> order to obta<strong>in</strong> unequivocal<br />
discrim<strong>in</strong>ation. Some restriction enzymes commonly used are: Cfo<br />
I, Hae III, H<strong>in</strong>f I, Hpa II, Scr FI, Taq I, Nde II, Dde I, Dra I and Mbo<br />
II. Several hundred species of food and beverage yeasts have now been<br />
exam<strong>in</strong>ed by this method and databases of fragment profiles for the<br />
different restriction enzymes and yeast species have been established
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 75<br />
(Esteve-Zarzoso et al., 1999; Granchi et al., 1999; Arias et al., 2002;<br />
Heras-Vazquez et al., 2003; Dias et al., 2003; Naumova et al., 2003).<br />
PCR-RFLP analyses have several advantages that are attractive to<br />
quality assurance analysis <strong>in</strong> the food and beverage <strong>in</strong>dustries. Once a<br />
pure yeast culture has been obta<strong>in</strong>ed, identification to species level can<br />
be done <strong>in</strong> several hours. Essentially, DNA is extracted from the yeast<br />
biomass, amplified by specific PCR, amplicons are digested with the<br />
restriction nucleases and the products separated by gel electrophoresis.<br />
The work load and equipment needs are m<strong>in</strong>imal and data are<br />
generally reproducible. The expense and time for sequenc<strong>in</strong>g are<br />
avoided.<br />
Although PCR-RFLP analysis of the ITS1-5.8S-ITS2 region has<br />
attracted most study to date, there is <strong>in</strong>creas<strong>in</strong>g <strong>in</strong>terest <strong>in</strong> the PCR-<br />
RFLP analysis of other ribosomal segments. These <strong>in</strong>clude the 18S-<br />
ITS region, 18S-ITS-5.8S region, the 26S and NTS regions. It is not<br />
evident at this stage whether target<strong>in</strong>g these regions offers any advantage<br />
over the ITS1-5.8S-ITS2 region and further studies evaluat<strong>in</strong>g the<br />
different approaches are required. Table 2 lists some key reports on the<br />
application of the PCR-RFLP analysis of ribosomal DNA regions to<br />
food and beverage yeasts.<br />
2.3. Nucleic acid probes and species-specific primers<br />
Nucleic acid probes are short, s<strong>in</strong>gle-stranded nucleotides (usually<br />
20-100 bases) that are designed to complement a specific sequence <strong>in</strong><br />
the DNA/RNA of the target organism. They are usually labelled with<br />
a marker molecule to enable their detection. Probes are used <strong>in</strong><br />
hybridization reactions, and are applied <strong>in</strong> a number of formats (Hill<br />
and J<strong>in</strong>neman, 2000; Cox and Fleet, 2003).<br />
In whole cell hybridization protocols (FISH, CISH), yeasts are<br />
directly visualised <strong>in</strong> situ, and identified with fluorescently-labelled, specific<br />
probes that b<strong>in</strong>d to rRNA, located <strong>in</strong> the ribosomes. In ecological<br />
studies, this technique is particularly useful for identify<strong>in</strong>g morphological<br />
types, for quantify<strong>in</strong>g target species and monitor<strong>in</strong>g microbial community<br />
structure and dynamics, for example, <strong>in</strong> exam<strong>in</strong><strong>in</strong>g the spatial<br />
relationships on surfaces of leaves and <strong>in</strong> biofilms (Table 3).<br />
Probes are also used <strong>in</strong> dot blot, slot blot and colony blot hybridisation<br />
assays of yeast biomass on membranes. Detection is achieved<br />
by a labeled DNA probe that hybridises to the DNA/RNA of the<br />
immobilised sample (Hill and J<strong>in</strong>neman, 2000). Another strategy is<br />
to coat the probe onto a solid substrate such as the wells of a<br />
microtitre tray, and use a modified ELISA format to detect the target
76 Ai L<strong>in</strong> Beh et al.<br />
Table 2. Application of PCR-restriction fragment length polymorphism (RFLP) to the identification of food and beverage yeasts<br />
Method: region; primers;<br />
restriction enzymes Applications References<br />
ITS2; (primers ITS3 and ITS4); AseI, Medically-important yeast species Chen et al. (2000)<br />
BanI, EcoRI, H<strong>in</strong>cII, StyI<br />
ITS1-5.8S rRNA-ITS2; (primers ITS1 Yeast species from w<strong>in</strong>e fermentations Guillamón et al. (1998), Ganga and<br />
and ITS4); CfoI, HaeIII, H<strong>in</strong>fI Mart<strong>in</strong>ez. (2004), Comb<strong>in</strong>a et al. (2005)<br />
Yeast species from Irish cider fermentations Morrissey et al. (2004)<br />
Yeast species associated with orange juice Arias et al. (2002)<br />
ITS1-5.8S rRNA-ITS2; (primers ITS1 132 species from food and beverages Esteve-Zarzoso et al. (1999)<br />
and ITS4); CfoI, HaeIII, H<strong>in</strong>fI, DdeI<br />
Yeast species from w<strong>in</strong>e fermentations Granchi et al. (1999), Rodríguez et al.<br />
(2004), Clemente-Jimenez et al. (2004)<br />
Yeast species dur<strong>in</strong>g fermentation and Esteve-Zarzoso et al. (2001)<br />
age<strong>in</strong>g of sherry w<strong>in</strong>es<br />
Yeast species from orange fruit and orange juice Heraz-Vazquez et al. (2003)<br />
1) ITS1-5.8S rRNA-ITS2; (primers ITS1 Yeast species from Sicilian sourdoughs Pulvirenti et al. (2001)<br />
and ITS4); H<strong>in</strong>fI, RsaI, NdeII, HaeIII<br />
2) NTS 2; (primers r-1234 and r-2156);<br />
AluI, BanI<br />
ITS1-5.8S rRNA-ITS2; (primers ITS1 Saccharomyces sensu stricto stra<strong>in</strong>s from Naumova et al. (2003)<br />
and ITS4); HaeIII, HpaII, ScrFI, TaqI African sorghum beer<br />
ITS1-5.8S rRNA-ITS2; (primers ITS1 Differentiation of S. bayanus, S. cerevisiae, Antunovics et al. (2005)<br />
and ITS4); HaeIII, MaeI S. paradoxus isolates from botrytised grape must<br />
ITS1-5.8S rRNA-ITS2; (primers ITS1 Species with<strong>in</strong> the genera of Hanseniaspora Cadez et al. (2003)<br />
and ITS4); H<strong>in</strong>fI, DdeI, MboII and Kloeckera
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 77<br />
1) ITS; (primers ITS1 and NL2); Discrim<strong>in</strong>ation/diversity of S. cerevisiae stra<strong>in</strong>s Baleiras Couto et al. (1996a)<br />
MseI, TaqI<br />
2) NTS; (primers JV51ET and JV52ET);<br />
MseI, TaqI<br />
18S rRNA-ITS1; (primers NS1 128 species from food, w<strong>in</strong>e, beer and soft dr<strong>in</strong>ks Dlauchy et al. (1999)<br />
and ITS2); HaeIII, MspI, AluI, RsaI<br />
18S rRNA-ITS1; (primers NS1 Yeast species from Hungarian dairy products Vasd<strong>in</strong>yei and Deak (2003)<br />
and ITS2); HaeIII, MspI<br />
1) 18S rRNA; (primers p108 and Discrim<strong>in</strong>ation of C. stellata, M. pulcherrima, Capece et al. (2003)<br />
M3989); HaeIII, MspI K. apiculata and S. pombe<br />
2) NTS; (primers NTSF and NTSR);<br />
HaeIII, MspI<br />
18SrDNA and ITS1; (primers NS1 Differentiation of S. cerevisiae and Redzepovic et al. (2002)<br />
and ITS2); HaeIII, MspI S. paradoxus isolates from Croatian v<strong>in</strong>eyards<br />
18SrDNA and ITS1; (primers NS1 Separation of Saccharomyces sensu stricto Smole Moz<strong>in</strong>a et al. (1997)<br />
and ITS2); CfoI, HaeIII, H<strong>in</strong>fI, MspI and Torulaspora species<br />
ITS1-5.8S rRNA-ITS2-part 18SrRNA ; Differentiation of brewery yeasts; Barszczewski and Robak (2004)<br />
(primers NS3 and ITS4); ScrFI, S. carlsbergensis, S. pastorianus, S. bayanus,<br />
HaeIII, MspI S. cerevisiae, S. brasiliensis, S. exiguus<br />
3′ ETS and IGS; (primers 5S2 and Discrim<strong>in</strong>ation of S. cerevisiae, S. carlsbergensis Mol<strong>in</strong>a et al. (1993)<br />
ETS2); MspI, ScrFI and S. pastorianus<br />
MET2 ; EcoRI, PstI Differentiation of S. uvarum and Demuyter et al. (2004)<br />
S. cerevisiae from w<strong>in</strong>e<br />
Separation of S. bayanus, S. cerevisiae and Antunovics et al. (2005)<br />
S. paradoxus w<strong>in</strong>e stra<strong>in</strong>s<br />
26S rRNA; (primers NL1 and NL4); AluI Yeast species from w<strong>in</strong>e fermentations van Keulen et al. (2003)<br />
26S rRNA; (primers NL1 and LRS); Yeast species from w<strong>in</strong>e fermentations Baleiras Couto et al. (2005)<br />
MseI, ApaI, H<strong>in</strong>fI
78 Ai L<strong>in</strong> Beh et al.<br />
Table 3. Application of nucleic acid probes and specific primers for the detection of food and beverage yeasts<br />
Probe/primer Format Application References<br />
Whole cell hybridization; Fluorescent/chemilum<strong>in</strong>escent <strong>in</strong> situ hybridization (FISH/CISH)<br />
FISH; PNA probe <strong>in</strong> D1/D2 26S rRNA D. bruxellensis isolates from w<strong>in</strong>e Stender et al. (2001), Dias et al. (2003)<br />
CISH; PNA probe <strong>in</strong> D1/D2 26S rRNA D. bruxellensis isolates from w<strong>in</strong>ery air samples Connell et al. (2002)<br />
CISH; PNA probes <strong>in</strong> 18S rRNA S. cerevisiae, Z. bailii, D. bruxellensis colonies Perry-O’Keefe et al. (2001)<br />
and 26S rRNA on filter membranes<br />
FISH; DNA probes <strong>in</strong> 18S rRNA S. cerevisiae, P. anomala, D. bruxellensis and Kosse et al. (1997)<br />
D. hansenii isolates, detection <strong>in</strong> yoghurts<br />
FISH; DNA probes <strong>in</strong> 18S rRNA Detection and quantification of A. pullulans Spear et al. (1999), Andrews et al. (2002)<br />
on leaf surfaces<br />
Dot blot/slot blot hybridization<br />
Dot blot hybridization D. hansenii isolates from cheese, differentiation Corredor et al. (2000)<br />
of hansenii and fabryii varieties.<br />
RNA slot blot hybridization Candida sp. EJ1 <strong>in</strong> w<strong>in</strong>e samples Mills et al. (2002)<br />
B. bruxellensis <strong>in</strong> w<strong>in</strong>e Cocol<strong>in</strong> et al. (2004)<br />
PCR-ELISA<br />
Probes <strong>in</strong> D1/D2 region Detection of mar<strong>in</strong>e yeast species; Kiesl<strong>in</strong>g et al. (2001)<br />
Probes immobilized on plates, PCR P. guillermondii, R. diobovatum,<br />
with biot<strong>in</strong>ylated primers R. sphaerocarpum, K.thermotolerans-like,<br />
Probes <strong>in</strong> ITS2 region C. parapsilosis, C. tropicalis, D. hansenii<br />
Probes labelled with DIG Detection of C. albicans, C. tropicalis, Fujita et al. (1995)<br />
C. krusei <strong>in</strong> blood<br />
Capture PCR amplicons on Identification of 18 Candida species Elie et al. (1998)<br />
strepavid<strong>in</strong> coated plates
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 79<br />
Species-specific primers<br />
Universal and species-specific Pathogenic yeasts C. neoformans, Fell (1995)<br />
primers (V3 region of LSU) T. cutaneum, R. mucilag<strong>in</strong>osa<br />
Universal and species-specific primers Detection of several Candida species Mannarelli and Kurtzman (1998)<br />
(D1/D2 region of LSU)<br />
1) species-specific primer pairs Identification of Z. bailii, Z. bisporus, Sancho et al. (2000)<br />
Z. rouxii and T. delbrueckii isolates from fruit<br />
2) one species-specific, the other<br />
universal (ITS region)<br />
Nested PCR D. bruxellensis stra<strong>in</strong>s from isolates and sherry Ibeas et al. (1996)<br />
Multiplex PCR 5 primers; 1 universal, Identification of Dekkera isolates; Egli and Henick-Kl<strong>in</strong>g (2001)<br />
4 species-specific (ITS region) differentiation of B. bruxellensis,<br />
B. anomala, B. custersianus, B. naardenensis<br />
PCR and RT-PCR Detection of B. bruxellensis/B. anomalus Cocol<strong>in</strong> et al. (2004)<br />
Specific primers (D1/D2 26S LSU) from w<strong>in</strong>e samples<br />
RT-PCR; Specific primer pairs (cs 1) gene Detection of viable C. krusei <strong>in</strong> fruit juice Casey and Dobson (2003)<br />
RT-PCR; Specific primers (ITS Identification of S. cerevisiae and Josepa et al. (2000)<br />
and LSU region) S. bayanus/pastorianus isolates<br />
PCR, Multiplex PCR; 4 primers; Detection of S. cerevisiae, S. bayanus Torriani et al. (2004)<br />
2 species-specific pairs (YBR033w region) and S. pastorianus<br />
Real time PCR<br />
Primer pairs (D1/D2 LSU) Detection and enumeration of Phister and Mills (2003)<br />
D. bruxellensis <strong>in</strong> w<strong>in</strong>es<br />
Primer pairs (rad4 gene) Detection and quantification of Delaherche et al. (2004)<br />
B. bruxellensis <strong>in</strong> w<strong>in</strong>es<br />
Specific primer pairs (cs 1) gene Quantification of C. krusei from fruit juice Casey and Dobson (2004)<br />
Universal primer pairs ITS3 and ITS4 Differentiation of Z. bailii, Z. rouxii, C. krusei, Casey and Dobson (2004)<br />
(5.8S and ITS2) R. glut<strong>in</strong>is and S. cerevisiae by difference <strong>in</strong> Tm
80 Ai L<strong>in</strong> Beh et al.<br />
DNA <strong>in</strong> PCR amplicons (Kiesl<strong>in</strong>g et al., 2002) (Table 3). Speciesspecific<br />
primers are used <strong>in</strong> PCR assays to generate amplicons.<br />
Production of the amplicon means that the particular target species<br />
is present <strong>in</strong> the sample. Qualitative detection of the target amplicon<br />
is done by its visualisation <strong>in</strong> gel electrophoresis. Real time PCR systems<br />
are now be<strong>in</strong>g applied to yeasts, and allow the simultaneous<br />
detection and quantification of the target species, omitt<strong>in</strong>g the electrophoresis<br />
step (Table 3).<br />
Nucleic acid probes and specific primers have ga<strong>in</strong>ed widespread<br />
use <strong>in</strong> the detection of bacterial species. Their application to the detection<br />
of yeast species has not been that extensive and further development<br />
is needed. Most probes reported to date have been developed<br />
around specific sequences <strong>in</strong> ribosomal DNA, and it would be worthwhile<br />
to identify other species-specific genes that could be targeted for<br />
probe development.<br />
2.4. Differentiation of Stra<strong>in</strong>s With<strong>in</strong> a Species<br />
The dist<strong>in</strong>ctive character and appeal of many foods and beverages<br />
(eg. bread, beer, w<strong>in</strong>e) produced by fermentation with yeasts are frequently<br />
attributable to the contribution and properties of particular<br />
stra<strong>in</strong>s. Stra<strong>in</strong> typ<strong>in</strong>g is also useful to trace the source of yeast contam<strong>in</strong>ation<br />
<strong>in</strong> outbreaks of food spoilage. The ability to differentiate<br />
stra<strong>in</strong>s with<strong>in</strong> a species is, therefore, an important requirement <strong>in</strong> quality<br />
assurance programs. Over the past 20 years, a diversity of molecular<br />
methods has been developed and applied to the differentiation of<br />
yeast stra<strong>in</strong>s, and some of these are sufficiently robust and convenient<br />
for rout<strong>in</strong>e use (Table 4).<br />
Electrophoretic karyotyp<strong>in</strong>g of genomic DNA us<strong>in</strong>g pulse field<br />
gel electrophoresis (PFGE) and RFLP analysis of genomic DNA<br />
have been widely applied to “f<strong>in</strong>gerpr<strong>in</strong>t” yeast stra<strong>in</strong>s with very<br />
good success and confidence, but they require significant attention<br />
to DNA preparation and extraction, as well as to subsequent electrophoretic<br />
analyses (Card<strong>in</strong>ali and Mart<strong>in</strong>i, 1994; Deak, 1995; van<br />
der Aa Kühle et al., 2001). Analysis of mitochondrial DNA by<br />
RFLP produces fragment profiles that give excellent stra<strong>in</strong> discrim<strong>in</strong>ation.<br />
Simplified methods for extraction and process<strong>in</strong>g of the<br />
mitochondrial DNA have greatly improved the convenience and<br />
reliability of this assay, and consequently it has found significant<br />
application to the analysis of food and beverage yeasts (Querol<br />
et al., 1992; López et al., 2001; see review of Loureiro and Malfieto-<br />
Ferreira, 2003).
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 81<br />
Table 4. Application of PCR-based methods for stra<strong>in</strong> and species differentiation of yeasts associated with foods and beverages<br />
Primers Application References<br />
AFLP<br />
EcoRI-C/Mse-AC Species and stra<strong>in</strong> differentiation of Saccharomyces de Barros Lopes et al. (1999, 2002)<br />
and non-Saccharomyces w<strong>in</strong>e yeasts<br />
MseI-C/PstI-AA, -AC, -AT<br />
EcoRI/MseI, n<strong>in</strong>e primer pairs Differentiation of w<strong>in</strong>e, brew<strong>in</strong>g, bakery and Azumi and Goto-Yamamoto (2000)<br />
sake stra<strong>in</strong>s of Saccharomyces species<br />
MseI-EcoRI four primer pairs Genetic analysis of S. cerevisiae w<strong>in</strong>e stra<strong>in</strong>s Gallego et al. (2005)<br />
EcoRI-C/MseI-AC Identification of pathogenic Candida species, Borst et al. (2003), Ball et al. (2004)<br />
subspecies of C. albicans and C. dubl<strong>in</strong>ensis<br />
EcoRI-MseI four primer pairs Intraspecific variability among A. pullulans De Curtis et al. (2004)<br />
RAPD and micro/m<strong>in</strong>isatellites<br />
(GTG) (CAG) and M13 Differentiation of S. cerevisiae, S. pastorianus, Lieckfeld et al. (1993)<br />
5 5<br />
S. bayanus, S. willianus<br />
(GTG) , (GACA) and phage Differentiation of C. neoformans, C. albidus, Meyer et al. (1993)<br />
5 4<br />
M13 core sequence C. laurentii and R. rubra species, and stra<strong>in</strong>s of<br />
C. neoformans<br />
Primers 15, 18, 20, 21 Differentiat<strong>in</strong>g S. cerevisiae and Z. bailii Baleiras Couto et al. (1994)<br />
Prelim<strong>in</strong>ary screen<strong>in</strong>g of primers Characterisation of w<strong>in</strong>e yeasts; R. mucilag<strong>in</strong>osa, Quesada and Cenis (1995)<br />
S. cerevisiae, S. exiguus, P. membranifaciens,<br />
P. anomala, T. delbrueckii, C. v<strong>in</strong>i stra<strong>in</strong>s<br />
Decamer 1; ACG GTG TTG G Dist<strong>in</strong>guish species with<strong>in</strong> genus Saccharomyces Molnár et al. (1995)<br />
Decamer 2; TGC CGA GCT G<br />
Decamer 3; GGG TAA CGC C Dist<strong>in</strong>guish species with<strong>in</strong> genus Metschnikowia Lopandic et al. (1996)<br />
Yeast species isolated from floral nectaries Herzberg et al. (2002)<br />
M13 Identify yeast species from cheese; D. hansenii, Prill<strong>in</strong>ger et al. (1999)<br />
S. cerevisiae, I. orientalis, K. marxianus, K. lactis,<br />
Y. lipolytica, C. catenulata, G. candidum<br />
Cont<strong>in</strong>ued
82 Ai L<strong>in</strong> Beh et al.<br />
Table 4. Application of PCR-based methods for stra<strong>in</strong> and species differentiation of yeasts associated with foods and beverages—cont’d<br />
Primers Application References<br />
Decamer 1; ACG GTG TTG G<br />
Decamer 2; TGC CGA GCT G<br />
Decamer 3; TGC AGC GTG G<br />
Decamer 4; GGG TAA CGC C<br />
M13 Yeast species from dairy products; S. cerevisiae, Andrighetto et al. (2000)<br />
K. marxianus, K. lactis, D. hansenii, Y. lipolytica,<br />
T. delbrueckii<br />
RF2<br />
Yeast species from sourdough products Forsh<strong>in</strong>o et al. (2004)<br />
M13V universal Yeast species from Greek sourdough; Paramithiotis et al. (2000)<br />
P. membranifaciens, S. cerevisiae, Y. lipolytica<br />
M13 Yeast species from artisanal Fiore Sado cheese; Fadda et al. (2004)<br />
C. zeylanoides, D. hansenii, K. lactis, C. lambica,<br />
G. candidum<br />
Stra<strong>in</strong> typ<strong>in</strong>g of D. hansenii and G. candidum Vasd<strong>in</strong>yei and Deak (2003)<br />
Primers 24, 28, OPA11 Discrim<strong>in</strong>ation of S. cerevisiae stra<strong>in</strong>s Baleiras Couto et al. (1996a)<br />
(GAC) , (GTG) 5 5<br />
(GTG) and (CAG) Differentiation of stra<strong>in</strong>s of Z. bailii and Z. bisporus Baleiras Couto et al. (1996b)<br />
5 5<br />
Separation of S. cerevisiae from K. apiculata Caruso et al. (2002)<br />
Separation of C. stellata, M. pulcherrima, Capece et al. (2003)<br />
K. apiculata and Schiz. pombe<br />
(GTG) , (CAG) and M13 Genotyp<strong>in</strong>g the R. glut<strong>in</strong>is complex Gadanho and Sampaio (2002)<br />
5 5<br />
Primer P24, (GTG) and (GAC) Typ<strong>in</strong>g of P. galeiformis stra<strong>in</strong>s <strong>in</strong> orange P<strong>in</strong>a et al. (2005)<br />
5 5<br />
juice production<br />
(GTG) (ATG) and M13 Differentiation of Hanseniaspora species Cadez et al. (2002)<br />
5 5<br />
OPA03, OPA 18
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 83<br />
ERIC1R, ERIC2 C. boid<strong>in</strong>ii, C. mesenterica, C. sake, C. stellata, Hierro et al. (2004)<br />
D. anomala, D. bruxellensis, H. uvarum, I. terricola,<br />
S. ludwigii, Schiz. pombe, T. debrueckii, Z. bailii,<br />
S. bayanus, S. cerevisiae from w<strong>in</strong>e<br />
REPIR1, REP2I<br />
Intron splice site primer EI1<br />
Intron splice primers Differentiation of commercial w<strong>in</strong>e S. cerevisiae de Barros Lopes et al. (1996, 1998)<br />
stra<strong>in</strong>s. Differentiation of non-Saccharomyces w<strong>in</strong>e<br />
species and stra<strong>in</strong>s; S. cerevisiae, S. bayanus,<br />
T. delbrueckeii, I. orientalis, H. uvarum,<br />
H. guillermondii, M. pulcherrima, P. fermentans,<br />
P. membranaefaciens<br />
EI1, EI2, LA1, LA2<br />
S. cerevisiae and sensu stricto stra<strong>in</strong>s<br />
Primers δ1 and δ2 Differentiation of S. cerevisiae stra<strong>in</strong>s, S. douglassi, Ness et al. (1993)<br />
S. chevalierii, S. bayanus<br />
Primers δ12 and δ2 Lavallée et al. (1994), Fernández-Esp<strong>in</strong>ar<br />
et al. (2001), Schuller et al. (2004)<br />
Identification/authentication of commercial<br />
w<strong>in</strong>e S. cerevisiae stra<strong>in</strong>s<br />
Characterisation of wild S. cerevisiae stra<strong>in</strong>s from Versavaud et al. (1995), Cappello et al.<br />
grapes and w<strong>in</strong>e fermentations (2004), Demuyter et al. (2004)<br />
Differentiat<strong>in</strong>g S. cerevisiae stra<strong>in</strong>s from sourdough Pulvirenti et al. (2001)<br />
Genetic relatedness between cl<strong>in</strong>ical and food de Llanos et al. (2004)<br />
S. cerevisiae stra<strong>in</strong>s<br />
Introns <strong>in</strong> COX1 Monitor w<strong>in</strong>e starter S. cerevisiae stra<strong>in</strong>s López et al. (2003)<br />
mitochondrial gene dur<strong>in</strong>g fermentation<br />
Cont<strong>in</strong>ued
84 Ai L<strong>in</strong> Beh et al.<br />
Table 4. Application of PCR-based methods for stra<strong>in</strong> and species differentiation of yeasts associated with foods and beverages—cont’d<br />
Primers Application References<br />
Microsatellite markers/Sequence-Tagged Site markers<br />
(ScTAT1) chromosome XIII Differentiation of S. cerevisiae stra<strong>in</strong>s Gallego et al. (1998)<br />
Locus SCYOR267, Locus SC8132X, Differentiation of <strong>in</strong>dustrial S. cerevisiae w<strong>in</strong>e stra<strong>in</strong>s González Techera et al. (2001)<br />
Locus SCPTSY7<br />
Locus SC8132X Monitor<strong>in</strong>g the populations of S. cerevisiae Howell et al. (2004)<br />
stra<strong>in</strong>s dur<strong>in</strong>g grape juice fermentation<br />
Multiplex 1; Loci ScAAT2,<br />
ScAAT3, ScAAT5 Differentiation of S. cerevisiae isolates from Pérez et al. (2001), Gallego et al. (2005)<br />
spontaneous w<strong>in</strong>e fermentations<br />
Multiplex 2; Loci ScAAT1,<br />
ScAAT4, ScAAT6 Differentiation of commercial S. cerevisiae Schuller et al. (2004)<br />
w<strong>in</strong>e stra<strong>in</strong>s<br />
M<strong>in</strong>isatellite core sequence (wild type phage) GAG GGT GGC GGT TCT; M13V universal; GTT TCC CCA GTC ACG AC; Phage M13 core<br />
sequence; GAG GGT GGX GGX TCT
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 85<br />
2.5. PCR-based F<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g<br />
PCR technology has provided new opportunities for develop<strong>in</strong>g<br />
faster, more convenient methods for typ<strong>in</strong>g yeasts and fungi. Two of<br />
the first methods developed were Random Amplified Polymorphic<br />
DNA (RAPD) analysis and Amplified Fragment Length<br />
Polymorphism (AFLP) analysis (Baleiras Couto et al., 1994, 1995,<br />
1996a; Vos et al., 1995; van der Vossen et al., 2003). These methods<br />
have the capability of analyz<strong>in</strong>g an extensive portion of the genome,<br />
and reveal polymorphisms that differentiate at both the species and<br />
stra<strong>in</strong> levels (Table 4).<br />
In the case of RAPD, DNA template is subject to PCR amplification<br />
with s<strong>in</strong>gle, short primers (10-15bp of random/arbitary<br />
sequences) that hybridize to a set of arbitary loci <strong>in</strong> the genome. Some<br />
primers used for this purpose are listed <strong>in</strong> van der Vossen et al. (2003).<br />
PCR cycles are performed under conditions of low str<strong>in</strong>gency. The<br />
amplicons produced are separated on gel electrophoresis and give profiles<br />
that f<strong>in</strong>gerpr<strong>in</strong>t the stra<strong>in</strong> or species. AFLP is a variation of<br />
RAPD. The approach firstly <strong>in</strong>volves digestion of genomic DNA with<br />
two restriction nucleases (usually EcoRI and MseI). The fragments are<br />
then ligated with end-specific adapters, followed by two successive<br />
rounds of PCR. Pre-selective PCR amplifies fragments us<strong>in</strong>g primers<br />
complimentary to the adapter sequences. A second, selective PCR is<br />
performed with primers conta<strong>in</strong><strong>in</strong>g additional nucleotide bases at the<br />
3′ end (selective bases are user def<strong>in</strong>ed). The result<strong>in</strong>g products are<br />
resolved by gel electrophoresis or by capillary electrophoresis. There<br />
are extra steps <strong>in</strong>volved <strong>in</strong> AFLP, but this method can potentially generate<br />
more extensive <strong>in</strong>formation from a s<strong>in</strong>gle restriction/ligation<br />
reaction than other PCR strategies (de Barros Lopes et al., 1999;<br />
Lopandic et al., 2005). Both RAPD and AFLP analyses gave excellent<br />
stra<strong>in</strong> and species differentiation. However, the ma<strong>in</strong> hurdles to rout<strong>in</strong>e<br />
application are the need for str<strong>in</strong>gent standardisation of conditions<br />
<strong>in</strong> order to obta<strong>in</strong> reproducible data, and the workload <strong>in</strong>volved.<br />
Micro- and m<strong>in</strong>isatellites are short repeat motifs of about 15-30 and<br />
2-10bp, respectively. Primers target<strong>in</strong>g these sequences are used <strong>in</strong><br />
PCR assays to generate an array of amplicons, the profile or “f<strong>in</strong>gerpr<strong>in</strong>t”<br />
of which reflects the polymorphism of these regions and the<br />
distance between them. Some commonly used primers for food yeasts<br />
are (GTG) 5 , (GAC) 5 and M13 phage core sequences. This method has<br />
had good success <strong>in</strong> differentiat<strong>in</strong>g stra<strong>in</strong>s of several food spoilage and<br />
w<strong>in</strong>e yeasts (Baleiras Couto, et al., 1996b), and for differentiat<strong>in</strong>g<br />
species of yeasts from dairy sources (Prill<strong>in</strong>ger et al., 1999) (Table 4).
86 Ai L<strong>in</strong> Beh et al.<br />
Intron splice sequences are also known for their polymorphism and<br />
have been exam<strong>in</strong>ed as sites that could give stra<strong>in</strong> differentiation. De<br />
Barros Lopes et al. (1996, 1998) reported a PCR method for the analysis<br />
of these sites. The method was simple, quick (several hours),<br />
robust, reproducible and gave profiles enabl<strong>in</strong>g the differentiation of<br />
w<strong>in</strong>emak<strong>in</strong>g stra<strong>in</strong>s of Saccharomyces. Build<strong>in</strong>g on this concept,<br />
López et al. (2002) developed a multiplex PCR assay based on <strong>in</strong>trons<br />
of the COX1 mitochondrial gene. The assay, which was simple and<br />
fast (8 hours), gave good differentiation of w<strong>in</strong>e stra<strong>in</strong>s of<br />
Saccharomyces. Other repetitive elements that are targeted <strong>in</strong> PCRmediated<br />
f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g techniques <strong>in</strong>clude the delta repeat, the repetitive<br />
extragenic pal<strong>in</strong>dromic (REP) and enterobacterial repetitive<br />
<strong>in</strong>tergenic consensus (ERIC) sequences (Hierro et al., 2004).<br />
The application of these PCR methods to food and beverage yeasts<br />
(Table 4) generally gives good species and stra<strong>in</strong> characterisation.<br />
However, the level of resolution achieved is greatly <strong>in</strong>fluenced by the<br />
choice of primers and the taxa under study.<br />
3. MOLECULAR STRATEGIES FOR<br />
MONITORING YEAST COMMUNITIES IN<br />
FOODS AND BEVERAGES<br />
The diversity of yeast species associated with foods and beverages<br />
is usually determ<strong>in</strong>ed by cultur<strong>in</strong>g homogenates of the product on<br />
plates of agar media (Fleet, 1992; Deak, 2003). Yeast colonies are then<br />
isolated and identified. As mentioned <strong>in</strong> previous sections, molecular<br />
methods have now found widespread application <strong>in</strong> identify<strong>in</strong>g these<br />
isolates. New, culture-<strong>in</strong>dependent methods based on PCR-denatur<strong>in</strong>g<br />
gel gradient electrophoresis (DGGE) and PCR-temperature gradient<br />
gel electrophoresis (TGGE) are now be<strong>in</strong>g used to determ<strong>in</strong>e the<br />
ecological profile of yeasts <strong>in</strong> foods and beverages (Muyzer and<br />
Smalla, 1998). The basic strategy is outl<strong>in</strong>ed <strong>in</strong> Figure 2. Total DNA<br />
is extracted from samples of the product. Us<strong>in</strong>g universal fungal<br />
primers (or genus-specific primers), yeast ribosomal DNA with<strong>in</strong> the<br />
extract is specifically amplified by PCR. Generally, the D1/D2 doma<strong>in</strong><br />
of the 26S subunit is targeted, but other regions such as the 18S subunit<br />
may be used. The amplicons produced by PCR are next separated<br />
by either DGGE or TGGE, which resolve the different DNA amplicons<br />
on the basis of their sequence/melt<strong>in</strong>g doma<strong>in</strong>s. DGGE uses a<br />
polyacrylamide gel conta<strong>in</strong><strong>in</strong>g a l<strong>in</strong>ear gradient of denaturant (mix-
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 87<br />
<strong>Food</strong> / Beverage<br />
Extract and purify DNA/RNA<br />
PCR-amplification of yeast rDNA<br />
DGGE/TGGE separation of DNA amplicons<br />
DNA bands<br />
correspond to<br />
different species<br />
Isolate and sequence bands to give species identity<br />
Figure 2. A culture-<strong>in</strong>dependent approach for determ<strong>in</strong><strong>in</strong>g the yeast ecology of foods<br />
and beverages us<strong>in</strong>g PCR-DGGE/TGGE analyses<br />
ture of urea and formamide), while TGGE uses a gradient of temperature<br />
to denature the strands of DNA amplicons. Usually, each DNA<br />
band found <strong>in</strong> the gel corresponds to a yeast species. The band is<br />
excised from the gel and sequenced to give the species identity. Thus,<br />
a profile of the species associated with the ecosystem is obta<strong>in</strong>ed,<br />
without the need for agar culture. It is believed that this molecular<br />
approach overcomes the bias of culture methods, and reveals species<br />
that might fail to produce colonies on agar media. Consequently, it is<br />
considered that a more accurate representation of the diversity of<br />
yeast species <strong>in</strong> the food product is obta<strong>in</strong>ed (Giraffa, 2004). Table 5<br />
lists a range of studies where PCR-DGGE/TGGE have been applied<br />
to the analysis of food and beverage yeasts.<br />
Generally, there has been good agreement between yeast species<br />
detected <strong>in</strong> foods and beverages by PCR-DGGE/TGGE and culture<br />
on agar media, but some discrepancies have been noted. In some cases,<br />
yeasts were recovered by DGGE/TGGE analyses but not by culture.<br />
These observations have led to suggestions that viable but nonculturable<br />
yeasts may be present (Mills et al., 2002; Meroth et al.,<br />
2003; Masoud et al., 2004; Prakitchaiwattana et al., 2004; Nielsen<br />
et al., 2005). However, DNA from non-viable yeast cells or DNA<br />
released from autolysed yeast cells could account for these f<strong>in</strong>d<strong>in</strong>gs,<br />
and caution is needed when <strong>in</strong>terpret<strong>in</strong>g the DGGE/TGGE data. To<br />
address this limitation, Mills et al. (2002) and Cocol<strong>in</strong> and Mills
88 Ai L<strong>in</strong> Beh et al.<br />
Table 5. Application of PCR-denaturation gradient gel electrophoresis and PCR-temperature gradient gel electrophoresis to ecological studies<br />
of yeasts <strong>in</strong> foods and beverages.<br />
Method: region/primers Application References<br />
PCR-DGGE Succession of yeast species <strong>in</strong> w<strong>in</strong>e fermentations;<br />
Nested PCR; NL1-NL4,<br />
NL1gc-LS2; 26S rDNA<br />
model w<strong>in</strong>e fermentation; Cocol<strong>in</strong> et al. (2000)<br />
commercial Dolce w<strong>in</strong>e fermentations; Cocol<strong>in</strong> et al. (2001)<br />
cont<strong>in</strong>uous w<strong>in</strong>e fermentation; Cocol<strong>in</strong> et al. (2002a)<br />
PCR-DGGE and RT PCR-DGGE Yeast species <strong>in</strong> Botrytis-affected w<strong>in</strong>e fermentations Mills et al. (2002)<br />
26S rDNA/rRNA; nested PCR; Inhibition of w<strong>in</strong>e yeast species by sulphur dioxide Cocol<strong>in</strong> and Mills (2003)<br />
NL1-NL4, NL1gc-LS2<br />
PCR-TGGE Discrim<strong>in</strong>ation of w<strong>in</strong>e yeast species Hernán-Gómez et al. (2000)<br />
18S rDNA;<br />
YUNIV1gc-YUNIV3 Diversity of yeast species <strong>in</strong> w<strong>in</strong>e fermentations Fernández-Gonzáles et al. (2001)<br />
PCR-DGGE Yeast species <strong>in</strong> sourdough starters mixtures, rye Meroth et al. (2003)<br />
flour and sourdough samples<br />
26S rDNA;<br />
U1gc-U2<br />
PCR-DGGE Yeast species on w<strong>in</strong>e grapes Prakitchaiwattana et al. (2004)<br />
26S rDNA; nested PCR;<br />
NL1-NL4, NL1gc-LS2
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 89<br />
PCR-DGGE Phyllospheric yeast species and fungicide Gildemacher et al. (2004)<br />
treatments on russett<strong>in</strong>g of Elstar apples<br />
18S rDNA; nested PCR;<br />
NS1-NS8, NS1gc-NS2+10<br />
PCR-DGGE Yeast species <strong>in</strong> raw milk Cocol<strong>in</strong> et al. (2002b)<br />
26S rDNA; NL1gc-LS2<br />
PCR-DGGE Yeast species <strong>in</strong>volved <strong>in</strong> coffee fermentation Masoud et al. (2004)<br />
26S rDNA; nested PCR;<br />
NL1-NL4, NL1gc-LS2; or NL1-LS2,<br />
NL1gc-LS2<br />
PCR-DGGE Yeast populations associated with Ghanaian Nielsen et al. (2005)<br />
26S rDNA; NL1gc-LS2 cocoa fermentations<br />
PCR-DGGE Differentiation of Saccharomyces sensu strictu stra<strong>in</strong>s; Manzano et al. (2004)<br />
S. cerevisiae, S. paradoxus and S. bayanus/S. pastorianus<br />
ITS2; Schafgc-Schar<br />
(Saccharomyces sensustricto<br />
specific)<br />
PCR-TGGE S. cerevisiae and S. paradoxus <strong>in</strong> w<strong>in</strong>e fermentation Manzano et al. (2005)<br />
ITS2; Schafgc-Schar
90 Ai L<strong>in</strong> Beh et al.<br />
(2003) have proposed the use of RT-PCR protocols that target RNA<br />
rather than DNA templates from the food or beverages.<br />
In several studies, culture methods have revealed the presence of<br />
yeast species not found by PCR-DGGE (Mills et al., 2002; Masoud<br />
et al., 2004; Prakitchaiwattana et al., 2004). Such yeasts were present<br />
at populations at less than 10 2 -10 3 CFU/ml or g of product and this is<br />
considered to be the lower limit of detection by PCR-DGGE.<br />
Nevertheless, there are reports where yeast species present at 10 4 -10 5<br />
CFU/ml or g product have not been detected by PCR-DGGE. This<br />
can occur when there is a mixture of different yeast species <strong>in</strong> the sample,<br />
with one species be<strong>in</strong>g numerically present at populations 100-<br />
1000 times more than other species. The DNA of the dom<strong>in</strong>ant<br />
species may b<strong>in</strong>d primers more favorably, result<strong>in</strong>g <strong>in</strong> weaker amplification<br />
of the m<strong>in</strong>ority species. Also, when universal primers are used,<br />
there may be stronger hybridization of the primers with the DNA of<br />
some species over others (Mills et al., 2002; Prakitchaiwattana et al.,<br />
2004; Nielsen et al., 2005).<br />
While the relative mobility of DNA bands on DGGE/TGGE gels<br />
can discrim<strong>in</strong>ate between closely related species (e.g. those <strong>in</strong><br />
Saccharomyces sensu stricto, Manzano et al., 2004), exceptions can<br />
occur. In some cases, different species have produced bands with<br />
similar mobilities (Hernán-Gómez et al., 2000; Gildemacher et al.,<br />
2004). There are several reports where multiple bands have been<br />
found for the one species (eg. two bands for Candida sp. (Mills et al.,<br />
2002), three bands for Pichia kluyveri (Masoud et al., 2004), and several<br />
bands for Metschnikowia pulcherrima (Prakitichaiwattana et al.,<br />
2004). The reasons for multiple band<strong>in</strong>g with<strong>in</strong> the one species are<br />
not well understood, but may reflect artefacts of PCR us<strong>in</strong>g primers<br />
with GC clamps and DNA denaturation k<strong>in</strong>etics dur<strong>in</strong>g electrophoresis.<br />
Multiple bands could arise from nucleotide variations<br />
among multiple rDNA copies with<strong>in</strong> a s<strong>in</strong>gle stra<strong>in</strong>, or could also<br />
<strong>in</strong>dicate the presence of different stra<strong>in</strong>s with<strong>in</strong> a species. To address<br />
these anomalies, it is good practice therefore, to isolate <strong>in</strong>dividual<br />
bands from DGGE/TGGE gels and confirm their identity by<br />
sequenc<strong>in</strong>g.<br />
F<strong>in</strong>ally, food samples often conta<strong>in</strong> large amounts of DNA from<br />
plants, animals and other microbial groups (eg. bacteria and filamentous<br />
fungi) that have the potential to <strong>in</strong>terfere with the specific PCRamplification<br />
of yeast DNA and compromise the reliability and<br />
quality of the data obta<strong>in</strong>ed by DGGE/TGGE. For example, we have<br />
experienced particular difficulty <strong>in</strong> detect<strong>in</strong>g yeasts <strong>in</strong> mould ripened<br />
cheeses with PCR-DGGE and PCR-TGGE because of the large
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 91<br />
amounts of fungal (Penicillium) DNA that occur <strong>in</strong> the cheese extracts<br />
(Nurl<strong>in</strong>awati, Cox and Fleet, unpublished data).<br />
4. FACTORS AFFECTING PERFORMANCE OF<br />
MOLECULAR METHODS FOR THE<br />
ANALYSIS OF YEASTS<br />
As mentioned previously, molecular methods need to meet certa<strong>in</strong><br />
performance and practical criteria before they will ga<strong>in</strong> acceptance<br />
for rout<strong>in</strong>e use <strong>in</strong> the quality assurance laboratories of the food and<br />
beverage <strong>in</strong>dustries. First, they will need to give accurate, reliable<br />
and reproducible data at the appropriate levels of sensitivity and<br />
selectivity, and meet the appropriate tolerances for false positive<br />
and false negative results. Second, they will need to be simple,<br />
convenient and <strong>in</strong>expensive to use, and give results relatively quickly<br />
(Cox and Fleet, 2003).<br />
PCR assays form the basis of most molecular methods used to<br />
analyse yeasts. Consequently, it is important to identify and understand<br />
the various factors which affect the performance of this technology.<br />
The pr<strong>in</strong>ciples of PCR can be found <strong>in</strong> many text books (eg.<br />
McPherson and Møller, 2000). More specific discussions of its application<br />
<strong>in</strong> microbiological analysis are given by Bridge et al. (1998),<br />
Hill and J<strong>in</strong>neman (2000), Sachse and Frey (2003) and Cox and Fleet<br />
(2003). The follow<strong>in</strong>g sections highlight some of the conceptual and<br />
practical variables that affect its performance as applied to the analysis<br />
of food and beverage yeasts. More general discussions of these factors<br />
are given by Edel (1998), Hill and J<strong>in</strong>neman (2000), Sachse<br />
(2003), Radström et al. (2003), Bretagne (2003) and Lübeck and<br />
Hoofar (2003).<br />
PCR assay <strong>in</strong>volves the follow<strong>in</strong>g operations; (i) sample preparation<br />
(ii) extraction and preparation of DNA (iii) amplification of DNA by<br />
PCR (iv) detection of PCR products and (v) process<strong>in</strong>g and <strong>in</strong>terpretation<br />
of the data.<br />
4.1. Sample Preparation and DNA Extraction<br />
For many applications, the sample is a pure culture of a yeast isolate.<br />
Consequently, sampl<strong>in</strong>g is not an issue provided that the culture<br />
has proven purity. Nevertheless, the culture needs to be grown to<br />
provide biomass for DNA extraction. Variables here <strong>in</strong>clude the
92 Ai L<strong>in</strong> Beh et al.<br />
culture medium and time of <strong>in</strong>cubation, and these have not been given<br />
proper consideration <strong>in</strong> previous literature. Carry over of media <strong>in</strong>gredients<br />
could <strong>in</strong>hibit the PCR assay (Rossen et al., 1992). The physiological<br />
age of the yeast cells (e.g. exponential, stationary phase,<br />
autolys<strong>in</strong>g, dead) at the time of assay can affect the efficiency of DNA<br />
extraction and the quality of DNA template for PCR. Depend<strong>in</strong>g on<br />
culture age, various cell prote<strong>in</strong>s, for example, may <strong>in</strong>teract with the<br />
genomic DNA, thereby affect<strong>in</strong>g primer anneal<strong>in</strong>g to the template, or<br />
they can affect the activity of the DNA polymerase (de Barros Lopes<br />
et al., 1996). Some basidiomycetous yeasts may have tougher cell walls<br />
than ascomycetous yeasts and require more vigorous procedures for<br />
equivalent DNA extraction (Prakitchaiwattana et al., 2004).<br />
The purity and concentration of template DNA become more critical<br />
when analys<strong>in</strong>g yeast cells associated with food or beverage<br />
matrices, as for example, <strong>in</strong> studies us<strong>in</strong>g DGGE or TGGE (Table 5).<br />
It is well known that plant polysaccharides, humic components and<br />
other polyphenolic materials can co-purify with DNA and <strong>in</strong>hibit<br />
the PCR reaction (Wilson, 1997; Marshall et al., 2003). The relative<br />
ratio of yeast DNA to other DNA species (e.g. that from filamentous<br />
fungi, bacteria, plants) is another factor that is not properly<br />
understood <strong>in</strong> the performance of PCR assays. The design of<br />
primers would be very important here to m<strong>in</strong>imise or prevent their<br />
b<strong>in</strong>d<strong>in</strong>g to non-target DNA.<br />
Many “<strong>in</strong>-house” methods have been described to extract and purify<br />
DNA from yeast cells, whether they be biomass orig<strong>in</strong>at<strong>in</strong>g from a<br />
pure culture or biomass extracted directly from the food matrix. The<br />
methods <strong>in</strong>clude freez<strong>in</strong>g and boil<strong>in</strong>g of cells, mechanical disruption<br />
by shak<strong>in</strong>g with glass beads or zirconium, digestion with lytic enzymes<br />
and extraction with chemical solvents (Hill and J<strong>in</strong>neman, 2000;<br />
Haugland et al., 2002). Ideally, this “front-end” part of the analytical<br />
process needs to be simple and convenient, but yield template DNA<br />
that will perform satisfactorily <strong>in</strong> PCR assays and give the required<br />
detection sensitivity. Critical evaluation and some degree of standardization<br />
of these methods are needed.<br />
4.2. PCR Amplification of Template DNA<br />
PCR assays are enzymatic reactions. Accord<strong>in</strong>gly, their performance<br />
and progress follow the basic pr<strong>in</strong>ciples of enzymology. The substrates<br />
are the template DNA, oligonucleotide primers and equimolar<br />
amounts of each nucleotide base; ATP, GTP, TTP and CTP, the<br />
enzyme to catalyze the reaction is DNA polymerase, and the product
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 93<br />
is newly synthesized DNA. Like all enzymatic processes, the reaction<br />
is highly specific, and its k<strong>in</strong>etics are determ<strong>in</strong>ed by factors such as<br />
pH, temperature, concentration of reactants, requirements for cofactors<br />
and the presence of any <strong>in</strong>hibitors. Magnesium ions are critical<br />
co-factors, the concentration of which determ<strong>in</strong>es the specificity,<br />
efficiency and fidelity of the reaction.<br />
Two factors make PCR more complex than other enzyme reactions.<br />
First, temperature control requires cyclic variation accord<strong>in</strong>g to the<br />
follow<strong>in</strong>g program; start<strong>in</strong>g at 91-97’C for the denaturation of double<br />
stranded DNA; decreas<strong>in</strong>g to 40-65’C for primer anneal<strong>in</strong>g to s<strong>in</strong>gle<br />
strands of template DNA; and <strong>in</strong>creas<strong>in</strong>g to 68-74’C for DNA strand<br />
extension by DNA polymerase. About 30-40 cycles are conducted.<br />
Second, the DNA product also becomes an enzyme substrate as the<br />
reaction progresses. The great diversity of PCR applications also<br />
<strong>in</strong>troduces specific variables that require understand<strong>in</strong>g and optimization.<br />
In particular, these are the length and sequence of primers, the<br />
amount of template DNA, the amount of non-target/background<br />
DNA, and carry-over material from the sample matrix that could<br />
<strong>in</strong>hibit primer anneal<strong>in</strong>g and DNA polymerase activity. Table 6 summarizes<br />
some of the key variables that affect the performance of PCR<br />
assays. Because of the broad range of PCR applications, it is difficult<br />
to prescribe one set of optimum conditions. Consequently, optimisation<br />
must be done on the basis of each application. The key aims of<br />
optimisation are to <strong>in</strong>crease diagnostic specificity and diagnostic sensitivity<br />
(detection limit). Some good discussions of these variables can<br />
be found <strong>in</strong> Edel (1998), McPherson and Møller (2000), Sachse (2003),<br />
Radström et al. (2003), Bretagne (2003) and Lübeck and Hoofar<br />
(2003). Wilson (1997) has reviewed various factors that <strong>in</strong>hibit and<br />
facilitate PCR.<br />
The conditions of electrophoresis used to separate and detect<br />
the DNA amplicons represent another suite of variables that need<br />
Table 6. Factors affect<strong>in</strong>g DNA amplification by PCR<br />
1. Primer to template ratio<br />
2. Efficiency of primer anneal<strong>in</strong>g<br />
3. Enzyme to template ratio<br />
4. Length and sequence of primers<br />
5. Concentration of non-target DNA<br />
6. Inhibitors from sample matrix<br />
7. Temperature cycl<strong>in</strong>g protocol<br />
8. Source of DNA polymerase<br />
9. Reaction facilitators
94 Ai L<strong>in</strong> Beh et al.<br />
to be optimised and managed. Variables, here <strong>in</strong>clude the gell<strong>in</strong>g<br />
agent (agarose or polyacrylamide) and its concentration, runn<strong>in</strong>g<br />
time, temperature and voltage, and composition of buffer (Andrews,<br />
1986; Hames, 1998). The extent of cross-l<strong>in</strong>k<strong>in</strong>g and pore size <strong>in</strong><br />
polyacrylamide gels affected the resolution of DNA bands and detection<br />
of yeast species on grapes by PCR-DGGE (Prakitchaiwattana<br />
et al., 2004).<br />
5. STANDARDIZATION OF MOLECULAR<br />
METHODS FOR ANALYSIS FOR YEASTS IN<br />
FOODS AND BEVERAGES<br />
The commercial significance of yeast <strong>in</strong> foods and beverages has<br />
major implications <strong>in</strong> national and <strong>in</strong>ternational trade (Fleet, 2001).<br />
Companies trad<strong>in</strong>g <strong>in</strong> foods and beverages will usually have contractual<br />
arrangements that specify criteria for the presence of yeasts. In<br />
this context, molecular methods used for yeast analyses will need to<br />
meet the rigours of legal or forensic scrut<strong>in</strong>y. They will need some<br />
form of standardization and <strong>in</strong>ternational acceptance. While there<br />
have been major advances <strong>in</strong> the harmonization and standardization<br />
of cultural methods for the analysis of microorganisms <strong>in</strong> food and<br />
beverages (<strong>Food</strong> Control, 1996; Scotter et al., 2001; Langton et al.,<br />
2002), this need rema<strong>in</strong>s a challenge for molecular methods.<br />
The lack of standardization and validation for molecular analyses<br />
of microorganisms <strong>in</strong> foods and beverages is well recognized and<br />
strategies are be<strong>in</strong>g developed to address this need, especially for bacteria<br />
of public health significance (Schafer et al., 2001; Lübeck and<br />
Hoofar, 2003; Lübeck et al., 2003). Hoofar and Cook (2003) and<br />
Malorny et al. (2003) have outl<strong>in</strong>ed the pr<strong>in</strong>ciples and protocols for<br />
achiev<strong>in</strong>g this goal, based upon the FOOD-PCR project of the<br />
European Commission (http://www.PCR.dk). These <strong>in</strong>itiatives<br />
equally apply to the molecular analyses of yeasts <strong>in</strong> foods and provide<br />
a framework upon which to develop similar projects for yeasts and<br />
other fungi. The first stage is to def<strong>in</strong>e the specific application to be<br />
evaluated. This is followed by evaluat<strong>in</strong>g and def<strong>in</strong><strong>in</strong>g the conditions<br />
of the assay (e.g. sample treatment and DNA extraction, primer selection,<br />
PCR conditions, detection limit), develop<strong>in</strong>g positive and negative<br />
control assays, select<strong>in</strong>g procedures for data analysis, and, f<strong>in</strong>ally,<br />
develop<strong>in</strong>g protocols for “<strong>in</strong>-house” and <strong>in</strong>ter-laboratory validation<br />
trials. From these evaluations, consensus and standardization should
Evaluation of Molecular Methods for the Analysis of Yeasts <strong>in</strong> <strong>Food</strong>s 95<br />
emerge. Leuschner et al. (2004) reported the results of an <strong>in</strong>terlaboratory<br />
evaluation of a PCR method for the detection and identification<br />
of probiotic stra<strong>in</strong>s of S. cerevisiae <strong>in</strong> animal feed. Good agreement<br />
(but not 100%) was obta<strong>in</strong>ed between laboratories and an “official”<br />
method for analys<strong>in</strong>g animal feed for these yeasts was proposed.<br />
6. REFERENCES<br />
Andrews, A. T., 1986, Electrophoresis Theory, Techniques and Biochemical and Cl<strong>in</strong>ical<br />
Applications, Oxford University Press, UK.<br />
Andrews, J. H., Spear, R. N., and Nordheim, E. V., 2002, Population biology of<br />
Aureobasidium pullulans on apple leaf surfaces, Can. J. Microbiol. 48:500-513.<br />
Andrighetto, G., Psomas, E., Tzanetakis, N., Suzzi, G., and Lombardi, A., 2000,<br />
Randomly amplified polymorphic DNA (RAPD) PCR for the identification of<br />
yeasts isolated from dairy products, Letters <strong>in</strong> Appl. Microbiol. 30:5-9.<br />
Antunovics, Z., Ir<strong>in</strong>yi, L., and Sipiczki, M., 2005, Comb<strong>in</strong>ed application of methods<br />
to taxonomic identification of Saccharomyces stra<strong>in</strong>s <strong>in</strong> ferment<strong>in</strong>g botrytized<br />
grape must, J. Appl. Microbiol. 98:971-979.<br />
Arias, C. R., Burns, J. K., Friedrich, L. M., Goodrich, R. M., and Parish, M. E., 2002,<br />
Yeast species associated with orange juice: Evaluation of different identification<br />
methods, Appl. Environ. Microbiol. 68:1955-1961.<br />
Azumi, M., and Goto-Yamamoto, N., 2001, AFLP analysis of type stra<strong>in</strong>s and laboratory<br />
and <strong>in</strong>dustrial stra<strong>in</strong>s of Saccharomyces sensu stricto and its application to<br />
phenetic cluster<strong>in</strong>g, Yeast. 18:1145-1154.<br />
Baleiras Couto, M. M., van der Vossen, J. M. B. M., Hofstra, H., and Huis <strong>in</strong>’t Veld,<br />
J. H. J., 1994, RAPD analysis: a rapid technique for differentiation of spoilage<br />
yeasts, Int. J. <strong>Food</strong> Microbiol. 24:249-260.<br />
Baleiras Couto, M. M., Vogels, J. T., Hofstra, H., Huis <strong>in</strong>’t Veld, J. H. J., and van der<br />
Vossen, J. M. B. M.,1995, Random amplified polymorphic DNA and restriction<br />
enzyme analysis of PCR amplified rDNA <strong>in</strong> taxonomy: two identification techniques<br />
for food-borne yeasts, J. Appl. Bacteriol. 79:525-535.<br />
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<strong>Food</strong>, Beneficial and Detrimental Aspects, T. Boekhout and V. Robert, eds, Behr’s-<br />
Verlag, Hamburg, pp. 123-138.
106 Ai L<strong>in</strong> Beh et al.<br />
Vasd<strong>in</strong>yei R., and Deák, T., 2003, Characterization of yeast isolates orig<strong>in</strong>at<strong>in</strong>g from<br />
Hungarian dairy products us<strong>in</strong>g traditional and molecular identification techniques,<br />
Int. J. <strong>Food</strong> Microbiol. 86:123-130.<br />
Versavaud A., Courcoux P., Roulland C., Dulau L., and Hallet J. N., 1995, Genetic<br />
diversity and geographical distribution of wild Saccharomyces cerevisiae stra<strong>in</strong>s<br />
from the w<strong>in</strong>e-produc<strong>in</strong>g area of Charentes, France. Appl. Environ. Microbiol.<br />
61:3521-3529.<br />
Villa-Carvajal, M., Coque, J. J. R., Álvarez-Rodríguez, M. L., Uruburu, F., and<br />
Belloch, C., 2004, Polyphasic identification of yeasts isolated from bark of<br />
cork oak dur<strong>in</strong>g the manufactur<strong>in</strong>g process of cork stoppers, FEMS Yeast Res.<br />
4:745-750.<br />
Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A.,<br />
Pot, J., Peleman, J., Kuiper, M., and Zabeau, M., 1995, AFLP: a new technique for<br />
DNA f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g, Nucleic Acids Res. 23:4407-4414.<br />
White, T. J., Bruns, T., Lee, S., and Taylor, J., 1990, Amplification and direct sequenc<strong>in</strong>g<br />
of fungal ribosomal RNA genes for phylogenentics, <strong>in</strong>: PCR Protocols:<br />
A Guide for Methods and Applications. M. A. Innis, D. H. Gelfand, J. J. Sn<strong>in</strong>sky<br />
and T. J. White, eds, Academic Press, San Diego, pp. 315-352.<br />
Wilson, I. G., 1997, Inhibition and facilitation of nucleic acid amplification. Appl.<br />
Environ. Microbiol. 63:3741-3751.
STANDARDIZATION OF METHODS FOR<br />
DETECTING HEAT RESISTANT FUNGI<br />
Jos Houbraken and Robert A. Samson *<br />
1. INTRODUCTION<br />
Heat resistant fungi can be def<strong>in</strong>ed as those capable of surviv<strong>in</strong>g<br />
temperatures at or above 75°C for 30 or more m<strong>in</strong>utes. The fungal<br />
structures which can survive these temperatures are ascospores, and<br />
sometimes chlamydospores, thick walled hyphae or sclerotia (Scholte<br />
et al., 2000). Dur<strong>in</strong>g the last three years, spoilage <strong>in</strong>cidents <strong>in</strong>volv<strong>in</strong>g<br />
heat resistant fungi occurred <strong>in</strong>creas<strong>in</strong>gly <strong>in</strong> various products exam<strong>in</strong>ed<br />
<strong>in</strong> our laboratory. Paecilomyces variotii, Fusarium oxysporum,<br />
Byssochlamys fulva, B. nivea, Talaromyces trachyspermus and<br />
Neosartorya species were often encountered <strong>in</strong> pasteurized fruit, dairy<br />
products and soft dr<strong>in</strong>ks. A questionnaire sent to many laboratories<br />
showed that <strong>in</strong>appropriate methods were used for the detection of<br />
heat resistant fungi, or that sometimes there was no special protocol<br />
at all. The use of <strong>in</strong>appropriate media, such as Sabouraud agar, wrong<br />
<strong>in</strong>cubation conditions and the analysis of <strong>in</strong>adequately sized samples<br />
were often encountered. In addition, accurate identification of the isolated<br />
colonies to species level often was not performed.<br />
In the literature many methods are described for the detection of<br />
heat resistant fungi (Murdock and Hatcher, 1978; Beuchat and Rice,<br />
1979; Beuchat and Pitt, 1992). Beuchat and Pitt (1992) described two<br />
methods: the Petri dish method or plat<strong>in</strong>g method and the direct <strong>in</strong>cubation<br />
method. In the first method, test tubes are used for the heat<strong>in</strong>g<br />
of the sample. Subsequently, the sample is poured <strong>in</strong>to large Petri<br />
* Centraalbureau voor Schimmelcultures, PO Box 85167, 3508 AD, Utrecht, The<br />
Netherlands. Correspondence to: houbraken@cbs.knaw.nl<br />
107
108 Jos Houbraken and Robert A. Samson<br />
dishes (diameter 14 cm) and agar media is added. In the second<br />
method, flat-sided bottles are used for heat<strong>in</strong>g and these bottles are<br />
<strong>in</strong>cubated directly at 30°C. The direct <strong>in</strong>cubation method has<br />
the advantage that contam<strong>in</strong>ation from aerial spores is m<strong>in</strong>imized; the<br />
disadvantage of this method is that the colonies grow<strong>in</strong>g <strong>in</strong> the<br />
bottle have to be transferred onto agar media for identification.<br />
Dur<strong>in</strong>g recent years a great variety of products have been <strong>in</strong>vestigated<br />
<strong>in</strong> our laboratory for the presence of heat resistant fungi. Some<br />
products were liquids (juices, colourants), some with a high viscosity<br />
(fruit concentrates), or solid products such as soil, pect<strong>in</strong>, liquorice,<br />
strawberries and cardboard. For several of these products the recommended<br />
methods are not suitable. A modified detection method us<strong>in</strong>g<br />
Stomacher bags is therefore proposed here.<br />
2. METHOD FOR EXAMINATION FOR HEAT<br />
RESISTANT MOULDS<br />
2.1. Ma<strong>in</strong> Modifications<br />
The modified method proposed here is based onto the protocol of<br />
Pitt and Hock<strong>in</strong>g (1997). The major modification is the use of<br />
Stomacher bags for the heat<strong>in</strong>g step which is the important step <strong>in</strong> the<br />
isolation of heat resistant fungi. The product can be easily homogenized<br />
<strong>in</strong> Stomacher bags, with a low risk of aerial contam<strong>in</strong>ation. The<br />
ascospores of many species of heat resistant fungi require heat activation<br />
before they will germ<strong>in</strong>ate (Katan, 1985; L<strong>in</strong>gappa and Sussman,<br />
1959). The heat treatment also <strong>in</strong>activates vegetative cells of fungi and<br />
bacteria, as well as less heat resistant spores (Beuchat and Pitt, 1992).<br />
Some protocols require that bottles be used for the heat<strong>in</strong>g of the sample.<br />
If bottles that are circular <strong>in</strong> cross section rather than flat-sided are<br />
used, the heat penetration <strong>in</strong>to the sample is particularly slow. When a<br />
Stomacher bag conta<strong>in</strong><strong>in</strong>g 250 ml of sample is sealed half way along its<br />
length, the thickness of the bag with the sample will be little more than<br />
1 cm, provid<strong>in</strong>g much faster heat penetration than <strong>in</strong> a 250 ml bottle.<br />
2.2. Description of the Modified Method<br />
Because the concentration of the heat resistant fungi <strong>in</strong> a sample is<br />
normally very low, the analysis of a large amount of sample is recom-
Standardization of Methods for Detect<strong>in</strong>g Heat Resistant Fungi 109<br />
mended. At least 100 g of sample should be exam<strong>in</strong>ed (Samson et al.,<br />
2000).<br />
Homogenize the sample before beg<strong>in</strong>n<strong>in</strong>g the analysis. Transfer 100<br />
g of sample <strong>in</strong>to a sterile Stomacher bag. Add sterile water (150 g) to<br />
the sample and homogenize us<strong>in</strong>g a Stomacher for 2 to 4 m<strong>in</strong>. If the<br />
sample is likely to conta<strong>in</strong> a higher concentration of heat resistant<br />
fungi (e.g. a soil sample), or the sample cannot be homogenized <strong>in</strong> 150<br />
grams of water (e.g. solid <strong>in</strong>gredients such as pect<strong>in</strong>), then a smaller<br />
amount of sample can be used. After the homogenization step, the<br />
Stomacher bag should be sealed about half way along its length us<strong>in</strong>g<br />
a heat-sealer, ensur<strong>in</strong>g that no air bubbles are present. After check<strong>in</strong>g<br />
the bag for leakage, heat treat the Stomacher bag for 30 m<strong>in</strong> at 75°C<br />
<strong>in</strong> a water bath (preferably one with the capability of shak<strong>in</strong>g or stirr<strong>in</strong>g).<br />
The water-bath should be at 75°C before the sample is <strong>in</strong>troduced.<br />
The sample should be placed <strong>in</strong> a horizontal position, totally<br />
submerged <strong>in</strong> the water.<br />
After the heat treatment, cool the samples to approximately 55°C.<br />
Aseptically transfer the contents of the Stomacher bag to a Schott<br />
bottle, or similar, (500 ml) with 250 ml melted double strength MEA<br />
conta<strong>in</strong><strong>in</strong>g chloramphenicol (200 mg/l, Oxoid) tempered to 55°C. Mix<br />
thoroughly and distribute the agar and sample mixture <strong>in</strong>to seven<br />
large plastic Petri dishes (diameter 14.5 cm). Place the Petri dishes <strong>in</strong>to<br />
a polyethylene bag to prevent dry<strong>in</strong>g and <strong>in</strong>cubate <strong>in</strong> an upright<br />
position at 30°C <strong>in</strong> darkness.<br />
The general procedure is illustrated <strong>in</strong> Figure 1.<br />
2.3. Incubation<br />
Many protocols require plates to be <strong>in</strong>cubated for at least 30 days.<br />
This period is too long for quality control <strong>in</strong> the food and beverage<br />
<strong>in</strong>dustry. In our experience, heat-resistant fungi will usually form<br />
colonies after 5 days and mature with<strong>in</strong> 14 days <strong>in</strong>cubation at 30°C, so<br />
check the Petri dishes for the presence of colonies after 7 and 14 days.<br />
Subculture if necessary, and identify all colonies us<strong>in</strong>g standard methods<br />
(Pitt and Hock<strong>in</strong>g, 1997; Samson et al., 2000). In some cases a<br />
prolonged <strong>in</strong>cubation period is necessary for the identification of<br />
the fungus. An overview of the <strong>in</strong>cubation time needed for some<br />
particular species is given <strong>in</strong> Figure 2.
110 Jos Houbraken and Robert A. Samson<br />
H 2 O<br />
Stomacher<br />
bag<br />
Sample<br />
1. Liquid samples: 100 ml + 150 ml water<br />
2. Pect<strong>in</strong>: 12.5 g + 230 ml water<br />
3. Fruit (concentrate): 100 g + 150 ml water<br />
4. Solid samples (eg cardboard, powdery<br />
<strong>in</strong>gredients, soil): 25 g + 225 ml water<br />
1. Incubate for 14 d. at 30°C<br />
2. Check every 7 days<br />
Homogenize<br />
and seal<br />
Mix throughly and disperse<br />
agar/product mass <strong>in</strong>to approx. 7-8<br />
petri-dishes (diam. 14.5 cm)<br />
Figure 1. Modified method for the detection of heat resistant fungi<br />
Talaromyces trachyspermus<br />
T. macrosporus, Hamigera sp.<br />
Heat treatment, 30 m<strong>in</strong> 75°C<br />
1. Cool the sample rapidly<br />
2. Mix the sample with 250 ml<br />
handwarm double strength<br />
MEA agar + chloramphenicol<br />
Eupenicillium species,<br />
Monascus ruber<br />
Byssochlamys sp., P. variotii s.l., Neosartorya sp.<br />
Air contam<strong>in</strong>ation or under pasteurization (Rhizopus, Humicola,<br />
Penicillia, A. niger / flavus (also sclerotial isolates)<br />
0 2 4 6 8 10 12 14<br />
<strong>in</strong>cubation time (days)<br />
Figure 2. Overview of the <strong>in</strong>cubation time needed to allow growth and development<br />
for particular species of heat resistant fungi<br />
3. REFERENCES<br />
Beuchat, L. R., and Rice, S. L., 1979, Byssochlamys spp. and their importance <strong>in</strong><br />
processed fruit syrups, Trans. Br. Mycol. Soc. 68:65-71.<br />
Beuchat, L. R., and Pitt, J. I., 1992, Detection and enumeration of heat-resistant<br />
molds, <strong>in</strong>: Compendium for the Microbiological Exam<strong>in</strong>ation of <strong>Food</strong>s, C. Vanderzant<br />
and D. F. Splittstoesser, eds, American Public Health Association, Wash<strong>in</strong>gton D. C.,<br />
pp. 251-263.
Standardization of Methods for Detect<strong>in</strong>g Heat Resistant Fungi 111<br />
Katan, T., 1985, Heat activation of dormant ascospores of Talaromyces flavus, Trans.<br />
Br. Mycol. Soc. 84:748-750.<br />
L<strong>in</strong>gappa, Y., and Sussman, A. S., 1959, Changes <strong>in</strong> the heat resistance of ascospores<br />
of Neurospora upon germ<strong>in</strong>ation, Am. J. Botany 49:671-678.<br />
Murdock, D. I., and Hatcher, W. S., 1978, A simple method to screen fruit juices and<br />
concentrates for heat-resistant mold, J. <strong>Food</strong>. Prot. 41:254-256.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, 2nd edition, Blackie<br />
Academic and Professional, London.<br />
Samson, R. A., Hoekstra, E. S., Lund, F., Filtenborg, O., and Frisvad, J. C., 2000,<br />
Methods for the detection, isolation and characterization of food-borne fungi, <strong>in</strong>:<br />
Introduction to <strong>Food</strong>- and Airborne Fungi, 6th edition, R. A. Samson, E. S.<br />
Hoekstra, J. C. Frisvad and O. Filtenborg, eds, Centraalbureau voor<br />
Schimmelcultures, Utrecht, pp. 283-297.<br />
Scholte, R. P. M., Samson, R. A. and Dijksterhuis, J., 2000, Spoilage fungi <strong>in</strong> the<br />
<strong>in</strong>dustrial process<strong>in</strong>g of food, <strong>in</strong>: Introduction to <strong>Food</strong>- and Airborne Fungi, 6th edition,<br />
R. A. Samson, E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, eds,<br />
Centraalbureau voor Schimmelcultures, Utrecht, pp. 339-356.
Section 3.<br />
Physiology and ecology of<br />
mycotoxigenic fungi<br />
Ecophysiology of fumonis<strong>in</strong> producers <strong>in</strong> Fusarium section Liseola<br />
Vicente Sanchis, Sonia Marín, Naresh Magan, and Antonio J. Ramos<br />
Ecophysiology of Fusarium culmorum and mycotox<strong>in</strong> production<br />
Naresh Magan, Russell Hope and David Aldred<br />
<strong>Food</strong>-borne fungi <strong>in</strong> fruit and cereals and their production of mycotox<strong>in</strong>s<br />
Birgitte Andersen and Ulf Thrane<br />
Black Aspergillus species <strong>in</strong> Australian v<strong>in</strong>eyards: from soil to ochratox<strong>in</strong> A<br />
<strong>in</strong> w<strong>in</strong>e<br />
Su-l<strong>in</strong> L. Leong, Ailsa D. Hock<strong>in</strong>g, John I. Pitt, Benozir A. Kazi, Robert W.<br />
Emmett and Eileen S. Scott<br />
Ochratox<strong>in</strong> A produc<strong>in</strong>g fungi from Spanish v<strong>in</strong>eyards<br />
Marta Bau, M. Rosa Bragulat, M. Lourdes Abarca, Santiago M<strong>in</strong>guez, and<br />
F. Javier Cabañes<br />
Fungi produc<strong>in</strong>g ochratox<strong>in</strong> <strong>in</strong> dried fruits<br />
Beatriz T. Iamanaka, Marta H. Taniwaki, E. Vicente and Hilary C. Menezes<br />
An update on ochratoxigenic fungi and ochratox<strong>in</strong> A <strong>in</strong> coffee<br />
Marta H. Taniwaki<br />
Mycobiota, mycotoxigenic fungi, and citr<strong>in</strong><strong>in</strong> production <strong>in</strong> black olives<br />
Dilek Heperkan, Burçak E. Meriç, Gülç<strong>in</strong> Sismanoglu, Gözde Dalkiliç, and<br />
Funda K. Güler<br />
Byssochlamys: significance of heat resistance and mycotox<strong>in</strong> production<br />
Jos Houbraken, Robert A. Samson and Jens C. Frisvad<br />
Effect of water activity and temperature on production of aflatox<strong>in</strong> and<br />
cyclopiazonic acid by Aspergillus flavus <strong>in</strong> peanuts<br />
Graciela Vaamonde, Andrea Patriarca and Virg<strong>in</strong>ia E. Fernández P<strong>in</strong>to
ECOPHYSIOLOGY OF FUMONISIN<br />
PRODUCERS IN FUSARIUM SECTION<br />
LISEOLA<br />
Vicente Sanchis, Sonia Marín, Naresh Magan and Antonio<br />
J. Ramos *<br />
1. INTRODUCTION<br />
Fumonis<strong>in</strong>s were first described as be<strong>in</strong>g produced by Fusarium<br />
Section Liseola species (Gelderblom et al., 1988; Marasas et al., 1988),<br />
and subsequently a number of toxicological studies have demonstrated<br />
their role <strong>in</strong> animal health problems caused by consumption of<br />
contam<strong>in</strong>ated feeds. A relationship has been postulated between frequent<br />
<strong>in</strong>gestion of fumonis<strong>in</strong> conta<strong>in</strong><strong>in</strong>g maize and <strong>in</strong>cidence of<br />
esophageal cancer by humans <strong>in</strong> certa<strong>in</strong> areas of the world. Thus<br />
fumonis<strong>in</strong>s have been classified as possible human carc<strong>in</strong>ogens<br />
(Group 2B), by IARC (1993). High levels of fumonis<strong>in</strong>s have been<br />
reported <strong>in</strong> maize <strong>in</strong> Africa, Asia and South America (Chu and Li,<br />
1994; Doko et al., 1996; Kedera et al., 1999; Ono et al., 1999; Med<strong>in</strong>a-<br />
Mart<strong>in</strong>ez and Mart<strong>in</strong>ez, 2000), sometimes co-occurr<strong>in</strong>g with other<br />
mycotox<strong>in</strong>s. Surveys have shown that much of the maize <strong>in</strong>tended for<br />
human consumption is contam<strong>in</strong>ated with fumonis<strong>in</strong>s to some extent<br />
(Pittet et al., 1992; Sanchis et al., 1994), and these mycotox<strong>in</strong>s may<br />
contam<strong>in</strong>ate a wide range of corn-based foods <strong>in</strong> our diet<br />
(Weidenboerner, 2001).<br />
* V. Sanchis (vsanchis@tecal.udl.es), S. Marín and A. J. Ramos, <strong>Food</strong> Technology<br />
Dept, Lleida University, 25198 Lleida, Spa<strong>in</strong>; N. Magan, Cranfield Biotechnology<br />
Centre, Cranfield University, Silsoe, England.<br />
115
116 Vicente Sanchis et al.<br />
2. IMPORTANCE OF THE<br />
ECOPHYSIOLOGICAL STUDIES<br />
The importance of the widespread contam<strong>in</strong>ation of foods and<br />
feeds by fumonis<strong>in</strong>s has led to a proliferation of studies aimed at<br />
understand<strong>in</strong>g the ecophysiology of the Fusarium spp. <strong>in</strong>volved, particularly<br />
F. verticillioides and F. proliferatum, and the del<strong>in</strong>eation of<br />
environmental conditions that allow production of fumonis<strong>in</strong>s. An<br />
understand<strong>in</strong>g of the <strong>in</strong>fluence of biotic and abiotic factors on germ<strong>in</strong>ation,<br />
growth and fumonis<strong>in</strong> production by these species is important<br />
<strong>in</strong> manag<strong>in</strong>g the problem of fumonis<strong>in</strong> contam<strong>in</strong>ation <strong>in</strong> the food<br />
supply. This study focuses on the impact of different abiotic factors<br />
<strong>in</strong>clud<strong>in</strong>g substrate, water activity (a w ), temperature and preservatives,<br />
and biotic factors such as the natural mycoflora present <strong>in</strong> the gra<strong>in</strong>.<br />
3. INFLUENCE OF ABIOTIC FACTORS ON<br />
FUNGAL DEVELOPMENT<br />
3.1. Substrate<br />
Fusarium Section Liseola species are much more commonly found<br />
<strong>in</strong> maize than other gra<strong>in</strong>s, such as wheat and barley. Studies carried<br />
out by Marín et al. (1999a) have shown that even though Fusarium<br />
verticillioides and F. proliferatum are able to grow on a wide variety of<br />
substrates, <strong>in</strong>clud<strong>in</strong>g wheat, barley and maize, high fumonis<strong>in</strong> biosynthesis<br />
only occurs <strong>in</strong> maize. The assayed isolates were able to grow <strong>in</strong><br />
the different cereal gra<strong>in</strong>s under similar conditions of temperature and<br />
a w , but negligible amounts of fumonis<strong>in</strong> B 1 (FB 1 ) were detected.<br />
Although fumonis<strong>in</strong>s are found ma<strong>in</strong>ly <strong>in</strong> corn and corn-based<br />
foods and feeds, there are a few reports of fumonis<strong>in</strong>s from other substrates<br />
such as ‘black oats’ animal feed from Brazil (Sydenham et al.,<br />
1992), New Zealand forage grass (Scott, 1993), Indian sorghum<br />
(Shetty and Bhat, 1997), rice (Abbas et al., 1998), asparagus (Logrieco<br />
et al., 1998), beer (Torres et al., 1998), wheat/barley/soybeans (Castella<br />
et al., 1999), and tea (Mart<strong>in</strong>s et al., 2001).<br />
3.2. Water Activity and Temperature<br />
Our results show that germ<strong>in</strong>ation of F. verticillioides and F. proliferatum<br />
is possible between 5-37°C at a w values above 0.88, but the
Ecophysiology of Fumonis<strong>in</strong> Producers <strong>in</strong> Fusarium Section Liseola 117<br />
range for growth is slightly narrower (7-37°C above 0.90 a w ) (Figure<br />
1). The lag phases were shorter at 25-30°C and 0.94-0.98 a w , and they<br />
<strong>in</strong>creased to 10-500 h at marg<strong>in</strong>al temperatures (5-10°C). There were<br />
some differences between stra<strong>in</strong>s. FB 1 production <strong>in</strong> gra<strong>in</strong> was<br />
observed between 10-37°C but only at a w of 0.93 and above. The<br />
optimum conditions for fumonis<strong>in</strong> production by F. verticillioides and<br />
F. proliferatum were 15-30°C at 0.97 a w (Marín et al., 1996; Marín<br />
et al., 1999b). These two environmental conditions (temperature and<br />
moisture availability) are the ma<strong>in</strong> factors which control fumonis<strong>in</strong><br />
production <strong>in</strong> gra<strong>in</strong>.<br />
The authors have developed detailed two-dimensional profiles of<br />
conditions that allow the production of FB 1 (Marín et al., 1999b)<br />
(Figure 2).<br />
3.3. Preservatives<br />
Gra<strong>in</strong> preservatives based on propionates have shown some activity<br />
<strong>in</strong> controll<strong>in</strong>g the growth of the Fusarium spp., and FB 1 production.<br />
Growth rates decreased as preservative concentration <strong>in</strong>creased,<br />
regardless of a w , while fumonis<strong>in</strong> production decreased only when a w<br />
was 0.93 or lower. In general, only a concentration ≥ 0.07% propionate<br />
was effective. In the presence of low propionate concentrations<br />
(0.03%), FB 1 production was sometimes stimulated, possibly due to<br />
assimilation of these compounds by the moulds. The <strong>in</strong>hibitory effect<br />
of the preservatives is significantly affected by the water activity and<br />
temperature of the gra<strong>in</strong> (Marín et al., 1999c).<br />
Growth rate (mm d -1 )<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0 10 20 30 40<br />
Temperature (°C)<br />
Figure 1. Effect of temperature and water activity on growth rate of F. verticillioides.<br />
a w : 0.98 (●), 0.96 (▲), 0.94 (■), 0.92 (●), 0.90 (▲), 0.88 (■)
118 Vicente Sanchis et al.<br />
Water activity<br />
1<br />
0.99<br />
0.98<br />
0.97<br />
0.96<br />
0.95<br />
0.94<br />
0.93<br />
0.92<br />
1000<br />
10<br />
50<br />
100<br />
5<br />
1<br />
5 10 15 20 25 30 35 40<br />
Temperature (°C)<br />
Figure 2. Water activity/temperature profiles for FB 1 accumulation by F. verticillioides<br />
after 28 days of <strong>in</strong>cubation on irradiated maize (from Marín et al., 1999b).<br />
Essential oils from plants have been also tested for their antimycotoxigenic<br />
activity and found to <strong>in</strong>hibit FB 1 accumulation <strong>in</strong> maize<br />
under moist conditions. Resveratrol, a compound known for its<br />
antioxidant properties, decreased FB 1 accumulation <strong>in</strong> maize at a concentration<br />
as low as 23 ppm (Fanelli et al., 2003).<br />
4. INFLUENCE OF BIOTIC FACTORS ON<br />
FUNGAL DEVELOPMENT<br />
In general, the presence of other fungal species <strong>in</strong> mixed cultures<br />
<strong>in</strong>hibited the growth of F. verticillioides and F. proliferatum.<br />
Aspergillus niger and A. flavus were particularly effective <strong>in</strong> decreas<strong>in</strong>g<br />
the competitiveness of the Fusaria (Marín et al., 1998). However, their<br />
effectiveness depended on abiotic factors. In general, the Fusarium<br />
spp. competed better at higher a w levels (0.98), and temperatures close<br />
to 15°C. Interest<strong>in</strong>gly, FB 1 production was stimulated by certa<strong>in</strong><br />
species at high water availabilities (0.98 a w ), ma<strong>in</strong>ly when compet<strong>in</strong>g<br />
with A. niger for occupation of the same niche (Table 1).<br />
Fumonis<strong>in</strong>s are secondary metabolites: if Fusarium is an endophyte,<br />
fumonis<strong>in</strong> production may be more important for reta<strong>in</strong><strong>in</strong>g its niche<br />
than for occupy<strong>in</strong>g it. However, if contam<strong>in</strong>ation occurs from the air
Ecophysiology of Fumonis<strong>in</strong> Producers <strong>in</strong> Fusarium Section Liseola 119<br />
Table 1. Influence of water activity and temperature on production of fumonis<strong>in</strong> B (ppm) by Fusarium spp. on maize gra<strong>in</strong> <strong>in</strong> the presence<br />
1<br />
of compet<strong>in</strong>g mycoflora after a 4-weeks <strong>in</strong>cubation period<br />
Temperature (°C) 15°C 25°C<br />
Water activity 0.93 0.95 0.98 0.93 0.95 0.98<br />
F. verticillioides 0.8±0.1 7.1±1.3 54.1±10.7 29.3±0.4 5.5±5.9 4.8±5.2<br />
+ A. niger 0.2±0.1 0.4±0.1 137.9±1.4 0.5±0.0 5.1±0.3 360.9±9.2<br />
+ A. ochraceus 0.6±0.0 1.1±0.1 84.3±1.8 0.3±0.2 47.3±2.9 40.2±0.6<br />
+ A. flavus 12.2±6.5 15.8±18.6 12.8±6.0 0.2±0.1 0.2±0.0 10.4±7.1<br />
+ P. implicatum 0.1±1.0 0.2±0.1 3.8±1.6 11.5±15.6 0.7±0.1 48.0±11.9<br />
F. proliferatum 17.2±4.8 34.0±8.8 22.8±6.7 5.6±1.0 3.6±4.7 0.7±0.5<br />
+ A. niger 19.1±26.4 33.5±6.3 1084.4±33.5 0.2±0.1 3.0±1.4 0.8±0.1<br />
+ A. ochraceus 0.1±0.0 1.7±0.1 48.8±0.5 0.9±0.0 2.9±0.1 0.2±0.0<br />
+ A. flavus 6.8±2.7 105.9±1.6 602.7±8.1 6.8±8.3 9.8±3.6 64.1±32.9<br />
+ P. implicatum 3.6±4.5 284.3±401.4 234.4±308.6 3.3±3.0 21.3±28.5 11.6±6.6
120 Vicente Sanchis et al.<br />
or leaves, establishment may be assisted by fumonis<strong>in</strong> production.<br />
However, field data do not support the hypothesis that F. verticillioides<br />
ga<strong>in</strong>s a competitive advantage via FB 1 (Reid et al., 1999).<br />
5. CONCLUSION<br />
Most fumonis<strong>in</strong> is produced <strong>in</strong> maize pre-harvest, but fumonis<strong>in</strong><br />
control <strong>in</strong> maize post-harvest can be achieved by effective control of<br />
the moisture content (a w ). Temperature control and periodic aeration,<br />
along with the natural microflora may act as additional controls.<br />
6. ACKNOWLEDGMENTS<br />
The authors are grateful to the Spanish Government (CICYT,<br />
Comision Interm<strong>in</strong>isterial de Ciencia y Tecnologia, grant ALI98 0509-<br />
C04-01) and the European Union (QLRT-1999-996) for the f<strong>in</strong>ancial<br />
support.<br />
7. REFERENCES<br />
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Chu, F. S., and Li, G. Y., 1994, Simultaneous occurrence of fumonis<strong>in</strong> B 1 and other<br />
mycotox<strong>in</strong>s <strong>in</strong> moldy corn collected from the People’s Republic of Ch<strong>in</strong>a <strong>in</strong><br />
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Vleggaar, R., and Kriek, N. P. J., 1988, Fumonis<strong>in</strong>s -novel mycotox<strong>in</strong>s with cancerpromot<strong>in</strong>g<br />
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to humans; some naturally occurr<strong>in</strong>g substances: food items and constituents, heterocyclic<br />
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for Research on Cancer, Lyon, pp. 26-32.<br />
Logrieco, A., Doko, M. B., Moretti, A., Frisullo, S., and Visconti, A., 1998,<br />
Occurrence of fumonis<strong>in</strong> B 1 and B 2 <strong>in</strong> Fusarium proliferatum <strong>in</strong>fected asparagus<br />
plants, J. Agric. <strong>Food</strong> Chem. 46:5201-5204.<br />
Marasas, W. F. O., Jaskiewicz, K., Venter, F. S., and van Schalkwyk, D. J., 1988,<br />
Fusarium moniliforme contam<strong>in</strong>ation of maize <strong>in</strong> oesophageal cancer areas <strong>in</strong><br />
Transkei, Sth Afr. Med. J., 74:110-114.<br />
Marín, S., Sanchis, V., Teixido, A., Saenz, R., Ramos, A. J., V<strong>in</strong>as, I., and Magan, N.,<br />
1996, Water and temperature relations and microconidial germ<strong>in</strong>ation of Fusarium<br />
moniliforme and F. proliferatum from maize, Can. J. Microbiol. 42:1045-1050.<br />
Marín, S., Sanchis, V., Rull, F., Ramos, A. J., and Magan, N., 1998, Colonization of<br />
maize gra<strong>in</strong> by Fusarium moniliforme and Fusarium proliferatum <strong>in</strong> the presence<br />
of compet<strong>in</strong>g fungi and their impact on fumonis<strong>in</strong> production, J. <strong>Food</strong> Prot. 61:<br />
1489-1496.<br />
Marín, S., Magan, N., Serra, J., Ramos, A. J., Canela, R., and Sanchis V., 1999a,<br />
Fumonis<strong>in</strong> B 1 production and growth of Fusarium moniliforme and Fusarium proliferatum<br />
on maize, wheat, and barley gra<strong>in</strong>, J. <strong>Food</strong> Sci. 64:921-924.<br />
Marín, S., Magan, N., Belli, N., Ramos, A. J., Canela, R., and Sanchis V., 1999b, Two<br />
dimensional profiles of fumonis<strong>in</strong> B 1 production by Fusarium moniliforme and<br />
Fusarium proliferatum <strong>in</strong> relation to environmental factors and potential for modell<strong>in</strong>g<br />
tox<strong>in</strong> formation <strong>in</strong> maize gra<strong>in</strong>, Int. J. <strong>Food</strong> Microbiol. 51:159-167.<br />
Marín, S., Sanchis, V., Sanz, D., Castel, I., Ramos, A. J., Canela, R., and Magan, N.,<br />
1999c, Control of growth and fumonis<strong>in</strong> B 1 production by Fusarium verticillioides<br />
and Fusarium proliferatum isolates <strong>in</strong> moist maize with propionate preservatives,<br />
<strong>Food</strong> Addit. Contam. 16:555-563.<br />
Mart<strong>in</strong>s, M. L., Mart<strong>in</strong>s, H. M., and Bernardo, F., 2001, Fumonis<strong>in</strong>s B 1 and B 2 <strong>in</strong><br />
black tea and medic<strong>in</strong>al plants, J. <strong>Food</strong> Prot. 64:1268-1270.<br />
Med<strong>in</strong>a-Mart<strong>in</strong>ez, M. S., and Mart<strong>in</strong>ez, A. J., 2000, Mold occurrence and aflatox<strong>in</strong><br />
B 1 and fumonis<strong>in</strong> B 1 determ<strong>in</strong>ation <strong>in</strong> corn samples <strong>in</strong> Venezuela, J. Agric. <strong>Food</strong><br />
Chem. 48:2833-2836.<br />
Ono, E. Y. S., Sugiura, Y., Homech<strong>in</strong>, M., Kamogae, M., Vizzoni, E., Ueno, Y., and<br />
Hirooka, E.Y., 1999, Effect of climatic conditions on natural mycoflora and<br />
fumonis<strong>in</strong>s <strong>in</strong> freshly harvested corn of the state of Parana, Brazil,<br />
Mycopathologia, 147:139-148.<br />
Pittet, A., Parisod, V., and Schellenberg, M., 1992, Occurrence of fumonis<strong>in</strong>s B 1<br />
and B 2 <strong>in</strong> corn-based products from the Swiss market, J. Agric. <strong>Food</strong> Chem.<br />
40:1445-1453.<br />
Reid, L. M., Nicol, R. W., Ouellet, T., Savard, M., Miller, J. D., Young, J. C., Stewart,<br />
D. W., and Schaafsma, A. W., 1999, Interaction of Fusarium gram<strong>in</strong>earum and<br />
F. moniliforme <strong>in</strong> maize ears: disease progress, fungal biomass, and mycotox<strong>in</strong><br />
accumulation, Phytopathology, 89:1028-1037.<br />
Sanchis, V., Abadias, M., Onc<strong>in</strong>s, L., Sala, N., V<strong>in</strong>as, I., and Canela, R., 1994,<br />
Occurrence of fumonis<strong>in</strong>s B 1 and B 2 <strong>in</strong> corn-based products from the Spanish<br />
market, Appl. Environ. Microbiol. 60:2147-2148.
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Scott, P. M., 1993, Fumonis<strong>in</strong>s, Int. J. <strong>Food</strong> Microbiol. 18:257-270.<br />
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co-occurrence with aflatox<strong>in</strong> B 1 <strong>in</strong> Indian sorghum, maize and poultry feeds,<br />
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1992, Fumonis<strong>in</strong> concentrations <strong>in</strong> Brazilian feeds associated with field outbreaks<br />
of confirmed and suspected animal mycotoxicoses, J. Agric. <strong>Food</strong> Chem. 40:<br />
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262-273.
ECOPHYSIOLOGY OF FUSARIUM<br />
CULMORUM AND MYCOTOXIN<br />
PRODUCTION<br />
Naresh Magan, Russell Hope and David Aldred *<br />
1. INTRODUCTION<br />
Fusarium ear blight is a cereal disease responsible for significant<br />
reduction <strong>in</strong> yield and quality of wheat gra<strong>in</strong> throughout the world.<br />
In addition to degradation <strong>in</strong> gra<strong>in</strong> quality, Fusarium species produce<br />
an array of mycotox<strong>in</strong>s which may contam<strong>in</strong>ate the gra<strong>in</strong>. This<br />
mycotox<strong>in</strong> production occurs preharvest and dur<strong>in</strong>g the early stages<br />
of dry<strong>in</strong>g (Botallico and Perrone, 2002; Magan et al., 2002). F. culmorum<br />
is the most common cause of Fusarium ear blight <strong>in</strong> the United<br />
K<strong>in</strong>gdom and some other countries and can produce trichothecenes<br />
<strong>in</strong>clud<strong>in</strong>g deoxynivalenol (DON) and nivalenol (NIV). DON and NIV<br />
are harmful to both animals and humans, caus<strong>in</strong>g a wide range of<br />
symptoms of vary<strong>in</strong>g severity, <strong>in</strong>clud<strong>in</strong>g immunosuppression.<br />
Germ<strong>in</strong>ation of macroconidia of F. culmorum can occur over a<br />
wide range of temperatures (5-35°C) with a m<strong>in</strong>imum a w near 0.86 at<br />
20-25°C based on an <strong>in</strong>cubation period of about 40 days (Magan and<br />
Lacey, 1984a). Longer term <strong>in</strong>cubations on other media have<br />
suggested limits for germ<strong>in</strong>ation of about 0.85 a w (Snow, 1949).<br />
The ecological strategies used by F. culmorum to occupy and dom<strong>in</strong>ate<br />
<strong>in</strong> the gra<strong>in</strong> niche are not understood. Fungi can have combative<br />
(C-selected), stress (S-selected) or ruderal (R-selected) strategies or<br />
* Applied <strong>Mycology</strong> Group, Biotechnology Centre, Cranfield University, Silsoe,<br />
Bedford MK45 4DT, U.K. Correspondence to n.magan@cranfield.ac.uk<br />
123
124 Naresh Magan et al.<br />
merged secondary strategies (C-R, S-R, C-S, C-S-R; Cooke and<br />
Whipps, 1993). Thus primary resource capture of nutritionally rich<br />
food matrices such as gra<strong>in</strong> by F. culmorum may depend on germ<strong>in</strong>ation<br />
and growth rate, enzyme production, sporulation and the capacity<br />
for produc<strong>in</strong>g secondary metabolites to enable effective<br />
competition with other fungi.<br />
Attempts to control F. culmorum and other Fusarium species have<br />
relied on the application of fungicides preharvest coupled with effective<br />
storage regimens. However, the tim<strong>in</strong>g and application of these<br />
control measures are critical. Some fungicides are <strong>in</strong>effective aga<strong>in</strong>st<br />
Fusarium ear blight and may <strong>in</strong> some cases result <strong>in</strong> a stimulation of<br />
mycotox<strong>in</strong> production, particularly under suboptimal fungal growth<br />
conditions and low fungicide doses (D’Mello et al., 1999; Jenn<strong>in</strong>gs et<br />
al., 2000; Magan et al., 2002). It has been shown that moisture conditions<br />
at anthesis are crucial <strong>in</strong> determ<strong>in</strong><strong>in</strong>g <strong>in</strong>fection and mycotox<strong>in</strong><br />
production by F. culmorum on wheat dur<strong>in</strong>g ripen<strong>in</strong>g. Few studies<br />
have been carried out to determ<strong>in</strong>e the effect that key environmental<br />
factors such as water activity (a w ), temperature and time have on fungal<br />
growth and mycotox<strong>in</strong> production. Some studies have identified<br />
the a w range for germ<strong>in</strong>ation and growth of F. culmorum and other<br />
Fusarium species (Sung and Cook, 1981; Magan and Lacey 1984a;<br />
Magan and Lacey 1984b). The comb<strong>in</strong>ed effect of a w and temperature<br />
has been found to be significant for growth and fumonis<strong>in</strong> production<br />
by Fusarium verticillioides and F. proliferatum which <strong>in</strong>fect maize<br />
(Mar<strong>in</strong> et al. 1999).<br />
Knowledge of the threshold limits for growth and mycotox<strong>in</strong> production<br />
are very important <strong>in</strong> controll<strong>in</strong>g the entry of mycotox<strong>in</strong>s <strong>in</strong>to<br />
the food cha<strong>in</strong>. The objective of this study was to exam<strong>in</strong>e <strong>in</strong> detail the<br />
impact of water availability, temperature and time on growth and DON<br />
and NIV production by an isolate of F. culmorum on a medium based<br />
on wheat gra<strong>in</strong>s. Production of enzymes that may assist F. culmorum<br />
to dom<strong>in</strong>ate <strong>in</strong> the gra<strong>in</strong> niche was also exam<strong>in</strong>ed.<br />
2. MATERIALS AND METHODS<br />
2.1. Fungal Isolates and Media<br />
A representative stra<strong>in</strong> of Fusarium culmorum (98WW4.5FC,<br />
Rothamsted Research Culture Collection, Harpenden, Herts, UK)<br />
was chosen from a range exam<strong>in</strong>ed previously and isolated from UK
Ecophysiology of Fusarium culmorum and Mycotox<strong>in</strong> Production 125<br />
wheat gra<strong>in</strong>, with a known history of mycotox<strong>in</strong> production (Lacey<br />
et al., 1999; Magan et al., 2002). The stra<strong>in</strong> produced quantities of<br />
mycotox<strong>in</strong>s comparable with other stra<strong>in</strong>s from Europe.<br />
A 2% milled wheat gra<strong>in</strong> agar (Agar No. 3, Oxoid, Bas<strong>in</strong>gstoke,<br />
UK) was used as the basic medium. The a w was adjusted with glycerol<br />
<strong>in</strong> the range 0.995-0.850 as described by Dallyn (1978). The equivalent<br />
moisture contents of the a w treatments of 0.995, 0.98, 0.95, 0.90 and<br />
0.85 were 30%, 26%, 22%, 19% and 17.5% for wheat gra<strong>in</strong>. Glycerol<br />
was used because of its <strong>in</strong>herent a w stability over the temperature<br />
range 10-40°C. Media were sterilised by autoclav<strong>in</strong>g for 15 m<strong>in</strong> at<br />
120°C. Media were cooled to 50°C before pour<strong>in</strong>g <strong>in</strong>to 90 mm Petri<br />
plates. The a w of media was confirmed us<strong>in</strong>g an Aqualab <strong>in</strong>strument<br />
(Decagon Inc., Wash<strong>in</strong>gton State, USA). In all cases the a w levels were<br />
checked at both <strong>in</strong>cubation temperatures (15° and 25°C) and were<br />
with<strong>in</strong> 0.003 of the desired levels.<br />
For studies <strong>in</strong> whole gra<strong>in</strong>, w<strong>in</strong>ter wheat was gamma irradiated at<br />
12kGy to remove contam<strong>in</strong>ant microorganisms, but conserve germ<strong>in</strong>ative<br />
capacity. No mycotox<strong>in</strong>s were found <strong>in</strong> the gra<strong>in</strong> lot used.<br />
Vary<strong>in</strong>g amounts of water were added to the gra<strong>in</strong> and an adsorption<br />
curve prepared to facilitate accurate modifications of the a w of the<br />
gra<strong>in</strong> comparable with the media-based studies. Gra<strong>in</strong> was placed <strong>in</strong><br />
sterile flasks and <strong>in</strong>oculated with appropriate volumes of sterile water<br />
to obta<strong>in</strong> the necessary treatments. The flasks were sealed and left for<br />
24-36 h to equilibrate at 4°C. The gra<strong>in</strong> was then decanted carefully<br />
<strong>in</strong>to 90 mm Petri dishes to obta<strong>in</strong> a monolayer of wheat gra<strong>in</strong>. In all<br />
cases the a w levels were checked at both temperatures as described<br />
above and were with<strong>in</strong> 0.003 of the desired levels.<br />
2.2. Inoculation and Growth Measurements<br />
For both agar and gra<strong>in</strong> based studies, replicates of each a w treatment<br />
were <strong>in</strong>oculated centrally with a 5µl drop of a 10 5 cfu/ml F. culmorum<br />
macroconidial suspension obta<strong>in</strong>ed from a 7 d colony grown<br />
on 2% milled wheat agar. Conidia were obta<strong>in</strong>ed by flood<strong>in</strong>g the<br />
culture with 5 ml sterile distilled water conta<strong>in</strong><strong>in</strong>g 0.5% Tween 80 and<br />
agitat<strong>in</strong>g the colony surface with a sterile glass rod. Replicates of the<br />
same treatment were enclosed <strong>in</strong> polyethylene chambers together with<br />
500 ml of a glycerol/water solution of the same a w , closed and <strong>in</strong>cubated<br />
at 15° or 25°C for up to 40 days. Growth measurements were<br />
taken throughout the <strong>in</strong>cubation period, by tak<strong>in</strong>g two diametric<br />
measurements of the colonies at right angles. Colonisation rates were<br />
determ<strong>in</strong>ed subsequently by l<strong>in</strong>ear regression of the radial extension
126 Naresh Magan et al.<br />
rates. Three replicates per treatment were removed after 10, 20, 30 or<br />
40 d and analysed for DON and NIV (agar media) and for DON<br />
(wheat gra<strong>in</strong>). The experiments were repeated once.<br />
2.3. Mycotox<strong>in</strong> Extraction and Analyses<br />
Mycotox<strong>in</strong> extraction was adapted from Cooney et al. (2001). The<br />
entire agar and mycelial culture or gra<strong>in</strong> sample from each replicate<br />
sample was placed <strong>in</strong> acetonitrile/methanol (14:1; 40 ml) and shaken<br />
for 12 h. Aliquots (2 ml) were taken for DON/NIV analysis and<br />
passed through a cleanup cartridge compris<strong>in</strong>g a 2 ml syr<strong>in</strong>ge (Fisher<br />
Ltd.) packed with a disc of filter paper (No. 1 Whatman International<br />
Ltd.), a 5 ml luger of glass wool and 300 mg of alum<strong>in</strong>a/activated carbon<br />
(20:1). The sample was allowed to gravity feed through the<br />
cartridge and residues <strong>in</strong> the cartridge washed out with acetonitrile/methanol/water<br />
(80:5:15; 500 µl). The comb<strong>in</strong>ed eluate was<br />
evaporated (compressed air, 50°C) and then resuspended <strong>in</strong><br />
methanol/water (5:95; 500 µl).<br />
Quantification of DON/NIV was accomplished by HPLC, us<strong>in</strong>g a<br />
Luna C18 reverse phase column (100 mm × 4.6 mm i.d.)<br />
(Phenomenex, Macclesfield, U.K.). Separation was achieved us<strong>in</strong>g<br />
an isocratic mobile phase of methanol/water (12:88) at an elution<br />
rate of 1.5 ml/m<strong>in</strong>. Eluates were detected us<strong>in</strong>g a UV detector set at<br />
220 nm with an attenuation of 0.01 AUFS. The retention times<br />
for NIV and DON were 3.4 and 7.5 m<strong>in</strong> respectively. External standards<br />
were used for quantification (Sigma-Aldrich, Poole, Dorset,<br />
U.K.). The limit for quantification was 5 ng/g for DON and 2.5 ng/g<br />
for NIV.<br />
2.4. Hydrolytic Enzyme Profiles <strong>in</strong> Gra<strong>in</strong><br />
For enzyme extraction subsamples of gra<strong>in</strong> (2 g) were placed <strong>in</strong><br />
4 ml potassium phosphate extraction buffer (10 mM; pH 7.2). The<br />
bottles were shaken on a wrist action shaker for 1 h at 4°C. Wash<strong>in</strong>gs<br />
were decanted <strong>in</strong>to plastic Eppendorf tubes (1.5 ml) and centrifuged<br />
<strong>in</strong> a bench microcentrifuge for 15 m<strong>in</strong>. The supernatant was<br />
decanted and stored <strong>in</strong> aliquots at −20°C for total and specific<br />
enzyme activity determ<strong>in</strong>ations. The total activity of seven<br />
hydrolytic enzyme activities was assayed us<strong>in</strong>g ρ-nitrophenyl substrates<br />
(Sigma Chemical Co., UK). Enzyme extract (40 µl), substrate
Ecophysiology of Fusarium culmorum and Mycotox<strong>in</strong> Production 127<br />
solution (40 µl) and the appropriate buffer (20 µl) were pipetted <strong>in</strong>to<br />
the wells of the microtitre plate and <strong>in</strong>cubated at 37°C for 1 h along<br />
with the appropriate controls. The reaction was stopped by the addition<br />
of 5 µl 1M sodium carbonate solution and left for 3 m<strong>in</strong>. The<br />
enzyme activity was measured, us<strong>in</strong>g a MRX multiscan plate reader<br />
(Dynex Technologies Ltd., Bill<strong>in</strong>ghurst, UK), by the <strong>in</strong>crease <strong>in</strong><br />
optical density at 405 nm caused by the liberation of ρ-nitrophenol<br />
by enzymatic hydrolysis of the substrate. Enzyme activity was calculated<br />
from a calibration curve of absorbance at 405 nm vs ρ-nitrophenol<br />
concentration and expressed as µmol ρ-nitrophenol<br />
released/m<strong>in</strong>.<br />
For specific activity determ<strong>in</strong>ations the prote<strong>in</strong> concentration was<br />
obta<strong>in</strong>ed us<strong>in</strong>g a Bic<strong>in</strong>chon<strong>in</strong>ic acid prote<strong>in</strong> assay kit (Sigma-Aldrich<br />
Ltd, Poole, Dorset, UK). This kit consisted of bic<strong>in</strong>chon<strong>in</strong>ic acid<br />
solution, copper (II) sulphate pentahydrate 4% solution and album<strong>in</strong><br />
standard (conta<strong>in</strong><strong>in</strong>g bov<strong>in</strong>e serum album<strong>in</strong> (BSA) at a concentration<br />
of 1.0 mg/ml). Prote<strong>in</strong> reduces alkal<strong>in</strong>e Cu (II) to Cu (I), which forms<br />
a purple complex with bic<strong>in</strong>chon<strong>in</strong>ic acid (a highly specific chromogenic<br />
reagent). The resultant absorbance at 550 nm is directly proportional<br />
to the prote<strong>in</strong> concentration. The work<strong>in</strong>g reagent was<br />
obta<strong>in</strong>ed by the addition of 1 part copper (II) sulphate solution to<br />
50 parts bic<strong>in</strong>chon<strong>in</strong>ic acid solution. The reagent is stable for one day<br />
provided it is stored <strong>in</strong> a closed conta<strong>in</strong>er at room temperature.<br />
Aliquots (10 µl) of each standard or enzyme extracts were placed <strong>in</strong><br />
the appropriate microtitre plate wells. Potassium phosphate extraction<br />
buffer 10 mM pH 7.2 (10 µl) was pipetted <strong>in</strong>to the blank wells. The<br />
work<strong>in</strong>g reagent (200 µl) was added to each well, shaken and plates<br />
<strong>in</strong>cubated at 37°C for 30 m<strong>in</strong>. The plates were allowed to cool to room<br />
temperature before measur<strong>in</strong>g the absorbance at 550 nm us<strong>in</strong>g a MRX<br />
multiscan plate reader. The prote<strong>in</strong> concentrations <strong>in</strong> the enzyme<br />
extracts were obta<strong>in</strong>ed from the calibration curve of absorbance at<br />
550 nm aga<strong>in</strong>st BSA concentration. These values were used to calculate<br />
the specific activity of the enzymes <strong>in</strong> nmol ρ-nitrophenol released<br />
per m<strong>in</strong> per µg prote<strong>in</strong>.<br />
2.5. Statistical Analyses<br />
The data were analysed us<strong>in</strong>g ANOVA (SigmaStat, SPSS Inc.),<br />
with significance values of
128 Naresh Magan et al.<br />
3. RESULTS<br />
3.1. Impact of Environment on Germ<strong>in</strong>ation and<br />
Growth<br />
The growth rate of F. culmorum on wheat-based media compared<br />
with that on monolayers of wheat gra<strong>in</strong> is shown <strong>in</strong> Figure 1. Growth<br />
was very similar over the whole range at 15° and 25°C, with a limit of<br />
about 0.90 a w . Mycelial colonisation of gra<strong>in</strong> was much faster at 25°<br />
than 15°C, over the range 0.995-0.96 a w . Previous studies on wheatbased<br />
media have identified m<strong>in</strong>imum limits for germ<strong>in</strong>ation and<br />
growth as be<strong>in</strong>g at 0.86 and 0.88 a w at 20-25°C, respectively (Magan<br />
and Lacey, 1984a,b). However, germ<strong>in</strong>ation and growth occurred over<br />
a wide temperature range (5-35°C). Studies on F. gram<strong>in</strong>earum, also<br />
an important wheat pathogen, suggest a similar range of conditions<br />
for isolates from both Europe and South America (Hope, 2003;<br />
Ramirez et al., 2004).<br />
3.2. Impact of Environment on Production<br />
of Deoxynivalenol and Nivalenol<br />
Studies on wheat-based media show that the time course of production<br />
of DON varies with temperature and a w (Figure 2). DON was<br />
produced over a narrower range of conditions (0.97-0.995 a w ) at 25°<br />
than 15°C (0.95-0.995 a w ). Concentrations produced were similar over<br />
a range of a w levels at 15°C, while at 25°C amounts 100 times greater<br />
were produced but over a narrower a w range. The pattern of production<br />
of NIV was different from DON, but aga<strong>in</strong> less NIV was produced<br />
at 15° that 25°C (Figure 3). Optimum NIV was produced at<br />
<strong>in</strong>termediate a w levels at 15°C (0.95-0.98 a w ). In contrast, at 25°C a significantly<br />
higher concentration was produced but only at 0.98 a w after<br />
40 d <strong>in</strong>cubation.<br />
On wheat gra<strong>in</strong>, maximum production occurred after 40 days at<br />
both 15° and 25°C over a similar a w range to that on milled wheat<br />
media (Figure 4). Aga<strong>in</strong>, higher concentrations of DON were produced<br />
at 25°C, with about a 5-10 fold reduction at 15°C. However, the<br />
a w range for rapid DON production was wider at 15° than 25°C.<br />
Comparisons can be made with production of fumonis<strong>in</strong>s by<br />
Fusarium Section Liseola. Studies by Mar<strong>in</strong> et al. (1999) showed<br />
that the limits for fumonis<strong>in</strong> production on maize gra<strong>in</strong> were<br />
about 0.91-0.92 a w for both F. verticillioides and F. proliferatum with<br />
temperature ranges of 15-30°C.
Ecophysiology of Fusarium culmorum and Mycotox<strong>in</strong> Production 129<br />
Kr (mm day -1)<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
(a)<br />
9<br />
8<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
(b)<br />
25°C<br />
15°C<br />
0.99 0.98 0.97 0.96 0.95 0.9 0.85<br />
0.99 0.98 0.97 0.96 0.95 0.9 0.85<br />
Water activity<br />
Figure 1. Comparison of growth rates of F. culmorum on milled wheat agar (a) and<br />
on monolayers of wheat gra<strong>in</strong> (b) at 15° and 25°C (adapted from Hope and Magan,<br />
2003 and Hope, 2003).<br />
For F. culmorum the available data from the literature and present<br />
work have been comb<strong>in</strong>ed to provide a two dimensional profile of the<br />
comb<strong>in</strong>ed impact that a w and temperature have on DON production<br />
on wheat. Data on germ<strong>in</strong>ation (Snow, 1949; Magan and Lacey,
130 Naresh Magan et al.<br />
Deoxynivalenol (ppm)<br />
0.40<br />
0.35<br />
0.30<br />
0.25<br />
0.20<br />
0.15<br />
0.10<br />
0.05<br />
35<br />
30<br />
25<br />
20<br />
0<br />
15<br />
10<br />
5<br />
0<br />
0.99<br />
0.98<br />
0.97<br />
15°C<br />
0.96<br />
25°C<br />
1984a), growth (Magan and Lacey, 1984b) and on DON production<br />
(Hope, 2003; Hope and Magan, 2003) have been comb<strong>in</strong>ed to produce<br />
these profiles (Figure 5). This shows that the range of conditions is<br />
broader for germ<strong>in</strong>ation and growth than for DON production. This<br />
trend is similar to that observed for other mycotoxigenic fungi<br />
(Northolt et al., 1976; 1979) for aflatox<strong>in</strong>s and A. flavus group, and for<br />
Penicillium verrucosum and ochratox<strong>in</strong>.<br />
0.95<br />
0.94<br />
0.99 0.98 0.97 0.96 0.95 0.94 0.93<br />
Water Activity<br />
0.93<br />
0<br />
0<br />
20<br />
40<br />
Time<br />
(days)<br />
40<br />
20 Time<br />
(days)<br />
Figure 2. Effect of a w and time on deoxynivalenol production by F. culmorum on<br />
milled wheat agar at 15° and 25°C (from Hope and Magan, 2003).
Ecophysiology of Fusarium culmorum and Mycotox<strong>in</strong> Production 131<br />
Nivalenol (ppm)<br />
0.40<br />
0.35<br />
0.30<br />
0.25<br />
0.20<br />
0.15<br />
0.10<br />
0.05<br />
4.5<br />
0<br />
4.0<br />
3.5<br />
3.0<br />
2.5<br />
2.0<br />
1.5<br />
1.0<br />
0.05<br />
0<br />
0.99<br />
0.99<br />
0.98<br />
0.98<br />
0.97<br />
0.97<br />
158C<br />
0.96<br />
258C<br />
0.96<br />
Water Activity<br />
0.95<br />
0.95<br />
0.94 0.93<br />
0.94 0.93<br />
0<br />
0<br />
20<br />
20<br />
40<br />
40<br />
Time<br />
(days)<br />
Time<br />
(days)<br />
Figure 3. Effect of a w and time on nivalenol production by F. culmorum on milled<br />
wheat agar at 15° and 25°C (from Hope and Magan, 2003).
132 Naresh Magan et al.<br />
Deoxynivalenol (ppm)<br />
0.35<br />
0.3<br />
0.25<br />
0.15<br />
0.05<br />
20<br />
0.2<br />
18<br />
16<br />
14<br />
0.1<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
0<br />
0.99<br />
0.99<br />
0.98 0.97 0.96<br />
0.98<br />
15°C<br />
25°C<br />
0.97 0.96 0.95<br />
Water activity<br />
0.95<br />
0.94 0.93<br />
0.94 0.93<br />
0<br />
0<br />
20<br />
20<br />
40<br />
40<br />
Time<br />
(days)<br />
Time<br />
(days)<br />
Figure 4. Effect of a w and time on deoxynivalenol production by F. culmorum on<br />
monolayers of wheat gra<strong>in</strong> at 15° and 25°C (Hope, 2003).<br />
3.3. Competition and Colonisation by F. culmorum<br />
Figure 6 shows that many hydrolytic enzymes are produced by<br />
F. culmorum which may enable rapid utilization of nutritional
Ecophysiology of Fusarium culmorum and Mycotox<strong>in</strong> Production 133<br />
Water activity/Moisture content<br />
>30%<br />
M.C.<br />
21-22%<br />
18-19%<br />
21-22%<br />
18-19%<br />
0.99<br />
0.97<br />
0.95<br />
0.93<br />
0.91<br />
0.89<br />
0.87<br />
0.99<br />
0.97<br />
0.95<br />
0.93<br />
0.91<br />
0.89<br />
0.87<br />
(a) Growth rate (mm day -1 )<br />
(b) Deoxynivalenol (ppm)<br />
0.85<br />
0 10 20 30 40<br />
Temperature (�C)<br />
Figure 5. Comparison of profiles and limits for (a) germ<strong>in</strong>ation ( ) and growth<br />
(mm/day) and (b) DON (mg/kg) production by Fusarium culmorum on wheat gra<strong>in</strong><br />
(compiled from Magan and Lacey, 1984a,b; Magan, 1988; Hope, 2003; Hope and<br />
Magan, 2003).<br />
resources over a range of a w and temperature conditions. Previous<br />
studies have demonstrated that F. culmorum produces a significant<br />
amount of cellulases over a range of a w levels (Magan and Lynch,<br />
1986). These hydrolytic enzymes may, when comb<strong>in</strong>ed with secondary<br />
3.0<br />
1.0<br />
0.1<br />
1.0<br />
0.1<br />
5.0<br />
0.25<br />
0.01<br />
10
134 Naresh Magan et al.<br />
µmol 4-nitrophenol m<strong>in</strong>−1 µg−1 gra<strong>in</strong><br />
nmol 4-nitrophenol m<strong>in</strong>−1 µg−1 prote<strong>in</strong><br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
(a)<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
(b)<br />
β-D-fucosidase<br />
α-Dgalactosidase<br />
β-Dglucosidase<br />
α-Dmanno<br />
sidase<br />
β-Dxylosidase<br />
N-acetyl-α-<br />
D-Glucosam<strong>in</strong>idase<br />
N-acetyl-β-<br />
D-Glucosam<strong>in</strong>idase<br />
Figure 6. Production of total (a) and specific (b) activity of seven different enzymes<br />
by F. culmorum on irradiated wheat gra<strong>in</strong> <strong>in</strong>cubated for 14 days at 0.99 a w and 25°C<br />
(Hope, 2003).
Ecophysiology of Fusarium culmorum and Mycotox<strong>in</strong> Production 135<br />
metabolite production and tolerance to <strong>in</strong>termediate moisture conditions<br />
facilitate competitiveness <strong>in</strong> food raw materials. Studies of F. verticillioides<br />
and F. proliferatum have also shown that production<br />
of some hydrolytic enzymes by these species dur<strong>in</strong>g colonisation of<br />
maize may be used as an <strong>in</strong>dicator and diagnosis of early <strong>in</strong>fection by<br />
these species (Mar<strong>in</strong> et al., 1998).<br />
It is of <strong>in</strong>terest that F. culmorum appears less competitive than<br />
F. gram<strong>in</strong>earum when the two species <strong>in</strong>teract <strong>in</strong> vitro on agar media or<br />
on wheat gra<strong>in</strong> (Magan et al., 2003), as both occupy similar ecological<br />
niches.<br />
4. CONCLUSIONS<br />
Growth, competitiveness, dom<strong>in</strong>ance and mycotox<strong>in</strong> production <strong>in</strong><br />
food matrices are <strong>in</strong>fluenced by complex <strong>in</strong>teractions between the<br />
environment, the prevail<strong>in</strong>g fungal community, and external factors.<br />
The role of mycotox<strong>in</strong>s is still unclear, but they may enable fungi to<br />
occupy a particular niche, or assist <strong>in</strong> exclud<strong>in</strong>g other competitors<br />
from the same niche.<br />
6. REFERENCES<br />
Bottalico, A., and Giancarlo, P., 2002, Toxigenic Fusarium species and mycotox<strong>in</strong>s<br />
associated with head blight <strong>in</strong> small-gra<strong>in</strong> cereals <strong>in</strong> Europe, Eur. J. Plant Pathol.<br />
108:611-624.<br />
Cooke, R. C., and Whipps, J. M., 1993. Ecophysiology of Fungi, Blackwell Scientific<br />
Publications, Oxford UK. 337 pp.<br />
Cooney, J. M., Lauren, D. R., and di Menna, M. E., 2001, Impact of competitive<br />
fungi on trichothecene production by Fusarium gram<strong>in</strong>earum, J. Agric. <strong>Food</strong> Chem.<br />
49:522-526.<br />
Dallyn, H., 1978, Effect of substrate, water activity on the growth of certa<strong>in</strong><br />
xerophilic fungi, PhD thesis, Polytechnic of the South Bank London, Council for<br />
National Academic Awards.<br />
D’Mello, J. P. F., Plac<strong>in</strong>ta, C. M., and Macdonald, A. M. C., 1999, Fusarium mycotox<strong>in</strong>s:<br />
a review of global implications for animal health, welfare and productivity,<br />
Animal <strong>Food</strong> Sci. Technol. 80:183-205.<br />
Jenn<strong>in</strong>gs, P., Turner J. A., and Nicholson, P., 2000, Overview of Fusarium ear blight<br />
<strong>in</strong> the UK -effect of fungicide treatment on disease control and mycotox<strong>in</strong> production,<br />
The British Crop Protection Council. Pests and Diseases 2000 2:707-712.<br />
Hope, R., 2003, Ecology and control of Fusarium species and mycotox<strong>in</strong>s <strong>in</strong> wheat<br />
gra<strong>in</strong>, PhD thesis, Institute of BioScience and Technology, Cranfield University,<br />
Silsoe, Bedford, U. K.
136 Naresh Magan et al.<br />
Hope, R. and Magan, N., 2003, Two-dimensional environmental profiles of growth,<br />
deoxynivalenol and nivalenol production by Fusarium culmorum on a wheat-based<br />
substrate, Lett. Appl. Microbiol. 37:70-74.<br />
Lacey J., Bateman G. L., and Mirocha C. J., 1999, Effects of <strong>in</strong>fection time and moisture<br />
on development of ear blight and deoxynivalenol production by Fusarium spp.<br />
<strong>in</strong> wheat, Annals Appl. Biol. 134:277-283.<br />
Magan, N., 1988, Effect of water potential and temperature on spore germ<strong>in</strong>ation and<br />
germ tube growth <strong>in</strong> vitro and on straw leaf sheaths, Trans. Br. Mycol. Soc. 90:<br />
97-107.<br />
Magan, N., and Lacey, J., 1984a, The effect of temperature and pH on the water relations<br />
of field and storage fungi, Trans. Br. Mycol. Soc. 82:71-81.<br />
Magan, N., and Lacey, J., 1984b, Water relations of some Fusarium species from<br />
<strong>in</strong>fected wheat ears and gra<strong>in</strong>, Trans. Br. Mycol. Soc. 83:281-285.<br />
Magan, N., and Lynch, J.M., 1986, Water potential, growth and cellulolysis of soil<br />
fungi <strong>in</strong>volved <strong>in</strong> decomposition of crop residues, J. Gen. Microbiol. 132:1181-<br />
1187.<br />
Magan, N., Hope, R., Colleate, A., and Baxter, E. S., 2002, Relationship between<br />
growth and mycotox<strong>in</strong> production by Fusarium species, biocides and environment,<br />
Eur. J. Plant Pathol. 108:685-690.<br />
Magan, N., Hope, R., Cairns, V., and Aldred, D., 2003, Post-harvest fungal ecology:<br />
impact of fungal growth and mycotox<strong>in</strong> accumulation <strong>in</strong> stored gra<strong>in</strong>, Eur. J. Plant<br />
Pathol. 109:723-730.<br />
Mar<strong>in</strong>, S., Sanchis, V., and Magan, N., 1998, Effect of water activity on hydrolytic<br />
enzyme production by F. moniliforme and F. proliferatum dur<strong>in</strong>g early stages of<br />
growth on maize, Int. J. <strong>Food</strong> Microbiol. 42:1-10<br />
Mar<strong>in</strong>, S., Sanchis, V., Ramos, A. J., and Magan, N., 1999, Two-dimensional profiles<br />
of fumonis<strong>in</strong> B 1 production by Fusarium moniliforme and F. proliferatum <strong>in</strong> relation<br />
to environmental factors and potential for modell<strong>in</strong>g tox<strong>in</strong> formation <strong>in</strong> maize<br />
gra<strong>in</strong>, Int. J. <strong>Food</strong> Microbiol. 51:159-167.<br />
Northolt, M. D., van Egmond, H. P., and Paulsch, W. E., 1979, Ochratox<strong>in</strong> A production<br />
by some fungal species <strong>in</strong> relation to water activity and temperature,<br />
J. <strong>Food</strong> Prot. 42:485-490.<br />
Northolt, M. D., Verhulsdonk, C. A. H., Soentoro, P. S. S. and Paulsch, W. E., 1976,<br />
Effect of water activity and temperature on aflatox<strong>in</strong> production by Aspergillus<br />
parasiticus, J. Milk <strong>Food</strong> Technol. 39:170-174.<br />
Ramirez, M. L., Chulze, S. N., and Magan, N., 2004, Impact of environmental factors<br />
and fungicides on growth and deox<strong>in</strong>ivalenol production by Fusarium gram<strong>in</strong>earum<br />
isolates from Argent<strong>in</strong>ian wheat, Crop Prot. 23:117-125.<br />
Snow, D., 1949, Germ<strong>in</strong>ation of mould spores at controlled humidities, Annals Appl.<br />
Biol. 36:1-13.<br />
Sung, J. M., and Cook, R. J., 1981, Effect of water potential on reproduction<br />
and spore germ<strong>in</strong>ation of Fusarium roseum “gram<strong>in</strong>earum”, “culmorum” and<br />
“avenaceum,” Phytopathology 71:499-504.
FOOD-BORNE FUNGI IN FRUIT AND<br />
CEREALS AND THEIR PRODUCTION OF<br />
MYCOTOXINS<br />
Birgitte Andersen and Ulf Thrane*<br />
1. INTRODUCTION<br />
The growth of filamentous fungi <strong>in</strong> foods and food products<br />
results <strong>in</strong> waste and is costly as well as sometimes hazardous. Many<br />
different fungal species can spoil food products or produce mycotox<strong>in</strong>s<br />
or both. As each fungal species produces its own specific, limited<br />
number of metabolites and is associated with particular types of food<br />
products, the number of mycotox<strong>in</strong>s potentially present <strong>in</strong> a particular<br />
product is limited (Filtenborg et al., 1996). If physical changes<br />
occur <strong>in</strong> a product, changes <strong>in</strong> the association of fungal species found<br />
<strong>in</strong> the product will also occur. With current understand<strong>in</strong>g it is possible<br />
to predict which fungi and mycotox<strong>in</strong>s a given product may conta<strong>in</strong>,<br />
when the type of food product and the history of production and<br />
storage are known.<br />
In Europe, fruit has received m<strong>in</strong>or attention <strong>in</strong> relation to fungal<br />
spoilage, whereas fungal spoilage of cereals has been studied extensively,<br />
but often with the focus on only one or two fungal genera.<br />
Apple juice is one of the few commodities that has caught the<br />
attention of the European authorities and regulation of patul<strong>in</strong> will be<br />
<strong>in</strong> force by the end of 2003 <strong>in</strong> Denmark (EC, 2004).<br />
* Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of<br />
Denmark, DK-2800 Kgs. Lyngby, Denmark. Correspondence to: ba@biocentrum.<br />
dtu.dk<br />
137
138 Birgitte Andersen and Ulf Thrane<br />
Knowledge of the composition and succession of the mycobiota<br />
<strong>in</strong> cereal gra<strong>in</strong>s and fruit dur<strong>in</strong>g maturation, harvest and storage is<br />
an important step towards the prediction of possible mycotox<strong>in</strong><br />
contam<strong>in</strong>ation. Some major spoilage genera on stored apples and<br />
cherries, e.g. Botrytis, Cladosporium and Rhizopus are not known to<br />
produce significant mycotox<strong>in</strong>s, while others <strong>in</strong>clud<strong>in</strong>g Alternaria,<br />
Aspergillus, Fusarium and Penicillium <strong>in</strong>clude species capable of produc<strong>in</strong>g<br />
a wide range of mycotox<strong>in</strong>s (Pitt and Hock<strong>in</strong>g, 1997). The<br />
production of mycotox<strong>in</strong>s is often species specific (Frisvad et al.,<br />
1998), so accurate identification of fungi to species is of major<br />
importance. Historically, identifications have not always been accurate,<br />
and <strong>in</strong>correct identifications have resulted <strong>in</strong> confusion and mis<strong>in</strong>terpretations<br />
(Marasas et al., 1984; Thrane, 2001; Andersen et al.,<br />
2004). In the case of Alternaria, where many taxa are still undescribed<br />
(Simmons and Roberts, 1993; Andersen et al., 2002), identification<br />
is only possible to a species-group level for many isolates.<br />
Before a contam<strong>in</strong>ated sample is analysed for mycotox<strong>in</strong>s, it is important<br />
to know which mycotox<strong>in</strong>s are likely to be present. Metabolite<br />
profiles from known species grown <strong>in</strong> pure culture can provide valuable<br />
<strong>in</strong>formation about the mycotox<strong>in</strong>s that may be found <strong>in</strong> cereals<br />
and fruit and their products, once the fungi normally associated with<br />
those products are known.<br />
Dur<strong>in</strong>g the last 15 years our group has analysed numerous cereal<br />
and fruit samples and recorded the fungal species found. Analysis of<br />
that large amount of data has shown that similar fungal species occur<br />
on the same product types year after year. The purpose of this paper<br />
is to present a list of the fungal species found on apples, cherries, barley<br />
and wheat from the Northern temperate zone together with a list<br />
of mycotox<strong>in</strong>s known to be produced by these fungi.<br />
2. MATERIALS AND METHODS<br />
2.1. Media<br />
Dichloran Rose Bengal Yeast Sucrose agar (DRYES; Frisvad,<br />
1983) and V8 juice agar (V8; Simmons, 1992) were used for fungal<br />
analyses of fruit, while Czapek Dox Iprodione Dichloran agar (CZID;<br />
Abildgren et al., 1987), Dichloran 18% Glycerol agar (DG18;<br />
Hock<strong>in</strong>g and Pitt, 1980), DRYES, and V8 were used for analysis of<br />
cereals. CZID plates were <strong>in</strong>cubated <strong>in</strong> alternat<strong>in</strong>g light/dark cycle
Fungi and Mycotox<strong>in</strong>s <strong>in</strong> Fruit and Cereals 139<br />
consist<strong>in</strong>g of 12 hours of black fluorescent and cool white daylight<br />
and 12 hours darkness at 20-23°C, while DG18 and DRYES plates<br />
were <strong>in</strong>cubated at 25°C <strong>in</strong> darkness and V8 plates <strong>in</strong> alternat<strong>in</strong>g cool<br />
white daylight (8 hours light/16 hours darkness) at 20-23°C. Alternaria<br />
and other dematiaceous hyphomycetes were enumerated on DRYES<br />
and/or V8. Fusarium species were enumerated on CZID and/or V8,<br />
while Eurotium, Aspergillus and Penicillium species and other hyal<strong>in</strong>e<br />
fungi were enumerated on DG18 and/or DRYES.<br />
A wide range of media were used for fungal identification. For<br />
Alternaria species and other black fungi, DRYES, Potato Carrot Agar<br />
(PCA; Simmons, 1992) and V8 were used; for Eurotium species, Malt<br />
Extract Agar (MEA, Pitt and Hock<strong>in</strong>g, 1997) and Czapek Dox agar<br />
(CZ; Samson et al., 2004) were used; for Fusarium species, Potato<br />
Dextrose agar (PDA; Samson et al., 2004) Yeast Extract Sucrose agar<br />
(YES; Samson et al., 2004) and Synthetischer nährstoffarmer agar<br />
(SNA: Nirenberg, 1976) were used. For Penicillium, Czapek Yeast<br />
extract Agar (CYA; Pitt and Hock<strong>in</strong>g, 1997), MEA, YES and<br />
Creat<strong>in</strong>e Sucrose agar (CREA; Samson et al., 2004) were used.<br />
2.2. Fruit<br />
Apple flowers with petals removed, apple peel and apples with fungal<br />
lesions were plated directly onto DRYES and V8. Sound apples<br />
were surfaced dis<strong>in</strong>fected by shak<strong>in</strong>g <strong>in</strong> freshly prepared 0.4 % NaOCl<br />
for 2 m<strong>in</strong>utes and then r<strong>in</strong>s<strong>in</strong>g with sterile water. The cores of the surface<br />
dis<strong>in</strong>fected apples were cut out with a sterile cork borer, cut <strong>in</strong>to<br />
5 pieces and plated on V8 and DRYES.<br />
Cherry flowers with petals and cherries with fungal lesions were<br />
plated directly onto DRYES and V8. Some cherries were surfaced<br />
dis<strong>in</strong>fected as described above. The stem and calyx ends of the surface<br />
dis<strong>in</strong>fected cherries were excised with a sterile scalpel and plated on V8<br />
and DRYES. The plates were <strong>in</strong>cubated as described above and after a<br />
week of <strong>in</strong>cubation the fungal colonies were enumerated. Representative<br />
colonies were then isolated and identified to species level.<br />
The development <strong>in</strong> the mycobiota at genus level was followed on<br />
apples (variety Jonagored) and two varieties of cherries (Vicky and<br />
Van) dur<strong>in</strong>g one growth season (2001 for cherries and 2002 for<br />
apples). The trees were sprayed for fungal diseases (grey mould,<br />
Botrytis spp.) several time before harvest. Flowers of both apples<br />
and cherries were exam<strong>in</strong>ed for fungal growth before the first spray<br />
application. Ten trees were selected from each orchard and sampled<br />
(10 units) from each tree. The ten samples from each orchard were
140 Birgitte Andersen and Ulf Thrane<br />
pooled (100 units per sample) and screened for the presence of<br />
Alternaria, Botrytis, Cladosporium, Fusarium and Penicillium species.<br />
2.3. Cereals<br />
Barley and wheat samples were plated directly with and without<br />
surface dis<strong>in</strong>fection. Kernels were surfaced dis<strong>in</strong>fected by shak<strong>in</strong>g <strong>in</strong><br />
freshly prepared 0.4 % NaOCl for 2 m<strong>in</strong>utes and then r<strong>in</strong>s<strong>in</strong>g with<br />
sterile water. Each sample consisted of 500 gra<strong>in</strong>s, that were not surface<br />
dis<strong>in</strong>fected. All samples were plates on DG18, DRYES, V8 and<br />
CZID. The plates were <strong>in</strong>cubated as described above and after a week<br />
of <strong>in</strong>cubation the fungal colonies were enumerated. Representative<br />
colonies were then isolated and identified to species level. They were<br />
screened for the presence of Alternaria, Bipolaris, Cladosporium,<br />
Eurotium/Aspergillus, Fusarium and Penicillium species.<br />
The development <strong>in</strong> the mycobiota at genus level was followed on<br />
wheat (variety Leguan) and barley (variety Ferment) dur<strong>in</strong>g one<br />
growth season (2002). One batch of barley seed was treated with fungicide<br />
before plant<strong>in</strong>g, while a second batch, and the wheat seed, were<br />
not treated.<br />
2.4. Fungal Species Associated with Fruit and Cereals<br />
In our laboratory, data on the occurrence of fungal contam<strong>in</strong>ation<br />
of fresh, stored, processed and mouldy samples of cereals and fruits<br />
have been collected, recorded and compiled for more than 15 years.<br />
Samples from the field, food factories and local supermarkets have<br />
been surveyed. A number of cultivars of apples (e.g. Cox’s Orange,<br />
Discovery, Gala, Jonagored), cherries (e.g. B<strong>in</strong>g, Van, Vicky), barley<br />
(e.g. Alexis, Chariot, Ferment, Krona) and wheat (e.g. Leguan) have<br />
been sampled and the result<strong>in</strong>g data analysed.<br />
2.5. Fungal Identification and Metabolite Profil<strong>in</strong>g<br />
Alternaria and Stemphylium isolates were transferred to DRYES<br />
and PCA, while other black fungi, <strong>in</strong>clud<strong>in</strong>g Cladosporium isolates,<br />
were transferred to DRYES and V8. Identifications of the black fungi<br />
were done accord<strong>in</strong>g to Andersen et al. (2002), Simmons and Roberts<br />
(1993), Samson et al. (2004) and Simmons (1967; 1969; 1986).<br />
Metabolite profil<strong>in</strong>g <strong>in</strong>volved extract<strong>in</strong>g n<strong>in</strong>e plugs of 14 day old<br />
DRYES cultures <strong>in</strong> ethyl acetate (1 ml) with formic acid (1%;<br />
Andersen et al., 2002) <strong>in</strong> an ultrasonic bath for 60 m<strong>in</strong>. The ethyl
Fungi and Mycotox<strong>in</strong>s <strong>in</strong> Fruit and Cereals 141<br />
acetate was evaporated and the dried sample redissolved <strong>in</strong> methanol<br />
(500 µl). Samples were then filtered and analysed on a HP1100 HPLC-<br />
DAD (Agilent, Germany) (Andersen et al., 2002).<br />
Fusarium isolates were transferred to SNA, PDA and YES and<br />
identified accord<strong>in</strong>g to Gerlach and Nirenberg (1982), Nirenberg<br />
(1989), Burgess et al. (1994) and Samson et al. (2004). Metabolites<br />
were profiled by extract<strong>in</strong>g n<strong>in</strong>e plugs of 14 day old PDA and YES<br />
cultures, each <strong>in</strong> dichloromethane: ethyl acetate (2:1 vol/vol; 1 ml) with<br />
formic acid (1%) <strong>in</strong> an ultrasonic bath for 60 m<strong>in</strong>. Then the organic<br />
phase was evaporated and the dried sample redissolved <strong>in</strong> methanol<br />
(500 µl). Samples were then filtered and analysed on a HP1100 HPLC-<br />
DAD (Smedsgaard, 1997).<br />
Penicillium isolates were transferred to CYA, MEA and YES and<br />
identified accord<strong>in</strong>g to Samson et al. (2004) and Samson and Frisvad<br />
(2004). Metabolites were profiled by extract<strong>in</strong>g three plugs of 7 day<br />
old CYA and YES cultures, then treated as for Fusarium extracts.<br />
2.6. Metabolites from Mouldy Fruit and Cereals<br />
Metabolites were extracted from the mouldy samples <strong>in</strong> the same<br />
way as for the pure fungal cultures. Each sample (100 g) was mixed<br />
with ethyl acetate (100 ml) conta<strong>in</strong><strong>in</strong>g formic acid (1%). The mixture<br />
was shaken regularly over a 2 hour period to extract the metabolites,<br />
then held overnight <strong>in</strong> a freezer. Extracts (14 ml) were decanted from<br />
the frozen water and sample matrix, evaporated to dryness, redissolved<br />
<strong>in</strong> methanol (500 µl) and analysed as before.<br />
3. RESULTS<br />
3.1. Fungal Development <strong>in</strong> Fruit<br />
The dom<strong>in</strong>ant fungal genera found <strong>in</strong> flowers, immature and mature<br />
fruit are given <strong>in</strong> Table 1. In apples, Cladosporium and Alternaria species<br />
constituted the major <strong>in</strong>fection <strong>in</strong> the flowers. Botrytis and Fusarium<br />
species were also found frequently, whereas Penicillium species were isolated<br />
only rarely. After the trees were sprayed with fungicide, Botrytis<br />
was elim<strong>in</strong>ated and the number of Cladosporium colonies was halved.<br />
The spray<strong>in</strong>g did not seem to effect the numbers of Alternaria, Fusarium<br />
or Penicillium colonies enumerated. The numbers of these three genera<br />
<strong>in</strong>creased with time and were highest at harvest.
142 Birgitte Andersen and Ulf Thrane<br />
Table 1. Fungal <strong>in</strong>fection (%) <strong>in</strong> Danish apples and cherries dur<strong>in</strong>g the growth seasons<br />
2002 and 2001 respectively<br />
Sour Sweet<br />
Apples cherries cherries<br />
(Jonagored) (Vicky) (Van)<br />
Fl. a Imm. b Mat. c Fl. Imm. Mat. Fl. Imm. Mat.<br />
Alternaria 77 80 88 68 33 60 66 22 18<br />
Botrytis 35 0 0 4 0 5 78 1 2<br />
Cladosporium 81 73 40 26 0 31 37 5 21<br />
Fusarium 20 53 64 2 0 7 0 1 6<br />
Penicillium 5 13 20 0 0 0 0 1 0<br />
a Fl. = flowers; b Imm. = immature; c Mat. = mature<br />
On the flowers of sweet cherries, Botrytis was the dom<strong>in</strong>ant genus,<br />
whereas it was isolated <strong>in</strong> very low numbers from the flowers of sour<br />
cherries (Table 1). In flowers of both cherry types, Alternaria and<br />
Cladosporium were found <strong>in</strong> high numbers and Fusarium was found <strong>in</strong><br />
low numbers. After spray<strong>in</strong>g, Botrytis was more or less elim<strong>in</strong>ated <strong>in</strong><br />
both immature and mature cherries. The number of Cladosporium<br />
colonies seen was greatly reduced <strong>in</strong> immature cherries after spray<strong>in</strong>g,<br />
but the numbers rose aga<strong>in</strong> as the cherries matured. Numbers of<br />
Alternaria isolated were somewhat reduced <strong>in</strong> immature cherries after<br />
spray<strong>in</strong>g, but the numbers rose aga<strong>in</strong> <strong>in</strong> sour cherries while it fell <strong>in</strong><br />
sweet cherries as they matured. A Penicillium species was found <strong>in</strong> only<br />
one sample of immature sweet cherries.<br />
The dom<strong>in</strong>ant toxigenic fungi on apples were Alt. tenuissima<br />
species-group, followed by Alt. arborescens species-group, F. avenaceum,<br />
F. lateritium, P. crustosum and P. expansum. On cherries, the<br />
dom<strong>in</strong>ant species were Alt. arborescens species-group followed by Alt.<br />
tenuissima species-group, F. lateritium and P. expansum.<br />
3.2. Fungal Development <strong>in</strong> Cereals<br />
The dom<strong>in</strong>ant genera found <strong>in</strong> seed, immature (harvested by hand)<br />
and mature (mach<strong>in</strong>e harvested) wheat and barley kernels are shown<br />
<strong>in</strong> Table 2. In untreated wheat seed, Penicillium constituted the major<br />
<strong>in</strong>fection <strong>in</strong> the seed together with Alternaria, Eurotium and<br />
Aspergillus. The same composition of fungi was seen <strong>in</strong> untreated barley<br />
seed, except that Penicillium counts were less than 50% of those <strong>in</strong><br />
treated seed. In barley seeds that had been treated with fungicides<br />
before sow<strong>in</strong>g, only two out of 500 gra<strong>in</strong>s were found to be <strong>in</strong>fected<br />
with fungi. The changes <strong>in</strong> mycobiota <strong>in</strong> immature kernels compared<br />
to the seed were most pronounced <strong>in</strong> barley, where the numbers of
Fungi and Mycotox<strong>in</strong>s <strong>in</strong> Fruit and Cereals 143<br />
Table 2. Fungal <strong>in</strong>fection (%) <strong>in</strong> Danish wheat and barley seeds and kernels without<br />
surface dis<strong>in</strong>fection dur<strong>in</strong>g the growth season 2002<br />
Wheat Barley Barley<br />
(Leguan) (Ferment) (Ferment)<br />
Seeda Imm. b Mat. c Seeda Imm. Mat. Seedd Imm. Mat.<br />
Alternaria 42 42 67 40 73 64 0 88 58<br />
Botrytis 0 0 9 5 43 37 0 22 31<br />
Eurotium/<br />
Aspergillus<br />
25 0 1 19 1 0 0 1 0<br />
Cladosporium 0 20 2 0 94 0 1 12 18<br />
Fusarium 0 82 90 0 59 83 0 78 98<br />
Penicillium 98 89 98 41 7 98 1 0 98<br />
a b c d Seed without fungicide treatment; Imm. = Immature; Mat. = Mature; Fungicide<br />
treated seed<br />
Fusarium and Alternaria rose markedly and the storage fungi disappeared.<br />
The change was less dramatic <strong>in</strong> wheat. The differences <strong>in</strong><br />
mycobiota from immature to mature kernels were m<strong>in</strong>or and mostly<br />
the number of fungal <strong>in</strong>fected kernels was stable or rose slightly. The<br />
number of Cladosporium found on immature and mature samples varied<br />
a great deal as high number of Fusarium and Penicillium colonies<br />
often obscured the smaller Cladosporium colonies. Surface dis<strong>in</strong>fection<br />
reduced the numbers of Penicillium and Eurotium colonies by<br />
80-90 %, and of Cladosporium and Fusarium by 40-50 %. Only 10-15%<br />
of Alternaria and Bipolaris could be removed, <strong>in</strong>dicat<strong>in</strong>g that the<br />
gra<strong>in</strong>s had <strong>in</strong>ternal <strong>in</strong>fections with these genera.<br />
Dom<strong>in</strong>ant fungi <strong>in</strong> common to both wheat and barley kernels were<br />
isolates of Alt. <strong>in</strong>fectoria species-group, F. avenaceum, P. aurantiogriseum,<br />
P. cyclopium and P. polonicum. However, barley had higher<br />
<strong>in</strong>fection rates with Bipolaris sorok<strong>in</strong>iana, P. hordei and P. verrucosum<br />
compared to wheat.<br />
3.3. Fungal Species Associated with Fruit and Cereals<br />
The frequencies of occurrence of fungal species <strong>in</strong> several cultivars<br />
of apples, cherries, barley and wheat are given <strong>in</strong> Tables 3 and 4. In our<br />
laboratory, such data have been compiled for more than 15 years. The<br />
differences <strong>in</strong> the mycobiota between cultivars were <strong>in</strong> most cases<br />
small. Most often the same fungal species were found and the variation<br />
was quantitative only. As can been seen from Tables 3 and 4, only<br />
a limited number of fungal species are found <strong>in</strong> both fruit and cereals.<br />
Newly harvested, undamaged apples and cherries were not usually<br />
<strong>in</strong>fected by fungi. When <strong>in</strong>fection was present, fungi mostly belonged
144 Birgitte Andersen and Ulf Thrane<br />
to the dematiaceous hyphomycetes (Table 3). The mycobiota of fresh<br />
apples consisted of ma<strong>in</strong>ly of Alt. tenuissima species-group, Botrytis<br />
and Cladosporium spp. In fresh cherries, Alt. arborescens speciesgroup<br />
and Stemphylium spp. constituted the mycobiota, along with<br />
Botrytis and Cladosporium spp. In storage, the mycobiota changed <strong>in</strong><br />
both types of fruit. In cherries, after only a few weeks <strong>in</strong> storage,<br />
Botrytis spp., P. expansum and Zygomycetes dom<strong>in</strong>ated the mycobiota<br />
and they often spoiled the cherries. In stored apples the same fungal<br />
species were found after months of storage together with P. solitum.<br />
Fungi were rarely found <strong>in</strong> juice made from apples or cherries, but<br />
when fungal growth was detected, Byssochlamys spp. and P. expansum<br />
were found <strong>in</strong> pasteurised and untreated juices, respectively.<br />
In contrast to fruit, samples of newly harvested, sound cereal gra<strong>in</strong>s<br />
always had some fungal <strong>in</strong>fections after surface dis<strong>in</strong>fection. Colonies<br />
of Alt. <strong>in</strong>fectoria species-group, Cladosporium spp., F. avenaceum and<br />
F. tric<strong>in</strong>ctum were always isolated from fresh barley and wheat.<br />
Epicoccum nigrum, F. culmorum, F, equiseti and F. poae were often<br />
seen also. As <strong>in</strong> fruit, the mycobiota changed <strong>in</strong> cereals <strong>in</strong> storage <strong>in</strong><br />
favour of Aspergillus and Penicillium species. In dry barley and wheat<br />
samples that had been stored for one year or more, Eurotium spp. and<br />
Table 3. Fungal occurrence (frequency) <strong>in</strong> apples and cherries from the north temperate<br />
zone: data accumulated over a 15 year period a<br />
Apples Cherries<br />
Fresh Stored Juice Fresh Stored Juice<br />
Alternaria arborescens<br />
sp.-grp.<br />
+ − − ++ + −<br />
Alt. <strong>in</strong>fectoria sp.-grp. (+) − − (+) − −<br />
Alt. tenuissima sp.-grp. ++ + − + − −<br />
Botrytis spp. ++ ++ − ++ ++ −<br />
Byssochlamys spp. − − + − − +<br />
Cladosporium spp. ++ + − ++ + −<br />
Fusarium avenaceum + + − − − −<br />
F. lateritium + (+) − + − −<br />
Monilia spp. (+) + − (+) + −<br />
Penicillium carneum (+) + − − − −<br />
P. crustosum + + − − − −<br />
P. expansum + ++ (+) + ++ (+)<br />
P. polonicum − (+) − − − −<br />
P. solitum (+) ++ − − − −<br />
Stemphylium spp. (+) − − ++ − −<br />
Zygomycetes − ++ − − ++ −<br />
a +++: Always present; ++: often present; +: sometimes present; (+): rarely present; −:<br />
never detected or found only once. Fresh <strong>in</strong>dicates direct plated, surface dis<strong>in</strong>fected<br />
sound samples; stored, direct plated dis<strong>in</strong>fected sound or visibly mouldy samples
Fungi and Mycotox<strong>in</strong>s <strong>in</strong> Fruit and Cereals 145<br />
P. aurantiogriseum always dom<strong>in</strong>ated the mycobiota. However, Alt.<br />
<strong>in</strong>fectoria species-group could still be isolated from barley that had<br />
been stored for two years. Aspergillus candidus, Asp. flavus, P. cyclopium,<br />
P. hordei, P. melanoconidium, P. polonicum, P. verrucosum and<br />
P. viridicatum were also often found <strong>in</strong> stored barley and <strong>in</strong> lower<br />
numbers <strong>in</strong> stored wheat.<br />
3.4. Production of Toxic Metabolites <strong>in</strong> Pure Culture<br />
Six genera out of the twelve listed <strong>in</strong> Tables 3 and 4 are regarded as<br />
be<strong>in</strong>g non-toxigenic, namely Botrytis, Cladosporium, Epicoccum,<br />
Table 4. Fungal occurrence (frequency) <strong>in</strong> barley and wheat from the north temperate<br />
zone: data accumulated over a 15 year period a<br />
Barley Wheat<br />
Fresh Stored Fresh Stored<br />
Alternaria arborescens sp.-grp. (+) − (+) −<br />
Alt. <strong>in</strong>fectoria sp.-grp. +++ ++ +++ ++<br />
Alt. tenuissima sp.-grp. + − + −<br />
Aspergillus candidus − ++ − ++<br />
Asp. flavus − ++ − ++<br />
Asp. niger − + − +<br />
Bipolaris sorok<strong>in</strong>iana + (+) (+) −<br />
Botrytis spp. (+) − (+) −<br />
Cladosporium spp. +++ (+) +++ (+)<br />
Epicoccum nigrum ++ − ++ −<br />
Eurotium spp. − +++ − +++<br />
Fusarium avenaceum +++ + +++ +<br />
F. culmorum ++ (+) ++ (+)<br />
F. equiseti ++ − ++ −<br />
F. gram<strong>in</strong>earum + − + −<br />
F. langsethiae (+) − − −<br />
F. lateritium (+) − − −<br />
F. poae ++ + ++ +<br />
F. sporotrichioides + − + −<br />
F. tric<strong>in</strong>ctum +++ + +++ +<br />
Penicillium aurantiogriseum (+) ++ (+) ++<br />
P. cyclopium (+) ++ (+) ++<br />
P. freii (+) + (+) ++<br />
P. hordei + ++ (+) +<br />
P. melanoconidium − ++ − ++<br />
P. polonicum − ++ − ++<br />
P. verrucosum + ++ + ++<br />
P. viridicatum − ++ − ++<br />
Stemphylium spp.<br />
aSee footnote to Table 3<br />
(+) − (+) −
146 Birgitte Andersen and Ulf Thrane<br />
Eurotium, Monilia and Stemphylium, together with the Zygomycetes<br />
(Pitt and Hock<strong>in</strong>g, 1997). Two genera which <strong>in</strong>clude toxigenic species,<br />
Bipolaris and Byssochlamys, were found only relatively rarely <strong>in</strong><br />
cereals and fruit, respectively. Alternaria, Aspergillus, Fusarium and<br />
Penicillium species were much more commonly isolated. These four<br />
genera <strong>in</strong>clude 25 toxigenic species found on a regular basis <strong>in</strong> either<br />
fruit or cereals. Only Alt. tenuissima species-group and F. avenaceum<br />
were regularly found <strong>in</strong> both fresh fruit and fresh cereals. In Table 5<br />
the mycotox<strong>in</strong>s that are produced <strong>in</strong> pure culture by the fungal species<br />
listed <strong>in</strong> Tables 3 and 4 are given. Of all of the species <strong>in</strong> Tables 3 and<br />
4, only Alt. <strong>in</strong>fectoria species-group and P. solitum are regarded as<br />
non-toxigenic. As can been seen from Table 5, several fungal species<br />
with<strong>in</strong> the same genus and found <strong>in</strong> the same product can produce the<br />
same mycotox<strong>in</strong>s (e.g. roquefort<strong>in</strong>e C and penitrem A by Penicillia <strong>in</strong><br />
fruit or culmor<strong>in</strong>s and trichothecenes by Fusaria <strong>in</strong> cereals). In fruit<br />
that has either been damaged <strong>in</strong> the orchard and/or stored poorly, one<br />
or more of the follow<strong>in</strong>g toxic metabolites might theoretically be<br />
found: altenuene, alternariols, chaetoglobos<strong>in</strong>s, citr<strong>in</strong><strong>in</strong>, patul<strong>in</strong>,<br />
roquefort<strong>in</strong>e C and tenuazonic acid. In fresh cereals that have been<br />
harvested dur<strong>in</strong>g a ra<strong>in</strong>y period, the follow<strong>in</strong>g toxic metabolites would<br />
be relevant: antibiotic Y, beauveric<strong>in</strong>, culmor<strong>in</strong>s, enniat<strong>in</strong>s, fusar<strong>in</strong> C,<br />
fusarochromanone, moniliform<strong>in</strong>, trichothecenes and zearalenone. In<br />
cereals that have been stored poorly and/or not dried down after harvest,<br />
the follow<strong>in</strong>g toxic metabolites should be considered: aflatox<strong>in</strong>s,<br />
aspergillic acid, citr<strong>in</strong><strong>in</strong>, cyclopiazonic acid, nephrotoxic glycopeptides,<br />
ochratox<strong>in</strong> A, penicillic acid, terphenyll<strong>in</strong>, verrucosid<strong>in</strong>,<br />
viomelle<strong>in</strong>, vioxanth<strong>in</strong>, viridic acid, xanthoasc<strong>in</strong> and xanthomegn<strong>in</strong>.<br />
However, it should be noted that mycotox<strong>in</strong>s produced <strong>in</strong> the field or<br />
at an early stage of storage always should be taken <strong>in</strong>to consideration,<br />
as mycotox<strong>in</strong>s <strong>in</strong> general are persistent through storage and processes<br />
for food and feed production.<br />
3.5. Production of Toxic Metabolites <strong>in</strong> Fruit and<br />
Cereals<br />
Analyses of extracts from pure fungal cultures can <strong>in</strong>dicate which<br />
fungal metabolites should be analysed, but the mycobiota of the<br />
actual sample needs to be determ<strong>in</strong>ed to make realistic recommendations.<br />
Mycotox<strong>in</strong>s that have been detected <strong>in</strong> naturally moulded<br />
apples, cherries, barley and wheat samples exam<strong>in</strong>ed <strong>in</strong> our laboratory<br />
are given <strong>in</strong> Table 6. Samples that were mouldy when received were<br />
extracted immediately, while sound samples were <strong>in</strong>cubated for a week
Fungi and Mycotox<strong>in</strong>s <strong>in</strong> Fruit and Cereals 147<br />
Table 5. Fungal species found <strong>in</strong> apples, cherries wheat and barley from the north<br />
temperate zone and some mycotox<strong>in</strong>s they are known to produce<br />
Toxigenic fungal species Mycotox<strong>in</strong>s produced <strong>in</strong> pure culture<br />
Alternaria arborescens sp.-grp. Altenuene, alternariols, altertox<strong>in</strong>s,<br />
tenuazonic acid<br />
Alt. tenuissima sp.-grp. Altenuene, alternariols, altertox<strong>in</strong>s,<br />
tenuazonic acid<br />
Aspergillus candidus Terphenyll<strong>in</strong>, xanthoasc<strong>in</strong><br />
Asp. flavus Aflatox<strong>in</strong>, aspergillic acid, cyclopiazonic acid<br />
Asp. niger Malform<strong>in</strong>s, naphtho-γ-pyrones<br />
Bipolaris sorok<strong>in</strong>iana Sterigmatocyst<strong>in</strong><br />
Byssochlamys spp. Byssochlamic acid, patul<strong>in</strong><br />
Fusarium avenaceum Antibiotic Y, aurofusar<strong>in</strong>, enniat<strong>in</strong>s, fusar<strong>in</strong> C,<br />
moniliform<strong>in</strong><br />
F. culmorum Aurofusar<strong>in</strong>, culmor<strong>in</strong>, fusar<strong>in</strong> C, trichothecenes,<br />
zearalenone<br />
F. equiseti Fusarochromanone, trichothecenes, zearalenone<br />
F. gram<strong>in</strong>earum Aurofusar<strong>in</strong>, culmor<strong>in</strong>, fusar<strong>in</strong> C, trichothecenes,<br />
zearalenone<br />
F. langsethiae Culmor<strong>in</strong>, enniat<strong>in</strong>s, trichothecenes<br />
F. lateritium Antibiotic Y, enniat<strong>in</strong>s,<br />
F. poae Beauveric<strong>in</strong>s, culmor<strong>in</strong>s, fusar<strong>in</strong> C, trichothecenes<br />
F. sporotrichioides Aurofusar<strong>in</strong>, beauveric<strong>in</strong>s, culmor<strong>in</strong>s, fusar<strong>in</strong> C,<br />
trichothecenes<br />
F. tric<strong>in</strong>ctum Antibiotic Y, aurofusar<strong>in</strong>, enniat<strong>in</strong>s, fusar<strong>in</strong> C,<br />
moniliform<strong>in</strong><br />
Penicillium aurantiogriseum Penicillic acid, verrucosid<strong>in</strong>, nephrotoxic<br />
glycopeptides<br />
P. carneum Patul<strong>in</strong>, isofumigaclav<strong>in</strong>, penitrem A,<br />
roquefort<strong>in</strong>e C<br />
P. crustosum Penitrem A, roquefort<strong>in</strong>e C<br />
P. cyclopium Penicillic acid, xanthomegn<strong>in</strong>, viomelle<strong>in</strong>,<br />
vioxanth<strong>in</strong><br />
P. expansum Citr<strong>in</strong><strong>in</strong>, chaetoglobos<strong>in</strong>s, communes<strong>in</strong>s, patul<strong>in</strong>,<br />
roquefort<strong>in</strong>e C<br />
P. freii Penicillic acid, xanthomegn<strong>in</strong>, viomelle<strong>in</strong>,<br />
vioxanth<strong>in</strong><br />
P. hordei Roquefort<strong>in</strong>e C<br />
P. melanoconidium Penicillic acid, penitrem A, xanthomegn<strong>in</strong><br />
P. polonicum Penicillic acid, verrucosid<strong>in</strong>, nephrotoxic<br />
glycopeptides<br />
P. verrucosum Citr<strong>in</strong><strong>in</strong>, ochratox<strong>in</strong> A<br />
P. viridicatum Penicillic acid, viridic acid, xanthomegn<strong>in</strong>,<br />
viomelle<strong>in</strong>
148 Birgitte Andersen and Ulf Thrane<br />
Table 6. Mycotox<strong>in</strong>s detected <strong>in</strong> naturally <strong>in</strong>fected samples<br />
Sample Metabolites detected<br />
Immature apples with mouldy core (1) Alternariols, antibiotic Y, aurofusar<strong>in</strong><br />
Immature apples with mouldy core (2) Altenuene, alternariols<br />
Mature apples with mouldy core (3) Alternariols, patul<strong>in</strong><br />
Mouldy apple pulp (4) Alternariols, antibiotic Y, aurofusar<strong>in</strong>,<br />
ascladiol,<br />
Mouldy apple on tree (5) Chaetoglobos<strong>in</strong> A<br />
Mouldy sweet cherries ‘June drop’ (6) Alternariols, antibiotic Y<br />
Mouldy, mature sweet cherries (7) Alternariols<br />
Mouldy cherry juice (8) Chaetoglobos<strong>in</strong>s, communes<strong>in</strong>s,<br />
roquefort<strong>in</strong>e C<br />
Mouldy barley (9) Ochratox<strong>in</strong> A<br />
Mouldy barley ‘hot spot’ (10) Ochratox<strong>in</strong> A<br />
Mouldy wheat (11) Antibiotic Y, ochratox<strong>in</strong> A, zearalenone<br />
Mouldy wheat (12) Aurofusar<strong>in</strong>, fusar<strong>in</strong> C, zearalenone<br />
or until mould could be seen to simulate a worst case. Fusarium and/or<br />
Alternaria metabolites were detected <strong>in</strong> samples of immature apples<br />
from the field with visible fungal growth (samples 1 and 2, worst cases),<br />
while the mature, fresh apple samples (samples 3 and 4, worst cases)<br />
also conta<strong>in</strong>ed Penicillium metabolites. One mature, mouldy apple<br />
(sample 5) still hang<strong>in</strong>g on the tree conta<strong>in</strong>ed only chaetoglobos<strong>in</strong> A.<br />
The cherry sample (sample 6), which consisted of immature cherries<br />
that had been shed by the trees, had 100 % <strong>in</strong>fection with Alternaria<br />
species. Extraction showed that it conta<strong>in</strong>ed high amounts of both<br />
Alternaria and Fusarium metabolites, whereas the mature cherries (sample<br />
7, worst case) only conta<strong>in</strong>ed Alternaria metabolites. The mouldy<br />
cherry juice (sample 8), on the other hand, conta<strong>in</strong>ed only Penicillium<br />
metabolites. The mouldy barley samples (samples 9 and 10), which had<br />
been stored without dry<strong>in</strong>g after harvest, conta<strong>in</strong>ed ochratox<strong>in</strong> A.<br />
Sample 10, sampled <strong>in</strong> the ‘green hot spot’ of the mouldy lot, conta<strong>in</strong>ed<br />
approximately 1000 times the amount of ochratox<strong>in</strong> A as sample 9,<br />
which had little visible fungal growth. In the mouldy wheat sample<br />
(sample 11) Fusarium metabolites as well as ochratox<strong>in</strong> A were<br />
detected, while sample 12 only conta<strong>in</strong>ed Fusarium metabolites.<br />
4. DISCUSSION<br />
Analyses of seasonal changes <strong>in</strong> apple mycobiota showed that<br />
many fungal species found <strong>in</strong> mature fruit already were present <strong>in</strong> the<br />
flowers and later colonized the immature fruit (Table 1). Spray<strong>in</strong>g with
Fungi and Mycotox<strong>in</strong>s <strong>in</strong> Fruit and Cereals 149<br />
fungicides decreased Botrytis <strong>in</strong>fection and had some effect on<br />
Cladosporium numbers, but no effect on Alternaria, Fusarium and<br />
Penicillium. The number of Alternaria, Fusarium and Penicillium<br />
<strong>in</strong>fected apples also <strong>in</strong>creased after spray<strong>in</strong>g and cont<strong>in</strong>ued to <strong>in</strong>crease<br />
until apples were picked. In cherries, the same fungal species were seen<br />
<strong>in</strong> mature cherries as <strong>in</strong> flowers (Table 1). Application of fungicides<br />
had an effect on all the fungal genera found <strong>in</strong> flowers; Alternaria<br />
<strong>in</strong>fections decreased by 50-65%. Alternaria and Cladosporium numbers,<br />
however, <strong>in</strong>creased aga<strong>in</strong> before harvest <strong>in</strong> sour cherries, while<br />
the numbers of Alternaria rema<strong>in</strong>ed constant <strong>in</strong> sweet cherries, probably<br />
due to the early drop of immature cherries, which had 100%<br />
<strong>in</strong>fection with Alternaria. Our results show that the mycobiota of<br />
apples and cherries are similar at genus level, but different <strong>in</strong> species<br />
composition. Alternaria tenuissima species-group, P. expansum and<br />
P. solitum dom<strong>in</strong>ate <strong>in</strong> apples, whereas A. arborescens species-group,<br />
and Stemphylium spp. dom<strong>in</strong>ate <strong>in</strong> cherries (Table 3).<br />
Analyses of the mycobiota <strong>in</strong> cereals from sow<strong>in</strong>g to harvest showed,<br />
<strong>in</strong> contrast to fruit, that the <strong>in</strong>itial mycobiota present <strong>in</strong> the untreated<br />
seed played only a small role <strong>in</strong> the subsequent mycobiota on mature<br />
kernels, though it may play a great role <strong>in</strong> the viability of the seed<br />
(Table 2). The two untreated seed samples conta<strong>in</strong>ed a high number of<br />
Alternaria, Penicillium and Eurotium species. Surface dis<strong>in</strong>fection<br />
removed 80-90% of the Penicillium and Eurotium numbers, while the<br />
same only could be done for 40-45% of the Alternaria. Furthermore,<br />
the Penicillium species <strong>in</strong> the seed and <strong>in</strong> the immature kernels were different.<br />
In the seed P. chrysogenum, P. cyclopium and P. freii were found,<br />
whereas P. aurantiogriseum, P. polonicum and P. verrucosum were found<br />
<strong>in</strong> mature kernels grow<strong>in</strong>g from the untreated seed. The only fungi that<br />
were found <strong>in</strong> larger amounts <strong>in</strong> seed and recovered <strong>in</strong> more than 50%<br />
of the harvested cereal samples belonged to A. <strong>in</strong>fectoria species-group.<br />
The numbers of Alternaria and Fusarium found <strong>in</strong> the three mature<br />
samples were low (less than 60%) and high (more than 80%), respectively,<br />
compared with other reports (Andersen et al., 1996; Kosiak<br />
et al., 2004). A very wet period <strong>in</strong> June and July 2002, dur<strong>in</strong>g the grow<strong>in</strong>g<br />
season <strong>in</strong> Western Denmark was probably responsible.<br />
Comparisons of the mycobiota from two mature barley samples grown<br />
from fungicide treated and untreated seed showed few differences.<br />
As fruits mature and are harvested, fungi such as Botrytis, Monilia<br />
and Zygomycetes are known to cause fruit spoilage <strong>in</strong> orchards as well<br />
as <strong>in</strong> storage, whereas Cladosporium and Epicoccum are known for their<br />
discolouration of cereals <strong>in</strong> the field. These fungi cause economical<br />
losses, but none of them are associated with production of mycotox<strong>in</strong>s.
150 Birgitte Andersen and Ulf Thrane<br />
Different species of Alternaria, Fusarium and Penicillium, on the other<br />
hand, all spoil fruit and cereals, but produce species specific mycotox<strong>in</strong>s<br />
(Table 5) and hundred mycotox<strong>in</strong>s and other biologically active<br />
metabolites from these three genera have been characterised with<strong>in</strong><br />
recent years (Nielsen and Smedsgaard, 2003) and it is reasonable to<br />
expect that more than the few <strong>in</strong>cluded <strong>in</strong> the legislation (aflatox<strong>in</strong>,<br />
ochratox<strong>in</strong>, deoxynivalenol, fumonis<strong>in</strong>s <strong>in</strong> cereals and patul<strong>in</strong> <strong>in</strong><br />
fruits) can be produced <strong>in</strong> mouldy foods.<br />
The results presented <strong>in</strong> this study show that Alternaria and Fusarium<br />
<strong>in</strong> fruit and cereals may pose a mycotox<strong>in</strong> risk. Dur<strong>in</strong>g spoilage of<br />
apples and cherries, P. expansum is known to produce patul<strong>in</strong>, which has<br />
been <strong>in</strong>corporated <strong>in</strong> the legislation on fruit produce. However, both<br />
Alternaria and Fusarium were able to produce additional metabolites <strong>in</strong><br />
mouldy fruit samples (Table 6, sample 4): alternariols, antibiotic Y and<br />
aurofusar<strong>in</strong>. In cereals, P. verrucosum is known to produce ochratox<strong>in</strong> A,<br />
which has also been <strong>in</strong>corporated <strong>in</strong> the legislation on raw cereal gra<strong>in</strong>.<br />
However, Fusarium was able to produce antibiotic Y and zearalenone <strong>in</strong><br />
addition to ochratox<strong>in</strong> A from P. verrucosum <strong>in</strong> mouldy wheat (Table 6,<br />
sample 11). For these lesser known metabolites no or very limited data<br />
are available on the toxicity on co-produced metabolites and their possible<br />
synergistic effects, which make risk assessment <strong>in</strong> food and food production<br />
systems difficult. In conclusion, we see the co-occurrence of<br />
these specific Alternaria and Fusarium metabolites and their potential<br />
toxicities as the major future challenge <strong>in</strong> food mycology.<br />
5. ACKNOWLEDGEMENTS<br />
The authors are grateful to Dr. Jens C. Frisvad for discussion of<br />
manuscript and identification of some of the Penicillium cultures. This<br />
work was partly supported by the Danish M<strong>in</strong>istry of <strong>Food</strong>,<br />
Agriculture and Fisheries through the program “<strong>Food</strong> Quality with a<br />
focus on <strong>Food</strong> Safety”, by LMC Centre for Advanced <strong>Food</strong> Studies<br />
and by the Danish Technical Research Council through ‘Program for<br />
Predictive Biotechnology’.<br />
6. REFERENCES<br />
Abildgren, M. P., Lund, F., Thrane, U., and Elmholt, S., 1987, Czapek-Dox agar conta<strong>in</strong><strong>in</strong>g<br />
iprodione and dicloran as a selective medium for the isolation of Fusarium<br />
species, Lett. Appl. Microbiol. 5:83-86.
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Andersen, B., Thrane, U., Svendsen, A., Rasmussen, I. A., 199, Associated field<br />
mycobiota on malt<strong>in</strong>g barley, Can. J. Bot. 74:854-858.<br />
Andersen, B., Krøger, E., and Roberts, R.G., 2002, Chemical and morphological<br />
segregation of Alternaria arborescens, A. <strong>in</strong>fectoria and A. tenuissima speciesgroups,<br />
Mycol. Res. 106:170-182.<br />
Andersen, B., Smedsgaard, J., and Frisvad, J. C., 2004, Penicillium expansum: consistent<br />
production of patul<strong>in</strong>, chaetoglobos<strong>in</strong>s and other secondary metabolites <strong>in</strong><br />
culture and their natural occurrence <strong>in</strong> fruit products, J. Agric. <strong>Food</strong> Chem.<br />
52:2421-2428.<br />
Burgess, L. W., Summerell, B. A., Bullock, S., Gott, K. P., and Backhouse, D., 1994,<br />
Laboratory Manual for Fusarium Research. 3rd Edition, University of Sydney,<br />
Sydney, Australia.<br />
EC (European Commission), 2004, European Commission Regulation 455/2004 of 11<br />
March, 2004. European Commission, http://europa.eu.<strong>in</strong>t/eur-lex/pri/en/oj/dat/2004/<br />
l_074/l_07420040312en00110011.pdf<br />
Filtenborg, O., Frisvad, J. C., and Thrane, U., 1996, Moulds <strong>in</strong> food spoilage, Int.<br />
J. <strong>Food</strong> Microbiol. 33:85-102.<br />
Frisvad, J. C., 1983, A selective and <strong>in</strong>dicative medium for groups of Penicillium viridicatum<br />
produc<strong>in</strong>g different mycotox<strong>in</strong>s on cereals, J. Appl. Bacteriol. 54:409-416.<br />
Frisvad, J. C., Thrane, U., and Filtenborg, O., 1998, Role and use of secondary<br />
metabolites <strong>in</strong> fungal taxonomy, <strong>in</strong>: Chemical Fungal Taxonomy, J. C. Frisvad,<br />
P. D. Bridge, and D. K. Arora, (eds), Marcel Dekker, New York, pp. 289-319.<br />
Gerlach, W., and Nirenberg, H., 1982, The genus Fusarium -A Pictorial Atlas,<br />
Mitteilungen aus der Biologische Bundesanstalt für Land-und Forstwirtschaft,<br />
Berl<strong>in</strong>-Dahlem 209:1-406.<br />
Hock<strong>in</strong>g, A. D., and Pitt, J. I., 1980, Dichloran-glycerol medium for enumeration of<br />
xerophilic fungi from low moisture foods, Appl. Environ. Microbiol. 39:488-492.<br />
Kosiak, B., Torp, M., Skjerve, E., and Andersen, B., 2004, Alternaria and Fusarium <strong>in</strong><br />
Norwegian gra<strong>in</strong>s of reduced quality -a matched pair sample study, Int. J. <strong>Food</strong><br />
Microbiol. 93:51-62.<br />
Marasas, W. F. O., Nelson, P. E., and Toussoun, T. A., 1984, Toxigenic Fusarium<br />
Species. Identity and Mycotoxicology, The Pennsylvania State University Press,<br />
University Park, pp. 1-328.<br />
Nielsen, K. F., and Smedsgaard, J., 2003, Fungal metabolite screen<strong>in</strong>g: database of<br />
474 mycotox<strong>in</strong>s and fungal metabolites for de-replication by standardised liquid<br />
chromatography-UV detection-mass spectrometry methodology, J. Chromatogr.<br />
A. 1002:111-136.<br />
Nirenberg, H., 1976, Untersuchungen über die morphologische und biologische<br />
Differenzierung <strong>in</strong> der Fusarium-Sektion Liseola, Mitteilungen aus der Biologische<br />
Bundesanstalt für Land-und Forstwirtschaft. Berl<strong>in</strong>-Dahlem 169:1-117.<br />
Nirenberg, H. I., 1989, Identification of Fusaria occurr<strong>in</strong>g <strong>in</strong> Europe on cereals and<br />
potatoes, <strong>in</strong>: Fusarium: Mycotox<strong>in</strong>s, Taxonomy and Pathogenicity, J. Chelkowski<br />
(ed.), Elsevier Science Publishers B.V., Amsterdam, pp. 179-193.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, Blackie Academic and<br />
Professional, London, pp. 1-593.<br />
Samson, R. A., Frisvad, J. C., 2004, Penicillium subgenus Penicillium: new taxonomic<br />
schemes, mycotox<strong>in</strong>s and other extrolites, Stud. Mycol. 49:1-257.<br />
Samson, R. A., Hoekstra, E. S., Frisvad, J. C., (eds), 2004, Introduction to <strong>Food</strong>and<br />
Airborne Fungi. 7th Edition. Centraalbureau voor Schimmelcultures, Utrecht,<br />
pp. 1-389.
152 Birgitte Andersen and Ulf Thrane<br />
Simmons, E. G., 1967, Typification of Alternaria, Stemphylium and Ulocladium,<br />
Mycologia 59:67-92.<br />
Simmons, E. G., 1969, Perfect states of Stemphylium, Mycologia 61:1-26.<br />
Simmons, E. G., 1986, Alternaria themes and variations (22-26), Mycotaxon 25:287-308.<br />
Simmons, E. G., 1992, Alternaria taxonomy: Current Status, viewpo<strong>in</strong>t, challenge, <strong>in</strong>:<br />
Alternaria Biology, Plant Diseases and Metabolites, J. Chelkowski and A. Visconti,<br />
eds, Elsevier, Amsterdam, pp. 1-35.<br />
Simmons, E. G., and Roberts, R. G., 1993, Alternaria themes and variations (73),<br />
Mycotaxon 48:109-140.<br />
Smedsgaard, J., 1997, Micro-scale extraction procedure for standardized screen<strong>in</strong>g of<br />
fungal metabolites production <strong>in</strong> cultures, J. Chromatogr. A 760:264-270.<br />
Thrane, U., 2001, Developments <strong>in</strong> the taxonomy of Fusarium species based on<br />
secondary metabolites, <strong>in</strong>: Fusarium. Paul E. Nelson Memorial Symposium. B. A.<br />
Summerell, J. F. Leslie, D. Backhouse, W. L. Bryden, and L. W. Burgess (eds), APS<br />
Press, St. Paul, M<strong>in</strong>nesota, pp. 29-49.
BLACK ASPERGILLUS SPECIES IN<br />
AUSTRALIAN VINEYARDS: FROM<br />
SOIL TO OCHRATOXIN A IN WINE<br />
Su-l<strong>in</strong> L. Leong, *‡ Ailsa D. Hock<strong>in</strong>g, * John I. Pitt, * Benozir<br />
A. Kazi, † Robert W. Emmett † and Eileen S. Scott ‡§<br />
1. INTRODUCTION<br />
Fungi classified <strong>in</strong> Aspergillus Section Nigri (the black Aspergilli)<br />
are ubiquitous saprophytes <strong>in</strong> soils around the world, particularly <strong>in</strong><br />
tropical and subtropical regions (Klich and Pitt, 1988; Pitt and<br />
Hock<strong>in</strong>g, 1997). Several species <strong>in</strong> this Section are common <strong>in</strong> v<strong>in</strong>eyards<br />
and are often associated with bunch rots (Amer<strong>in</strong>e et al., 1980).<br />
A. niger is reported to be the primary cause of Aspergillus rot <strong>in</strong><br />
grapes before harvest (Nair, 1985; Snowdon, 1990), while A. aculeatus<br />
(Jarvis and Traquair, 1984) and A. carbonarius (Gupta, 1956) have<br />
also been reported. The development of fungal bunch rots has been<br />
correlated with the splitt<strong>in</strong>g of grape berries (Barbetti, 1980), and<br />
Aspergillus counts on grapes grown for dry<strong>in</strong>g were greater dur<strong>in</strong>g seasons<br />
when ra<strong>in</strong> before harvest caused the berries to split (Figure 1)<br />
(Leong et al., 2004). Spores of black Aspergillus spp. are resistant<br />
to UV light (Rotem and Aust, 1991), which may account for their<br />
* Su-l<strong>in</strong> L. Leong, Ailsa D. Hock<strong>in</strong>g and John I. Pitt, CSIRO <strong>Food</strong> Science Australia,<br />
North Ryde, NSW 2113, Australia.<br />
† Benozir A. Kazi and Robert W. Emmett, Department of Primary Industries,<br />
Mildura, Victoria 3502, Australia.<br />
‡ Su-l<strong>in</strong> L. Leong and Eileen S. Scott, University of Adelaide, Glen Osmond, South<br />
Australia 5064, Australia.<br />
§ All authors, Cooperative Research Centre for Viticulture, Glen Osmond, South<br />
Australia 5064, Australia. Correspondence to: ailsa.hock<strong>in</strong>g@csiro.au<br />
153
154 Su-l<strong>in</strong> L. Leong et al.<br />
Mean black Aspergillus<br />
count (cfu/g fruit)<br />
1,000,000<br />
800,000<br />
600,000<br />
400,000<br />
200,000<br />
0<br />
1998 1999 2000<br />
Year<br />
A. aculeatus<br />
A. carbonarius<br />
A. niger<br />
Figure 1. Mean severity of <strong>in</strong>fection of fresh and dry<strong>in</strong>g grapes (comb<strong>in</strong>ed) by each<br />
of three black Aspergillus species over three successive harvest seasons. Ra<strong>in</strong> occurred<br />
before harvest <strong>in</strong> 1999 and 2000. The mean count was derived by summ<strong>in</strong>g the counts<br />
for each species of black Aspergillus from all the fruit samples, and divid<strong>in</strong>g that sum<br />
by the total number of samples. Reproduced from Leong, S. L., Hock<strong>in</strong>g. A. D., and<br />
Pitt, J. I., 2004, Australian Journal of Grape and W<strong>in</strong>e Research 10: 83-88 (with permission<br />
from the Australian Society of Viticulture and Oenology).<br />
persistence <strong>in</strong> v<strong>in</strong>eyards and on grape berries even after dry<strong>in</strong>g (K<strong>in</strong>g<br />
et al., 1981; Abdel-Sater and Saber, 1999; Abarca et al., 2003).<br />
With<strong>in</strong> Section Nigri, A. carbonarius and A. niger have been shown to<br />
produce the mycotox<strong>in</strong>, ochratox<strong>in</strong> A (OA) (Abarca et al., 1994; Téren<br />
et al., 1996; Heenan et al., 1998; reviewed <strong>in</strong> Abarca et al., 2001). OA is<br />
a demonstrated nephrotox<strong>in</strong>, which may also be carc<strong>in</strong>ogenic, teratogenic,<br />
immunogenic and genotoxic. It has been classified as Group 2B,<br />
a “possible human carc<strong>in</strong>ogen” (Castegnaro and Wild, 1995).<br />
OA has been detected <strong>in</strong> grapes and grape products <strong>in</strong>clud<strong>in</strong>g juice,<br />
w<strong>in</strong>e, dried v<strong>in</strong>e fruit and w<strong>in</strong>e v<strong>in</strong>egars (Zimmerli and Dick, 1996;<br />
MacDonald et al., 1999; Majerus et al., 2000; Markarki et al., 2001;<br />
Da Rocha Rosa et al., 2002; Sage et al., 2002; OA <strong>in</strong> w<strong>in</strong>e and grape<br />
juice reviewed by Bellí et al., 2002). A survey of 600 Australian w<strong>in</strong>es<br />
showed that OA was present only at low levels. Only 15% of samples<br />
had levels > 0.05 µg/l and 85% of these were < 0.2 µg/l. The maximum<br />
level found was 0.61 µg/l (Hock<strong>in</strong>g et al., 2003). In Europe,<br />
a limit of 2 µg/kg <strong>in</strong> table w<strong>in</strong>es is under discussion (Anon., 2003)<br />
and a limit of 10 µg/kg <strong>in</strong> dried v<strong>in</strong>e fruits has been set (European<br />
Commission, 2002).<br />
OA <strong>in</strong> grapes and grape products is produced by toxigenic A. carbonarius<br />
and A. niger species which have been isolated from grapes <strong>in</strong>
Black Aspergillus Species <strong>in</strong> Australian V<strong>in</strong>eyards 155<br />
France (Sage et al., 2002), South America (Da Rocha Rosa et al.,<br />
2002), Spa<strong>in</strong> (Cabañes et al., 2002), Italy (Battilani et al., 2003b),<br />
Portugal (Serra et al., 2003) and Greece (Tjamos et al., 2004). In an<br />
extensive study of Australian dried v<strong>in</strong>e fruit, stra<strong>in</strong>s of A. carbonarius<br />
were commonly isolated from semi-dried and dried v<strong>in</strong>e fruit <strong>in</strong> the<br />
field, and all were capable of produc<strong>in</strong>g OA <strong>in</strong> the laboratory (Leong<br />
et al., 2004). Hence A. carbonarius is thought to be the primary species<br />
responsible for OA production <strong>in</strong> grapes <strong>in</strong> Australia.<br />
Assum<strong>in</strong>g that OA production <strong>in</strong> grapes ceases at the commencement<br />
of process<strong>in</strong>g, typically a sterilisation step <strong>in</strong> <strong>in</strong>dustrial juice and<br />
w<strong>in</strong>e production (Roset, 2003), the concentration of OA <strong>in</strong> the f<strong>in</strong>al<br />
product is a function of the <strong>in</strong>itial concentration <strong>in</strong> the grapes and the<br />
effect of process<strong>in</strong>g. Processes which reduce OA can be classified <strong>in</strong>to<br />
two groups, physical removal and degradation.<br />
Physical removal of OA first <strong>in</strong>volves remov<strong>in</strong>g the site where OA has<br />
been produced, for example, the removal of visibly mouldy berries from<br />
table grapes. It is not well understood if OA is primarily associated with<br />
the sk<strong>in</strong>, pulp or juice of grape berries. However, a strong association<br />
with the sk<strong>in</strong> or pulp would suggest that a relatively small proportion of<br />
OA rema<strong>in</strong>s <strong>in</strong> the f<strong>in</strong>ished beverage. The high water content of grape<br />
berries may lead to the migration of OA from the zone of fungal growth<br />
to other parts of the berry (Engelhardt et al., 1999).<br />
A second aspect of physical removal of OA is the partition<strong>in</strong>g of the<br />
tox<strong>in</strong> between solid and liquid phases dur<strong>in</strong>g process<strong>in</strong>g. Fernandes<br />
et al. (2003) conducted microv<strong>in</strong>ification trials on crushed grapes<br />
spiked with OA, and reported reductions <strong>in</strong> OA of 50-95%. The most<br />
significant reductions resulted from solid-liquid separation steps, such<br />
as press<strong>in</strong>g the juice or w<strong>in</strong>e from the sk<strong>in</strong>s, and decant<strong>in</strong>g the w<strong>in</strong>e<br />
from precipitated solids. Many of the solids present <strong>in</strong> grape juice have<br />
an aff<strong>in</strong>ity for OA and will loosely b<strong>in</strong>d and precipitate the tox<strong>in</strong> from<br />
solution (Roset, 2003), as do some f<strong>in</strong><strong>in</strong>g agents added dur<strong>in</strong>g w<strong>in</strong>emak<strong>in</strong>g,<br />
such as activated charcoal (Dumeau and Trione, 2000;<br />
Castellari et al., 2001; Silva et al., 2003).<br />
Little is known about the degradation of OA by w<strong>in</strong>e yeasts dur<strong>in</strong>g<br />
fermentation, though this has been demonstrated dur<strong>in</strong>g beer fermentation<br />
(Baxter et al., 2001). Silva et al. (2003) reported reduction <strong>in</strong><br />
OA by lactic acid bacteria dur<strong>in</strong>g malolactic fermentation which<br />
follows the completion of primary (yeast) fermentation. However,<br />
Fernandes et al. (2003) argued that this is not a true degradation,<br />
rather, bacterial biomass b<strong>in</strong>d<strong>in</strong>g OA that later settles out of the<br />
w<strong>in</strong>e. The addition of sulphur dioxide and the pasteurisation of juice<br />
by heat<strong>in</strong>g have no effect on OA (Roset, 2003).
156 Su-l<strong>in</strong> L. Leong et al.<br />
This paper presents orig<strong>in</strong>al data on OA contam<strong>in</strong>ation of w<strong>in</strong>e, cover<strong>in</strong>g<br />
the source of A. carbonarius <strong>in</strong> Australian v<strong>in</strong>eyards, the survival<br />
of A. carbonarius spores on the surface of bunches, and the passage of<br />
OA throughout v<strong>in</strong>ification of grapes <strong>in</strong>oculated with A. carbonarius.<br />
2. MATERIALS AND METHODS<br />
2.1. Aspergillus carbonarius <strong>in</strong> the V<strong>in</strong>eyard<br />
Environment<br />
Substrates were collected from v<strong>in</strong>eyards <strong>in</strong> the grape-grow<strong>in</strong>g<br />
region centred around Mildura, Victoria, Australia. Substrates collected<br />
<strong>in</strong>cluded parts of v<strong>in</strong>es [green, yellow (senesc<strong>in</strong>g) and dead leaf<br />
tissue, green and dead berries, dead bunch remnants (dried rachides),<br />
tendrils, canes and bark] and materials from the v<strong>in</strong>eyard floor [green<br />
cover crop plants, dead cover crop trash, v<strong>in</strong>e trash and soil].<br />
Collections were made over three grow<strong>in</strong>g seasons, from six v<strong>in</strong>eyards<br />
<strong>in</strong> 2000-01 and from three v<strong>in</strong>eyards <strong>in</strong> 2001-02 and 2002-03. In 2000-<br />
01, samples of substrates were collected from three sites along a<br />
diagonal transect <strong>in</strong> each v<strong>in</strong>eyard 2 weeks after veraison and at harvest.<br />
In the latter two seasons, samples of substrates were collected<br />
from five sites along a diagonal transect <strong>in</strong> each v<strong>in</strong>eyard when berries<br />
were pea size, at 2 weeks after veraison and at harvest. To quantify<br />
A. carbonarius present on the surface of these substrates, samples were<br />
washed for 2 m<strong>in</strong> <strong>in</strong> sterile water conta<strong>in</strong><strong>in</strong>g Citowett® (BASF<br />
Australia Ltd, Victoria, Australia) as a wett<strong>in</strong>g agent, and aliquots of<br />
the solution were plated <strong>in</strong> duplicate onto Dichloran Rose Bengal<br />
Chloramphenicol Agar (DRBC) (Pitt and Hock<strong>in</strong>g, 1997). Serial dilutions<br />
were performed on soil samples, followed by plat<strong>in</strong>g onto<br />
DRBC. After plates were <strong>in</strong>cubated at 25°C for 5-7 days, colonies of<br />
A. carbonarius were identified and enumerated.<br />
The presence of A. carbonarius <strong>in</strong> v<strong>in</strong>eyard soils was also compared<br />
among four v<strong>in</strong>eyards <strong>in</strong> 2002-03 by dilution plat<strong>in</strong>g as described<br />
above. Thirty soil samples were collected from each v<strong>in</strong>eyard, ten<br />
samples at each stage of v<strong>in</strong>e growth, i.e. when berries were pea size,<br />
at 2 weeks after veraison and at harvest. The tillage practices of the<br />
v<strong>in</strong>eyards were noted. Soil was sampled directly under v<strong>in</strong>es and also<br />
between v<strong>in</strong>e rows. In one v<strong>in</strong>eyard, five soil sampl<strong>in</strong>g cores of 0.2 m 2<br />
were taken from under v<strong>in</strong>es. For each soil core, A. carbonarius was<br />
enumerated <strong>in</strong> soil at the surface, 5 cm and 15 cm below the surface.<br />
A. carbonarius was also enumerated <strong>in</strong> the air of this v<strong>in</strong>eyard.<br />
Colonies from 20 l of air sampled us<strong>in</strong>g a MAS-100® air sampler
Black Aspergillus Species <strong>in</strong> Australian V<strong>in</strong>eyards 157<br />
(Merck KGaA, Darmstadt, Germany) were enumerated on DRBC.<br />
Samples were taken on n<strong>in</strong>e occasions from air <strong>in</strong> the v<strong>in</strong>eyard at<br />
10 cm, 100 cm and 180 cm above the v<strong>in</strong>eyard floor.<br />
Statistical analyses were performed us<strong>in</strong>g Genstat (6th edition,<br />
Lawes Agricultural Trust, Rothamsted, UK).<br />
2.2. Survival of Aspergillus carbonarius Spores on the<br />
Surface of Bunches Preharvest<br />
A trial was conducted <strong>in</strong> the Hunter Valley, New South Wales,<br />
Australia to exam<strong>in</strong>e the survival of A. carbonarius spores on the surface<br />
of Chardonnay and Shiraz grapes (three replicate rows) dur<strong>in</strong>g<br />
the grow<strong>in</strong>g season <strong>in</strong> 2002-03. The v<strong>in</strong>es were over 25 years old,<br />
tra<strong>in</strong>ed onto horizontal wires and under drip irrigation.<br />
Spores of 7-14 day old cultures of A. carbonarius on Czapek Yeast<br />
Agar (CYA) (Pitt and Hock<strong>in</strong>g, 1997) plates were harvested <strong>in</strong>to sterile<br />
water conta<strong>in</strong><strong>in</strong>g Tween-80® (0.05% w/v; Merck, Victoria,<br />
Australia), and diluted to 2-4 × 10 5 colony form<strong>in</strong>g units per ml<br />
(cfu/ml). Bunches were <strong>in</strong>oculated by immersion <strong>in</strong> 1 l of suspension<br />
conta<strong>in</strong>ed with<strong>in</strong> a plastic bag. The same <strong>in</strong>oculum was used for up to<br />
40 bunches without decrease <strong>in</strong> the spore concentration. Twelve<br />
bunches <strong>in</strong> each row were <strong>in</strong>oculated at pre-bunch closure (berries<br />
green and pea size), veraison and 11-16 days preharvest. Two bunches<br />
were comb<strong>in</strong>ed <strong>in</strong>to a s<strong>in</strong>gle sample, result<strong>in</strong>g <strong>in</strong> six samples per replicate<br />
at each sampl<strong>in</strong>g stage. Inoculated bunches were sampled after<br />
the <strong>in</strong>oculum had dried to give an <strong>in</strong>itial value, at each of the subsequent<br />
stages and at harvest.<br />
Bunches were homogenised for 3 m<strong>in</strong> <strong>in</strong> a stomacher (BagMixer,<br />
Interscience, France) with the addition of sterile distilled water, and<br />
serial dilutions of the suspension were plated onto DRBC. After <strong>in</strong>cubation<br />
for 3 days at 25°C, colonies of A. carbonarius were enumerated.<br />
The average berry weight at each growth stage was calculated, and the<br />
number of A. carbonarius colonies was expressed as cfu per berry, <strong>in</strong><br />
order to compare the number of viable spores present dur<strong>in</strong>g each stage.<br />
2.3. W<strong>in</strong>emak<strong>in</strong>g<br />
2.3.1. Inoculation and V<strong>in</strong>ification of Grapes<br />
Berries were <strong>in</strong>oculated preharvest with a spore suspension of<br />
A. carbonarius (prepared as described <strong>in</strong> 2.2) at approximately 10 7 cfu/ml.
158 Su-l<strong>in</strong> L. Leong et al.<br />
Stra<strong>in</strong>s selected for <strong>in</strong>oculation were local to the region of the experimental<br />
v<strong>in</strong>eyard, and were strong producers of OA when screened on<br />
Coconut Cream Agar (CCA) (Heenan et al., 1998). A variety of <strong>in</strong>oculation<br />
techniques was employed, all <strong>in</strong>volv<strong>in</strong>g puncture damage to<br />
the berry sk<strong>in</strong> and subsequent contact with the spore suspension. In<br />
addition to the primary <strong>in</strong>oculation, a supplementary <strong>in</strong>oculation of<br />
additional fruit was often performed towards harvest to ensure sufficient<br />
fruit for v<strong>in</strong>ification. At harvest, <strong>in</strong>oculated and un<strong>in</strong>oculated<br />
fruit were mixed to simulate high, <strong>in</strong>termediate and low or absent<br />
levels of OA <strong>in</strong> fruit. Table 1 summarises the <strong>in</strong>oculation, <strong>in</strong>cubation<br />
and harvest details.<br />
Table 1. Preparation of OA-contam<strong>in</strong>ated grapes for w<strong>in</strong>emak<strong>in</strong>g<br />
Location, Mildura, Victoria, 2002 Hunter Valley, New South<br />
v<strong>in</strong>tage Wales, 2003<br />
A. carbonarius FRR 5374 a , FRR 5573, FRR 5682, FRR 5683<br />
stra<strong>in</strong>s FRR 5574<br />
Grape variety Chardonnay Shiraz Semillon Shiraz<br />
Method of Berries <strong>in</strong>jected Berries Berries Berries<br />
<strong>in</strong>oculation us<strong>in</strong>g syr<strong>in</strong>ge <strong>in</strong>jected us<strong>in</strong>g punctured <strong>in</strong>jected<br />
{berries syr<strong>in</strong>ge {sk<strong>in</strong> with a bed of us<strong>in</strong>g<br />
<strong>in</strong>jected scored us<strong>in</strong>g p<strong>in</strong>s dipped syr<strong>in</strong>ge<br />
us<strong>in</strong>g syr<strong>in</strong>ge} b grater and <strong>in</strong> spore<br />
sprayed with suspension<br />
spore {berries<br />
suspension} <strong>in</strong>jected<br />
us<strong>in</strong>g syr<strong>in</strong>ge}<br />
Period from 21 days 14 days 9 days 8 days<br />
primary <strong>in</strong>oc. {4 days} b {13 days} {3 days}<br />
until harvest<br />
High OA w<strong>in</strong>e: 53 kg 120 kg 25 kg 28 kg<br />
mass of grapes <strong>in</strong>oculated <strong>in</strong>oculated <strong>in</strong>oculated <strong>in</strong>oculated<br />
Intermediate 34 kg 46 kg 15 kg 11 kg<br />
OA w<strong>in</strong>e: mass <strong>in</strong>oculated <strong>in</strong>oculated <strong>in</strong>oculated <strong>in</strong>oculated<br />
of grapes + 23 kg + 73 kg + 13 kg + 16 kg<br />
un<strong>in</strong>oculated un<strong>in</strong>oculated un<strong>in</strong>oculated un<strong>in</strong>oculated<br />
Control w<strong>in</strong>e: 51 kg 118 kg 32 kg 27 kg<br />
mass of grapes un<strong>in</strong>oculated un<strong>in</strong>oculated un<strong>in</strong>oculated un<strong>in</strong>oculated<br />
Size of 4 l 16 l 2 l 4 l<br />
ferment <strong>in</strong>clud<strong>in</strong>g <strong>in</strong>clud<strong>in</strong>g<br />
sk<strong>in</strong>s sk<strong>in</strong>s<br />
aFRR numbers are from the culture collection of <strong>Food</strong> Science Australia, North<br />
Ryde, NSW, Australia.<br />
b {} bracketed text refers to supplementary <strong>in</strong>oculation of additional fruit.
Black Aspergillus Species <strong>in</strong> Australian V<strong>in</strong>eyards 159<br />
Harvested bunches were chilled at 4°C prior to crush<strong>in</strong>g. After<br />
crush<strong>in</strong>g, eight samples of must from each tox<strong>in</strong> level were collected <strong>in</strong><br />
order to establish the <strong>in</strong>itial total OA present <strong>in</strong> the berries. Samples<br />
were also collected throughout the v<strong>in</strong>ification process as described<br />
below.<br />
Dur<strong>in</strong>g white v<strong>in</strong>ification, the must was pressed, after which potassium<br />
metabisulphite was added to give 50 ppm SO 2 <strong>in</strong> the juice.<br />
Pect<strong>in</strong>ase was added <strong>in</strong> the form of Pomolase AC50 (0.05 ml/l<br />
juice; Enzyme Solutions, Victoria, Australia) or Pect<strong>in</strong>ase (0.5 g/l<br />
juice; Fermtech, Queensland, Australia). The juice was overlaid with<br />
nitrogen or carbon dioxide, and refrigerated at 4°C for at least 24 h to<br />
precipitate solids. In 2002, the juice was divided <strong>in</strong>to four replicate<br />
ferments at each tox<strong>in</strong> level before clarification; this division occurred<br />
after clarification <strong>in</strong> 2003. The pH was adjusted to approximately 3.3<br />
by the addition of tartaric acid to give a titratable acidity of 6.5-7.0<br />
g/l. The clarified juice was siphoned <strong>in</strong>to bottles filled with nitrogen or<br />
carbon dioxide and fitted with stoppers and air traps. The yeast QA23<br />
(Lallemand, Toulouse, France) was rehydrated and added at a rate<br />
equivalent to 0.2 g dry yeast/l juice. Diammonium phosphate was<br />
added at 0.5 g/l juice. The fermentation temperature was 19°C <strong>in</strong> 2002<br />
and 15°C <strong>in</strong> 2003. Diammonium phosphate was added dur<strong>in</strong>g fermentation<br />
as required and after fermentation was completed, the w<strong>in</strong>e<br />
was racked. Potassium metabisulphite was added at a rate equivalent<br />
to 50 ppm SO 2 to stabilise the w<strong>in</strong>e and prevent further fermentation.<br />
Bentonite (0.5 g/l; Fermtech, Queensland, Australia) and Liquif<strong>in</strong>e<br />
(2002: 1 ml/l; 2003: 0.6 ml/l; W<strong>in</strong>ery Supplies, Victoria, Australia) were<br />
added, and the bottles placed at 19°C (2002) or 15°C (2003) to allow<br />
precipitation of solids. A second rack<strong>in</strong>g was performed for all bottles,<br />
and potassium metabisulphite was added to br<strong>in</strong>g the free SO 2 to 20<br />
ppm. The bottles were placed at < 4°C for cold stabilisation for > 30<br />
days. The w<strong>in</strong>e was filtered <strong>in</strong>to 375 ml glass bottles with cork<br />
closures.<br />
Dur<strong>in</strong>g red v<strong>in</strong>ification, the must was divided <strong>in</strong>to 4 replicate fermentations<br />
at each tox<strong>in</strong> level. Potassium metabisulphite was added to<br />
give 50 ppm SO 2 <strong>in</strong> the must, diammonium phosphate was added at<br />
0.5 g/l must, and tartaric acid was added to br<strong>in</strong>g the titratable acidity<br />
to 6.5 g/l. The yeast D254 (Lallemand, Toulouse, France) was rehydrated<br />
and added at approximately 0.3 g/l must. The cap was plunged<br />
2-3 times daily. The must was pressed after 4 days of fermentation at<br />
room temperature <strong>in</strong> 2002, and after 6 days of fermentation at approximately<br />
20°C <strong>in</strong> 2003. Fermentation was f<strong>in</strong>ished <strong>in</strong> bottles at room<br />
temperature. Dur<strong>in</strong>g the first rack<strong>in</strong>g, 50 ppm SO 2 was added, after
160 Su-l<strong>in</strong> L. Leong et al.<br />
which the w<strong>in</strong>e was held at 19°C (2002) or 15°C (2003) to precipitate<br />
yeast cells and other solids. At the second rack<strong>in</strong>g, SO 2 was added to<br />
ma<strong>in</strong>ta<strong>in</strong> a f<strong>in</strong>al concentration of 50 ppm. The w<strong>in</strong>e was held at < 2°C<br />
for cold stabilisation, after which the pH was adjusted and the w<strong>in</strong>e<br />
bottled through a filtration l<strong>in</strong>e.<br />
Bottles were cellared at room temperature (approximately 22°C) <strong>in</strong><br />
2002, and at 15°C <strong>in</strong> 2003.<br />
2.3.2. OA Assays<br />
A new method was developed for the rapid analysis of OA <strong>in</strong> grape<br />
matrices. Samples were standardised by weight.<br />
Grape musts were homogenised and a 10 g subsample weighed <strong>in</strong>to<br />
a centrifuge tube. Methanol (10 ml), Milli-Q water (10 ml) and 10N<br />
HCl (≈ 0.15 ml) were added, and mixed thoroughly with the sample.<br />
For liquid samples, 10 g of sample was mixed with methanol (1.5 ml)<br />
and 10N HCl (≈ 0.15 ml). The mixture was centrifuged at 2500 rpm<br />
for 15 m<strong>in</strong>. A 900 mg C18 solid phase extraction cartridge (Maxi-<br />
Clean, Alltech, Deerfield, USA) was conditioned with 5 ml acetonitrile<br />
followed by 5 ml water, and the supernatant was passed dropwise<br />
through this cartridge under vacuum. The pellet was resuspended <strong>in</strong><br />
10 ml 10% methanol, then centrifuged for a further 15 m<strong>in</strong> at 2500<br />
rpm. This supernatant was also passed through the C18 cartridge. For<br />
must samples, an additional 10 ml water was washed through the C18<br />
cartridge at this stage.<br />
A 200 mg am<strong>in</strong>opropyl cartridge (4 ml Extract-Clean, Alltech,<br />
Deerfield, USA) was conditioned with 3 ml methanol. The C18 and<br />
am<strong>in</strong>opropyl cartridges were attached <strong>in</strong> series, and the sample was<br />
eluted from the C18 cartridge onto the am<strong>in</strong>opropyl cartridge with the<br />
addition of 10 ml methanol. The sample was eluted from the am<strong>in</strong>opropyl<br />
cartridge with 10 ml 35% ethyl acetate <strong>in</strong> cyclohexane conta<strong>in</strong><strong>in</strong>g<br />
0.75% formic acid.<br />
The eluate was dried under reduced pressure at 45°C and was resuspended<br />
<strong>in</strong> 1 ml mobile phase (35% acetonitrile conta<strong>in</strong><strong>in</strong>g 0.1% acetic<br />
acid) for analysis by HPLC (Hock<strong>in</strong>g et al., 2003).<br />
Aliquots of the w<strong>in</strong>e extracts were chromatographed on an<br />
Ultracarb (30) C18 4.6 × 250 mm, 5 µm column (Phenomenex,<br />
Torrance, USA). The mobile phase consisted of acetonitrile:<br />
water:acetic acid (50:49:1, v/v), and was delivered through the heated<br />
column (40°C) at a flow of 1.3 ml/m<strong>in</strong> us<strong>in</strong>g a Shimadzu 10A VP high<br />
pressure b<strong>in</strong>ary gradient solvent delivery system. Detection of OA<br />
was achieved by post column addition of ammonia (12.5% w/w,
Black Aspergillus Species <strong>in</strong> Australian V<strong>in</strong>eyards 161<br />
0.2 ml/m<strong>in</strong>) and monitor<strong>in</strong>g the natural fluorescence of OA at 435 nm<br />
after excitation at 385 nm (Shimadzu, RF-10AXL). Sample <strong>in</strong>jections<br />
were performed us<strong>in</strong>g a Shimadzu SIL-10Advp autosampler, and the<br />
typical <strong>in</strong>jection volume was 30 µl. OA <strong>in</strong> the w<strong>in</strong>e extracts was quantified<br />
by comparison with a calibration curve. Typical recoveries<br />
ranged from 80-100%. The results presented have not been corrected<br />
for recovery.<br />
3. RESULTS AND DISCUSSION<br />
3.1. Aspergillus carbonarius <strong>in</strong> the V<strong>in</strong>eyard<br />
Environment<br />
Counts of A. carbonarius were high <strong>in</strong> soil and <strong>in</strong> v<strong>in</strong>e trash on soil<br />
and relatively low on other substrates (Table 2). Hence, soil is likely to<br />
be the primary source of A. carbonarius <strong>in</strong> v<strong>in</strong>eyards.<br />
In the four v<strong>in</strong>eyards surveyed, soil beneath v<strong>in</strong>es conta<strong>in</strong>ed more<br />
A. carbonarius than soil between rows (P < 0.05, Figure 2). This<br />
association is likely to be due to damaged and dead berries fall<strong>in</strong>g<br />
onto the soil and provid<strong>in</strong>g a sugar-rich medium for the growth of<br />
<strong>in</strong>digenous saprophytic Aspergillus species. The concentration of<br />
A. carbonarius propagules was highest at the surface of soil, where<br />
this debris is found, and decreased deeper with<strong>in</strong> the soil profile of<br />
an untilled v<strong>in</strong>eyard (Figure 3). The v<strong>in</strong>eyard <strong>in</strong> which the soil profile<br />
was regularly disturbed by till<strong>in</strong>g conta<strong>in</strong>ed a higher concentration<br />
of A. carbonarius <strong>in</strong> the soil than v<strong>in</strong>eyards <strong>in</strong> which the soil was<br />
less disturbed (P < 0.05, Figure 2). In v<strong>in</strong>eyards with m<strong>in</strong>imal tillage,<br />
A. carbonarius may be one member of a complex and stable microbial<br />
community associated with the cover crop and other flora on the<br />
soil surface. One potential effect of regular tillage is to allow<br />
the <strong>in</strong>crease of a dom<strong>in</strong>ant species (Marfen<strong>in</strong>a and Mirch<strong>in</strong>k, 1989).<br />
Table 2. Aspergillus carbonarius on v<strong>in</strong>eyard materials. Results expressed as cfu/ml<br />
surface wash unless otherwise <strong>in</strong>dicated.<br />
Substrate 2000-01 2001-02 2002-03<br />
Canes (dead) 8 1 not recorded<br />
V<strong>in</strong>e bark 31 9 not recorded<br />
Bunch remnants 47 15 not recorded<br />
Cover trash (dead) 8 10 2<br />
V<strong>in</strong>e trash on soil 669 45 20<br />
Soil (cfu/g) 4,987 1,219 1,342
162 Su-l<strong>in</strong> L. Leong et al.<br />
Mean A. carbonarius<br />
count (cfu/g soil)<br />
5,000<br />
4,000<br />
3,000<br />
2,000<br />
1,000<br />
0<br />
a<br />
Between rows<br />
Under v<strong>in</strong>es<br />
A<br />
a<br />
1 2 3 4<br />
V<strong>in</strong>eyard<br />
Figure 2. Aspergillus carbonarius <strong>in</strong> soil under v<strong>in</strong>es and between v<strong>in</strong>e rows <strong>in</strong> v<strong>in</strong>eyards<br />
with m<strong>in</strong>imal and cont<strong>in</strong>ual tillage, n = 30. V<strong>in</strong>eyard 1 and 3: No tillage for the<br />
last 3 and 4 years, respectively. V<strong>in</strong>eyard floors were covered with wild grasses and/or<br />
weeds. V<strong>in</strong>eyard 2: Tilled once each year before sow<strong>in</strong>g a rye grass cover crop.<br />
V<strong>in</strong>eyard 4: Tilled monthly after a cover crop was established. Vertical bars with different<br />
letters differ significantly (LSD, P < 0.05).<br />
A. carbonarius may compete more effectively for sugars from fallen<br />
berries, and tillage would also distribute propagules throughout the<br />
soil profile. Other authors have observed higher levels of some fungi<br />
<strong>in</strong> tilled soil than <strong>in</strong> untilled soil. An example is the primary<br />
Mean A. carbonarius<br />
count (cfu/g soil)<br />
1,500<br />
1,000<br />
500<br />
0<br />
b<br />
B<br />
0-1 cm 5 cm 15 cm<br />
Depth<br />
Figure 3. Aspergillus carbonarius <strong>in</strong> untilled v<strong>in</strong>eyard soil at different depths <strong>in</strong> 2001-02,<br />
n = 5. Vertical bars with different letters differ significantly (LSD, P < 0.05).<br />
a<br />
a<br />
A<br />
a<br />
b<br />
C
Black Aspergillus Species <strong>in</strong> Australian V<strong>in</strong>eyards 163<br />
pathogen of root rot of wheat, Cochliobolus sativus (Diehl, 1979;<br />
Duczek, 1981; Diehl et al., 1982).<br />
A. carbonarius was present <strong>in</strong> v<strong>in</strong>eyard air, though the concentration<br />
of conidia decreased with height from the v<strong>in</strong>eyard floor (Figure 4).<br />
This suggests that w<strong>in</strong>d may distribute conidia of A. carbonarius from<br />
the soil onto berry surfaces.<br />
Dur<strong>in</strong>g the early stages of berry development from pre-bunch closure<br />
until veraison, A. carbonarius spores survived poorly and a n<strong>in</strong>e<br />
fold decrease <strong>in</strong> the number of viable propagules was observed<br />
(Figure 5). This suggests that the surface of green berries is a hostile<br />
environment for the survival of spores. The v<strong>in</strong>e canopy dur<strong>in</strong>g the<br />
early part of the season <strong>in</strong> 2002-2003 was sparse due to drought. Thus,<br />
the berries were not shielded from UV light by overhang<strong>in</strong>g leaves.<br />
A sparse canopy would also allow greater penetration of rout<strong>in</strong>e<br />
fungicide sprays, which, together with the UV light may have<br />
contributed to the death of the spores (Rotem and Aust, 1991).<br />
3.2. Survival of Aspergillus carbonarius Spores on<br />
Bunch Surfaces Preharvest<br />
For Shiraz bunches <strong>in</strong>oculated at pre-bunch closure, there was a<br />
consistent decrease <strong>in</strong> the counts of A. carbonarius between the <strong>in</strong>oculation<br />
time and veraison. This decrease cont<strong>in</strong>ued <strong>in</strong> some bunches<br />
between veraison and harvest (Figure 6). However, <strong>in</strong> other bunches,<br />
the count <strong>in</strong>creased due to <strong>in</strong>fection of berries and subsequent<br />
Mean A. carbonarius<br />
count (cfu/L air)<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
c<br />
b<br />
10 cm 100 cm 180 cm<br />
Height<br />
Figure 4. Aspergillus carbonarius <strong>in</strong> air at different levels above the v<strong>in</strong>eyard floor,<br />
n = 9. Vertical bars with different letters differ significantly (LSD, P < 0.05).<br />
a
164 Su-l<strong>in</strong> L. Leong et al.<br />
A. carbonarius count<br />
(log cfu per berry)<br />
3.25<br />
3<br />
2.75<br />
2.5<br />
2.25<br />
2<br />
1.75<br />
1.5<br />
Pre-bunch closure Veraison<br />
Figure 5. Spore death on berry surfaces between pre-bunch closure and veraison;<br />
n = 32, compris<strong>in</strong>g both Chardonnay and Shiraz varieties. 50% of the data are<br />
conta<strong>in</strong>ed with<strong>in</strong> the boxes, while the bar with<strong>in</strong> the box plot shows the median value.<br />
Maximum and m<strong>in</strong>imum values are <strong>in</strong>dicated by vertical l<strong>in</strong>es. The difference between<br />
Aspergillus carbonarius spore count per berry at pre-bunch closure and veraison was<br />
significant at P < 0.001.<br />
A. carbonarius count (cfu per berry)<br />
1,000,000<br />
100,000<br />
10,000<br />
1,000<br />
100<br />
10<br />
1<br />
Row A<br />
Row B<br />
Pre-bunch closure, day 0<br />
Veraison, day 27<br />
Preharvest, day 62<br />
Harvest, day 78<br />
Row C<br />
Figure 6. Survival of Aspergillus carbonarius <strong>in</strong>oculated on the surface of Shiraz<br />
berries at pre-bunch closure.
Black Aspergillus Species <strong>in</strong> Australian V<strong>in</strong>eyards 165<br />
sporulation by the mould. A slight but statistically significant decrease<br />
<strong>in</strong> the counts of A. carbonarius was also observed on Chardonnay<br />
bunches <strong>in</strong>oculated 11 days before harvest (Figure 7). There were no<br />
fungicide sprays applied dur<strong>in</strong>g this period, hence death of spores<br />
could be attributed to residual fungicide activity and/or exposure to<br />
UV light. Samples with <strong>in</strong>creased A. carbonarius counts had foci of<br />
<strong>in</strong>fection and sporulation on some berries.<br />
These results suggest that the critical time for the development of<br />
Aspergillus rots occurs from veraison onwards. Battilani et al. (2003a)<br />
and Serra et al. (2003) both noted black Aspergillus spp. were more<br />
frequently isolated from berries after veraison.<br />
3.3. W<strong>in</strong>emak<strong>in</strong>g<br />
HPLC analysis of samples taken throughout v<strong>in</strong>ification showed<br />
that the greatest reduction <strong>in</strong> OA was observed at press<strong>in</strong>g, as the<br />
mean concentration of OA <strong>in</strong> white juice was 26% of the concentration<br />
<strong>in</strong> crushed grapes (must); the correspond<strong>in</strong>g figure for Shiraz was<br />
28% (Figure 8). This suggests that there is a strong association of OA<br />
with the sk<strong>in</strong>s and seeds (marc) trapped dur<strong>in</strong>g press<strong>in</strong>g. Clarification<br />
of white juice with pect<strong>in</strong>ase and precipitation of solids overnight<br />
resulted <strong>in</strong> a mean reduction <strong>in</strong> OA of an additional 12%, with<strong>in</strong> the<br />
range observed for precipitation of must sediments dur<strong>in</strong>g <strong>in</strong>dustrial<br />
A. carbonarius count (cfu per berry)<br />
100,000<br />
10,000<br />
1,000<br />
100<br />
10<br />
1<br />
Row A<br />
Row B<br />
Preharvest, day 0<br />
Harvest, day 11<br />
Row C<br />
Figure 7. Survival of Aspergillus carbonarius <strong>in</strong>oculated on the surface of<br />
Chardonnay berries preharvest. The difference between A. carbonarius spore count per<br />
berry at preharvest and harvest was significant at P < 0.05.
166 Su-l<strong>in</strong> L. Leong et al.<br />
Proportion of OA relative to<br />
<strong>in</strong>itial concentration <strong>in</strong> grapes<br />
(a)<br />
Proportion of OA relative to<br />
<strong>in</strong>itial concentration <strong>in</strong> grapes<br />
(b)<br />
100%<br />
80%<br />
60%<br />
40%<br />
20%<br />
0%<br />
Variety, Tox<strong>in</strong> level (V<strong>in</strong>tage)<br />
Must Juice Clarified juice At first rack<strong>in</strong>g Bottled w<strong>in</strong>e W<strong>in</strong>e after 14 mth<br />
100%<br />
80%<br />
60%<br />
40%<br />
20%<br />
0%<br />
Chard, <strong>in</strong>termed<br />
(2002)<br />
Intermed (2002)<br />
Chard, high<br />
(2002)<br />
High (2002)<br />
Sem, <strong>in</strong>termed<br />
(2003)<br />
Intermed (2003)<br />
Tox<strong>in</strong> level (V<strong>in</strong>tage)<br />
Sem, high<br />
(2003)<br />
High (2003)<br />
Must After press<strong>in</strong>g At first rack<strong>in</strong>g Bottled w<strong>in</strong>e W<strong>in</strong>e after 14 mth<br />
Figure 8. Reduction <strong>in</strong> ochratox<strong>in</strong> A dur<strong>in</strong>g a. white and b. red w<strong>in</strong>e production. The<br />
white varieties used were Chardonnay (Chard) and Semillon (Sem) and the red variety<br />
was Shiraz. Berries which had been crushed and destemmed were termed “must”,<br />
and for this study, were deemed to conta<strong>in</strong> the total ochratox<strong>in</strong> A <strong>in</strong>itially present.<br />
Only w<strong>in</strong>es from 2002 v<strong>in</strong>tage had been stored for 14 months at the time of analysis,<br />
hence these results are absent for the 2003 v<strong>in</strong>tage.
Black Aspergillus Species <strong>in</strong> Australian V<strong>in</strong>eyards 167<br />
storage of grape juice (Roset, 2003). In most white and red ferments,<br />
rack<strong>in</strong>g (decant<strong>in</strong>g w<strong>in</strong>e after fermentation) also reduced OA content.<br />
These reductions may be due to loose <strong>in</strong>teractions between OA and<br />
the solids settl<strong>in</strong>g out of solution. The mean OA concentration of<br />
white and red w<strong>in</strong>es, respectively, at bottl<strong>in</strong>g was 4% and 13% of the<br />
<strong>in</strong>itial concentration <strong>in</strong> grapes. Slight reductions <strong>in</strong> OA were observed<br />
<strong>in</strong> red and white w<strong>in</strong>es after 14 months of storage. These trends <strong>in</strong><br />
reduction <strong>in</strong> OA were apparently unaffected by the <strong>in</strong>itial concentration<br />
of OA <strong>in</strong> the grapes, which varied <strong>in</strong> the two v<strong>in</strong>tages. Initial OA<br />
concentrations fell with<strong>in</strong> the range 2-66 ng/g for white grapes and<br />
2-114 ng/g for red grapes.<br />
The reduction <strong>in</strong> OA concentration at press<strong>in</strong>g was similar for both<br />
white and red w<strong>in</strong>es (approximately 70%), however, at bottl<strong>in</strong>g, red<br />
w<strong>in</strong>es reta<strong>in</strong>ed three fold more of the <strong>in</strong>itial OA concentration than<br />
white w<strong>in</strong>es. This difference may be <strong>in</strong>herent to the v<strong>in</strong>ification<br />
process. In white v<strong>in</strong>ification, the juice after press<strong>in</strong>g does not conta<strong>in</strong><br />
alcohol, and OA may readily b<strong>in</strong>d to prote<strong>in</strong>s and other solids <strong>in</strong> the<br />
juice, to be removed dur<strong>in</strong>g clarification. In red v<strong>in</strong>ification, the w<strong>in</strong>e<br />
after press<strong>in</strong>g conta<strong>in</strong>s alcohol and hence OA present is less bound to<br />
solids and more soluble <strong>in</strong> the liquid phase. Thus rack<strong>in</strong>g of red w<strong>in</strong>es<br />
after fermentation does not result <strong>in</strong> the same reduction <strong>in</strong> OA as<br />
clarification of white juice. Also, dur<strong>in</strong>g fermentation of red must on<br />
sk<strong>in</strong>s, there may be <strong>in</strong>creased partition<strong>in</strong>g of OA from the pulp and<br />
sk<strong>in</strong>s <strong>in</strong>to the alcohol produced dur<strong>in</strong>g fermentation.<br />
Fernandes et al. (2003) reported the opposite effect, with white<br />
w<strong>in</strong>es reta<strong>in</strong><strong>in</strong>g a greater proportion of <strong>in</strong>itial OA than red w<strong>in</strong>es<br />
(8-14% cf. 6%). This difference can be expla<strong>in</strong>ed by not<strong>in</strong>g that OA<br />
that has been spiked <strong>in</strong>to crushed grapes may <strong>in</strong>teract differently with<br />
grape solids compared with OA exuded directly <strong>in</strong> the berries from<br />
fungal hyphae. However, the importance of solid-liquid separations <strong>in</strong><br />
the removal of OA dur<strong>in</strong>g v<strong>in</strong>ification is clear, regardless of the means<br />
of OA contam<strong>in</strong>ation.<br />
3.4. Future Directions<br />
An understand<strong>in</strong>g of the source of A. carbonarius and other members<br />
of Section Nigri <strong>in</strong> v<strong>in</strong>eyards may aid <strong>in</strong> the development of management<br />
strategies to m<strong>in</strong>imise dispersal from the soil to bunches.<br />
Reduc<strong>in</strong>g the frequency of tillage may be one strategy to m<strong>in</strong>imise<br />
Aspergillus <strong>in</strong> the soil and air. The presence of A. carbonarius spores on<br />
bunches dur<strong>in</strong>g the early stages of berry development does not<br />
necessarily lead to the development of Aspergillus rots and subsequent
168 Su-l<strong>in</strong> L. Leong et al.<br />
production of OA, as the spores do not survive well on the surface of<br />
green berries. Berry soften<strong>in</strong>g from veraison onwards appears to<br />
<strong>in</strong>crease berry susceptibility to Aspergillus rots. Fungicide sprays are<br />
the primary tool for management of Aspergillus rots <strong>in</strong> matur<strong>in</strong>g<br />
bunches, however, Australia has strict guidel<strong>in</strong>es govern<strong>in</strong>g their use <strong>in</strong><br />
the weeks before harvest to m<strong>in</strong>imise chemical residues <strong>in</strong> the w<strong>in</strong>e.<br />
The efficacy and tim<strong>in</strong>g of these sprays is under <strong>in</strong>vestigation, as part<br />
of an overall strategy to reduce the <strong>in</strong>cidence of Aspergillus <strong>in</strong> v<strong>in</strong>eyards.<br />
Studies of the fate of OA dur<strong>in</strong>g v<strong>in</strong>ification are also cont<strong>in</strong>u<strong>in</strong>g,<br />
to <strong>in</strong>crease understand<strong>in</strong>g of the relative proportion of OA<br />
rema<strong>in</strong><strong>in</strong>g <strong>in</strong> w<strong>in</strong>e after v<strong>in</strong>ification under Australian conditions.<br />
This has implications for sett<strong>in</strong>g acceptable limits for OA <strong>in</strong> w<strong>in</strong>egrapes<br />
at harvest, and also for further process<strong>in</strong>g of waste streams<br />
from v<strong>in</strong>ification.<br />
4. ACKNOWLEDGEMENTS<br />
This research was supported by the Australian Government and<br />
Australian grapegrowers and w<strong>in</strong>emakers through their <strong>in</strong>vestment <strong>in</strong><br />
the Cooperative Research Centre for Viticulture and Horticulture<br />
Australia Ltd. Support from grape growers <strong>in</strong> the Sunraysia district,<br />
Victoria, and Glen Howard of Somerset V<strong>in</strong>eyard, Pokolb<strong>in</strong> (Hunter<br />
Valley, New South Wales) is gratefully acknowledged, as is the contribution<br />
from Syngenta Crop Protection Pty Ltd. Narelle Nancarrow,<br />
Kathy Clarke and Margaret Leong are thanked for their help with the<br />
field trials. W<strong>in</strong>emak<strong>in</strong>g was carried out with assistance from Mark<br />
Krstic, Glenda Kelly and Fred Hancock at the Victorian Department of<br />
Primary Industries, Mildura, Victoria, and Stephen W. White, Nai<br />
Tran-D<strong>in</strong>h and Nick Charley at <strong>Food</strong> Science Australia. The key role of<br />
Peter Varelis, Kylie McClelland, Shane Cameron, Kathy Schneebeli<br />
(Analytical Chemistry, <strong>Food</strong> Science Australia) <strong>in</strong> development of the<br />
OA assays is most gratefully acknowledged. Advice on the statistical<br />
analyses was provided by John Reynolds (formerly Senior Biometrician,<br />
Victorian Department of Primary Industries, Attwood, Victoria).<br />
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MacDonald, S., Wilson, P., Barnes, K., Damant, A., Massey, R., Mortby, E., and<br />
Shepherd, M. J., 1999, Ochratox<strong>in</strong> A <strong>in</strong> dried v<strong>in</strong>e fruit: method development and<br />
survey, <strong>Food</strong> Addit. Contam. 16:253-260.<br />
Majerus, P., Bresch, H., and Otteneder, H., 2000, Ochratox<strong>in</strong> <strong>in</strong> w<strong>in</strong>es, fruit juices and<br />
season<strong>in</strong>gs, Arch. Lebensmittelhyg. 51:81-128.<br />
Marfen<strong>in</strong>a, O. E., and Mirch<strong>in</strong>k, T. G., 1989, Effect of human activity on soil microfungi,<br />
Sov. Soil Sci. 21:40-47.<br />
Markarki, P., Delpont-B<strong>in</strong>et, C., Grosso, F., and Dragacci, S., 2001, Determ<strong>in</strong>ation of<br />
ochratox<strong>in</strong> A <strong>in</strong> red w<strong>in</strong>e and v<strong>in</strong>egar by immunoaff<strong>in</strong>ity high-pressure liquid chromatography,<br />
J. <strong>Food</strong> Prot. 64:533-537.<br />
Nair, N. G., 1985, Fungi associated wtih bunch rot of grapes <strong>in</strong> the Hunter Valley,<br />
Aust. J. Agric. Res. 36:435-442.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, 2nd edition, Blackie<br />
Academic and Professional, London, pp. 385-388, 510-511.<br />
Roset, M., 2003, Survey on ochratox<strong>in</strong> A <strong>in</strong> grape juice, Fruit Process. 13:167-172.<br />
Rotem, J., and Aust, H. J., 1991, The effect of ultraviolet and solar radiation and temperature<br />
on survival of fungal propagules, J. Phytopathol. 133:76-84.<br />
Sage, L., Krivobok, S., Delbos, É., Seigle-Murandi, F., and Creppy, E. E., 2002,<br />
Fungal flora and ochratox<strong>in</strong> A production <strong>in</strong> grapes and musts from France,<br />
J. Agric. <strong>Food</strong> Chem. 50:1306-1311.<br />
Serra, R., Abrunhosa, L., Kozakiewicz, Z., and Venâncio, A., 2003, Black Aspergillus<br />
species as ochratox<strong>in</strong> A producers <strong>in</strong> Portuguese w<strong>in</strong>e grapes, Int. J. <strong>Food</strong><br />
Microbiol. 88:63-68.<br />
Silva, A., Galli, R., Grazioli, B., and Fumi, M. D., 2003, Metodi di riduzione<br />
di residui di ocratoss<strong>in</strong>a A nei v<strong>in</strong>i, Ind. Bevande 32:467-472.<br />
Snowdon, A. L., 1990, A Colour Atlas of Post-Harvest Diseases and Disorders of Fruit<br />
and Vegetables. I. General Introduction and Fruits, Wolfe Scientific, London, p. 256.
Black Aspergillus Species <strong>in</strong> Australian V<strong>in</strong>eyards 171<br />
Téren, J., Varga, J., Hamari, Z., R<strong>in</strong>yu, E., and Kevei, F., 1996, Immunochemical<br />
detection of ochratox<strong>in</strong> A <strong>in</strong> black Aspergillus stra<strong>in</strong>s, Mycopathologia 134:<br />
171-176.<br />
Tjamos, S. E., Antoniou, P. P., Kazantzidou, A., Antonopoulos, D. F., Papageorgiou,<br />
I., and Tjamos, E. C., 2004, Aspergillus niger and Aspergillus carbonarius <strong>in</strong><br />
Cor<strong>in</strong>th rais<strong>in</strong> and w<strong>in</strong>e-produc<strong>in</strong>g v<strong>in</strong>eyards <strong>in</strong> Greece: population composition,<br />
ochratox<strong>in</strong> A production and chemical control, J. Phytopathol. 152:250-255.<br />
Zimmerli, B., and Dick, R., 1996, Ochratox<strong>in</strong> A <strong>in</strong> table w<strong>in</strong>e and grape-juice:<br />
occurrence and risk assessment, <strong>Food</strong> Addit. Contam. 13:655-668.
OCHRATOXIN A PRODUCING FUNGI FROM<br />
SPANISH VINEYARDS<br />
Marta Bau, M. Rosa Bragulat, M. Lourdes Abarca,<br />
Santiago M<strong>in</strong>guez and F. Javier Cabañes *<br />
1. INTRODUCTION<br />
Ochratox<strong>in</strong> A (OA) is a nephrotoxic mycotox<strong>in</strong> naturally occurr<strong>in</strong>g<br />
<strong>in</strong> a wide range of food commodities. It has been classified by IARC<br />
as a possible human renal carc<strong>in</strong>ogen (group 2B) (Castegnaro and<br />
Wild, 1995) and among other toxic effects, is teratogenic, immunotoxic,<br />
genotoxic, mutagenic and carc<strong>in</strong>ogenic (Creppy, 1999). W<strong>in</strong>e is<br />
considered the second major source of OA <strong>in</strong> Europe, with cereals<br />
be<strong>in</strong>g the primary source. S<strong>in</strong>ce the first report on the occurrence of<br />
OA <strong>in</strong> w<strong>in</strong>e (Zimmerli and Dick, 1996) its presence <strong>in</strong> w<strong>in</strong>e and grape<br />
juice have been reported <strong>in</strong> a broad variety of w<strong>in</strong>es from different<br />
orig<strong>in</strong>s. Maximum OA levels have been established for cereals and<br />
dried v<strong>in</strong>e fruits <strong>in</strong> the European Union, and it is possible that other<br />
commodities such as w<strong>in</strong>e and grape juices will be regulated before the<br />
end of 2003 (Anonymous, 2002).<br />
Until recently, Aspergillus ochraceus and Penicillium verrucosum<br />
were considered the ma<strong>in</strong> OA-produc<strong>in</strong>g species. P. verrucosum is usually<br />
found <strong>in</strong> cool temperate regions and has been reported almost<br />
exclusively <strong>in</strong> cereal and cereal products while A. ochraceus is found<br />
*Marta Bau, M. Rosa Bragulat, M. Lourdes Abarca and F. Javier Cabañes:<br />
Departament de Sanitat i d’Anatomia Animals, Universitat Autònoma de Barcelona,<br />
08193 Bellaterra, Barcelona, Spa<strong>in</strong>. Santiago M<strong>in</strong>guez: Institut Català de la V<strong>in</strong>ya i el<br />
Vi (INCAVI), Generalitat de Catalunya, Vilafranca del Penedés, Barcelona, Spa<strong>in</strong>.<br />
Correspondence to: Javier.Cabanes@uab.es<br />
173
174 Marta Bau et al.<br />
sporadically <strong>in</strong> different commodities <strong>in</strong> warmer and tropical climates<br />
(Pitt and Hock<strong>in</strong>g, 1997). The production of OA by species <strong>in</strong><br />
Aspergillus section Nigri has received considerable attention s<strong>in</strong>ce the<br />
first description of OA production by Aspergillus niger var. niger<br />
(Abarca et al., 1994) and by Aspergillus carbonarius (Horie, 1995).<br />
Recently, A. carbonarius and other black aspergilli belong<strong>in</strong>g to the<br />
A. Niger aggregate have been described as a ma<strong>in</strong> possible sources of<br />
OA contam<strong>in</strong>ation <strong>in</strong> grapes (Da Rocha Rosa et al., 2002; Sage et al.,<br />
2002; Battilani et al., 2003; Magnoli et al., 2003; Serra et al., 2003),<br />
w<strong>in</strong>e (Cabañes et al., 2002), and also <strong>in</strong> dried v<strong>in</strong>e fruits (Heenan et al.,<br />
1998; Abarca et al., 2003).<br />
The objective of this study was to identify the ochratoxigenic mycobiota<br />
of grapes from v<strong>in</strong>eyards ma<strong>in</strong>ly located along the<br />
Mediterranean coast of Spa<strong>in</strong>.<br />
2. MATERIALS AND METHODS<br />
2.1. Samples<br />
Dur<strong>in</strong>g the 2001 and 2002 seasons, fungi capable of produc<strong>in</strong>g<br />
ochratox<strong>in</strong> A were isolated from the grapes from seven Spanish v<strong>in</strong>eyards.<br />
The v<strong>in</strong>eyards were located ma<strong>in</strong>ly along the Mediterranean<br />
coast and belonged to five w<strong>in</strong>emak<strong>in</strong>g regions: Barcelona (two v<strong>in</strong>eyards),<br />
Tarragona (two v<strong>in</strong>eyards), Valencia (one v<strong>in</strong>eyard), Murcia<br />
(one v<strong>in</strong>eyard) and Cádiz (one v<strong>in</strong>eyard). In each v<strong>in</strong>eyard, from May<br />
to October, sampl<strong>in</strong>gs were made at four different times, co<strong>in</strong>cid<strong>in</strong>g<br />
with the follow<strong>in</strong>g developmental stages of the grape: sett<strong>in</strong>g, one<br />
month after berry-set, veraison and harvest<strong>in</strong>g. At each sampl<strong>in</strong>g<br />
time, 10 bunches were collected from 10 different plants located<br />
approximately along two cross<strong>in</strong>g diagonals of the v<strong>in</strong>eyard. Every<br />
bunch was collected <strong>in</strong> a separate paper bag and analyzed <strong>in</strong> the laboratory<br />
with<strong>in</strong> 24-48 h of collection.<br />
2.2. Mycological study<br />
Ten berries from each bunch were randomly selected, with five<br />
plated directly onto dichloran rose bengal chloramphenicol agar<br />
(DRBC) (Pitt and Hock<strong>in</strong>g, 1997) and five onto malt extract agar<br />
(MEA) (Pitt and Hock<strong>in</strong>g, 1997) supplemented with 100 ppm of chloramphenicol<br />
and 50 ppm of streptomyc<strong>in</strong>. In total, 5,600 berries were
Ochratox<strong>in</strong> A Produc<strong>in</strong>g Fungi from Spanish V<strong>in</strong>eyards 175<br />
analyzed. Plates were <strong>in</strong>cubated at 25°C for 7 days. All fungi belong<strong>in</strong>g<br />
to Aspergillus and Penicillium genera were isolated for identification<br />
to species level. (Raper and Fennell, 1965; Pitt, 1979; Klich and<br />
Pitt, 1988; Pitt and Hock<strong>in</strong>g, 1997).<br />
2.3. Ability of Fungal Isolates to Produce Ochratox<strong>in</strong><br />
Isolates belong<strong>in</strong>g to Aspergillus spp. and Penicillium spp. were<br />
evaluated us<strong>in</strong>g a previously described HPLC screen<strong>in</strong>g method<br />
(Bragulat et al., 2001). Briefly, the isolates were grown on Czapek<br />
Yeast extract Agar (CYA) and on Yeast extract Sucrose agar (YES)<br />
(Pitt and Hock<strong>in</strong>g, 1997) and <strong>in</strong>cubated at 25°C for 7 days. Isolates<br />
identified as A. carbonarius were grown on CYA for 10 days at 30°C<br />
because these <strong>in</strong>cubation conditions have been cited as optimal for<br />
detect<strong>in</strong>g OA production <strong>in</strong> this species (Cabañes et al., 2002; Abarca<br />
et al., 2003). From each isolate, three agar plugs were removed from<br />
different po<strong>in</strong>ts of the colony and extracted with 0.5 ml of methanol.<br />
The extracts were filtered and <strong>in</strong>jected <strong>in</strong>to the HPLC.<br />
2.4. Data analysis<br />
Data obta<strong>in</strong>ed were analyzed statistically by means of one-way<br />
analysis of variance test and Student’s test. All statistical analyses<br />
were performed us<strong>in</strong>g SPSS software (version 10.0).<br />
3. RESULTS AND DISCUSSION<br />
The occurrence of Aspergillus spp. <strong>in</strong> the 5,600 berries plated on<br />
the two culture media used are shown <strong>in</strong> Table 1. Although the number<br />
of isolates recovered on DRBC medium was higher than on MEA,<br />
the differences were not statistically significant. A total of 1,061 isolates<br />
belong<strong>in</strong>g to twenty Aspergillus spp. (<strong>in</strong>clud<strong>in</strong>g Emericella spp.<br />
and Eurotium amstelodami) were identified. Isolates of A. carbonarius<br />
and A. niger aggregate constituted 88.7% of the total Aspergillus isolates<br />
(Figure 1). Aspergillus niger aggregate were isolated from 14.2%<br />
of plated berries, and A. carbonarius from 2.6%. The occurrence of the<br />
rema<strong>in</strong><strong>in</strong>g Aspergillus spp. ranged from 0.02% to 0.5%.<br />
The distribution of the A. niger aggregate and A. carbonarius isolates<br />
<strong>in</strong> 2001 and 2002 seasons dur<strong>in</strong>g the development of berries is<br />
shown <strong>in</strong> Figure 2. Although they were recovered <strong>in</strong> all the stages
176 Marta Bau et al.<br />
Table 1. Occurrence of Aspergillus spp. <strong>in</strong> grapes from Spanish v<strong>in</strong>eyards exam<strong>in</strong>ed<br />
dur<strong>in</strong>g the 2001 and 2002 seasons<br />
No. (%) of positive berries<br />
Total DRBCa MEAa Species (n = 5,600) (n = 2,800) (n = 2,800)<br />
Aspergillus niger aggregate 797 (14.23) 438 359<br />
A. carbonarius 144 (2.57) 83 61<br />
A. ustus 26 (0.46) 15 11<br />
A. fumigatus 19 (0.34) 10 9<br />
A. flavus 11 (0.20) 6 5<br />
A. tamarii 9 (0.16) 4 5<br />
A. japonicus var. aculeatus 8 (0.14) 3 5<br />
A. ochraceus 8 (0.14) 8 0<br />
Emericella nidulans 6 (0.11) 4 2<br />
A. alliaceus 5 (0.09) 4 1<br />
A. terreus 5 (0.09) 4 1<br />
A. wentii 5 (0.09) 3 2<br />
A. melleus 4 (0.07) 4 0<br />
A. flavipes 3 (0.05) 2 1<br />
A. ostianus 3 (0.05) 3 0<br />
A. parasiticus 3 (0.05) 2 1<br />
Eurotium amstelodami 2 (0.04) 1 1<br />
Emericella astellata 1 (0.02) 0 1<br />
Emericella variecolor 1 (0.02) 0 1<br />
A. versicolor 1 (0.02) 0 1<br />
Total Aspergillus spp. 1061 594 467<br />
aDRBC: dichloran rose bengal chloramphenicol agar; MEA: malt extract agar.<br />
sampled, there was a statistically significant <strong>in</strong>crease at harvest<strong>in</strong>g.<br />
The number of isolates recovered <strong>in</strong> 2002 was lower than <strong>in</strong> 2001,<br />
probably due to different climatic conditions. Nevertheless <strong>in</strong> both<br />
seasons black Aspergilli showed the same tendency, with the highest<br />
levels of isolation at harvest<strong>in</strong>g.<br />
A total of 165 isolates belong<strong>in</strong>g to genus Penicillium were identified.<br />
The most frequent species were P. glabrum, P. brevicompactum,<br />
P. sclerotiorum, P. citr<strong>in</strong>um, P. chrysogenum and P. thomii. The occurrence<br />
of the rema<strong>in</strong><strong>in</strong>g Penicillium spp. was lower than 0.12%. OA production<br />
was not detected by any of the 165 Penicillium isolates. Only<br />
one isolate of P. verrucosum was identified. This isolates was able to<br />
produce citr<strong>in</strong><strong>in</strong> but did not produce OA.<br />
The ability of Aspergillus isolates to produce ochratox<strong>in</strong> A is shown<br />
<strong>in</strong> Table 2. All the A. carbonarius isolates (n = 144) were able to produce<br />
OA whereas only eight isolates of A. niger aggregate (n = 797) were<br />
toxigenic. None of the A. japonicus var. aculeatus (n = 8) produced OA.
Ochratox<strong>in</strong> A Produc<strong>in</strong>g Fungi from Spanish V<strong>in</strong>eyards 177<br />
Figure 1. Black aspergilli grow<strong>in</strong>g on plated berries from harvest<strong>in</strong>g time. (Note their<br />
high occurrence at this sampl<strong>in</strong>g time).<br />
No. of isolates<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
A. carbonarius 2001 A. carbonarius 2002<br />
A. niger aggregate 2001 A. niger aggregate 2002<br />
I II III IV<br />
Figure 2. Distribution of A. carbonarius and A. niger aggregate isolates at each developmental<br />
stages of the berries: I, berry set; II, one month after berry set; III, veraison;<br />
IV, harvest
178 Marta Bau et al.<br />
Table 2. Ochratox<strong>in</strong> A production (µg/g of culture medium) by Aspergillus spp. isolated<br />
from Spanish grapes<br />
No. of positive Mean<br />
Species isolates /Total concentration Range<br />
A. carbonarius 144 / 144 24.6 0.1 – 378.5<br />
A. niger aggregate 8 / 797 29.2 0.05 – 230.9<br />
A. alliaceus 5 / 5 351.4 197.6 – 715.4<br />
A. ochraceus 4 / 8 440.8 1.3 – 1026.7<br />
A. ostianus 3 / 3 1273.3 245.9 – 2514.1<br />
A. melleus 2 / 4 19.7 7.3 – 32.2<br />
OA production was also detected by other Aspergillus species outside<br />
section Nigri. Four of the eight isolates of A. ochraceus and two of<br />
the four isolates of A. melleus produced OA. All isolates classified as A.<br />
ostianus (n=3) and A. alliaceus (n=5) were able to produce OA. Some of<br />
these species were able to produce OA <strong>in</strong> large quantities <strong>in</strong> pure culture,<br />
but due to their low occurrence, they are probably a relatively unimportant<br />
source of this mycotox<strong>in</strong> <strong>in</strong> grapes.<br />
Although the possible participation of different OA produc<strong>in</strong>g<br />
species may occur, our results are strong evidence of the contribution<br />
of A. carbonarius to OA contam<strong>in</strong>ation <strong>in</strong> grapes, ma<strong>in</strong>ly at the f<strong>in</strong>al<br />
developmental stage of the berries, and consequently <strong>in</strong> w<strong>in</strong>e.<br />
4. ACKNOWLEDGEMENTS<br />
This research was supported by the European Union project<br />
QLK1-CT-2001-01761 (Quality of Life and Management of Liv<strong>in</strong>g<br />
Resources Programme (QoL), Key Action 1 on <strong>Food</strong>, Nutrition and<br />
Health). The f<strong>in</strong>ancial support of the M<strong>in</strong>isterio de Ciencia y<br />
Tecnología of the Spanish Government (AGL01-2974-C05-03) is also<br />
acknowledged.<br />
5. REFERENCES<br />
Abarca, M. L., Bragulat, M. R., Castellá, G., and Cabañes, F. J., 1994, Ochratox<strong>in</strong> A<br />
production by stra<strong>in</strong>s of Aspergillus niger var. niger, Appl. Environ. Microbiol.<br />
60:2650-2652.<br />
Abarca, M. L., Accensi, F., Bragulat, M. R., Castellá. G., and Cabañes, F. J., 2003,<br />
Aspergillus carbonarius as the ma<strong>in</strong> source of ochratox<strong>in</strong> A contam<strong>in</strong>ation <strong>in</strong> dried<br />
v<strong>in</strong>e fruits from the Spanish market, J. <strong>Food</strong> Prot. 66:504-506.
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Anonymous, 2002. Commission regulation (EC) no 472/2002 of 12 March 2002<br />
amend<strong>in</strong>g regulation (EC) no 466/2001 sett<strong>in</strong>g maximum levels for certa<strong>in</strong> contam<strong>in</strong>ants<br />
<strong>in</strong> foodstuffs, Off. J. Eur. Communities L75, 18-20.<br />
Battilani, P., Pietri, A., Bertuzzi, T., Languasco, L., Giorni, P., and Kozakiewicz, Z.,<br />
2003, Occurrence of ochratox<strong>in</strong> A-produc<strong>in</strong>g fungi <strong>in</strong> grapes grown <strong>in</strong> Italy,<br />
J. <strong>Food</strong> Prot. 66:633-636.<br />
Bragulat, M. R., Abarca, M. L., and Cabañes, F. J., 2001, An easy screen<strong>in</strong>g method<br />
for fungi produc<strong>in</strong>g ochratox<strong>in</strong> A <strong>in</strong> pure culture, Int J. <strong>Food</strong> Microbiol. 71:<br />
139-144.<br />
Cabañes, F. J., Accensi, F., Bragulat, M. R., Abarca, M. L., Castellá, G., M<strong>in</strong>guez, S.,<br />
and Pons, A., 2002, What is the source of ochratox<strong>in</strong> A <strong>in</strong> w<strong>in</strong>e?, Int J. <strong>Food</strong><br />
Microbiol. 79:213-215.<br />
Castegnaro, M., and Wild, C. P., 1995, IARC activities <strong>in</strong> mycotox<strong>in</strong> research, Natural<br />
Tox<strong>in</strong>s 3:327-331.<br />
Creppy, E. E., 1999. Human ochratoxicoses. J. Toxicol. – Tox<strong>in</strong> Rev. 18:273-293.<br />
Da Rocha Rosa, C. A., Palacios, V., Comb<strong>in</strong>a, M., Fraga, M. E., De Oliveira Rekson,<br />
A., Magnoli, C. E., amd Dalcero, A. M., 2002, Potential ochratox<strong>in</strong> A producers<br />
from w<strong>in</strong>e grapes <strong>in</strong> Argent<strong>in</strong>a and Brazil. <strong>Food</strong> Addit. Contam. 19:408-414.<br />
Heenan, C. N., Shaw, K. J., and Pitt, J. I., 1998, Ochratox<strong>in</strong> A production by<br />
Aspergillus carbonarius and A. niger isolates and detection us<strong>in</strong>g coconut cream<br />
agar, J. <strong>Food</strong> Mycol. 1:67-72.<br />
Horie, Y., 1995, Productivity of ochratox<strong>in</strong> A of Aspergillus carbonarius <strong>in</strong> Aspergillus<br />
section Nigri. Nippon K<strong>in</strong>gakukai Kaiho 36:73-76.<br />
Klich, M. A., and Pitt, J. I., 1988, A Laboratory Guide to Common Aspergillus Species<br />
and their Teleomorphs. CSIRO Division of <strong>Food</strong> Process<strong>in</strong>g, North Ryde, NSW.<br />
Magnoli, C., Violante, M., Comb<strong>in</strong>a, M., Palacio, G., and Dalcero, A., 2003,<br />
Mycoflora and ochratox<strong>in</strong>-produc<strong>in</strong>g stra<strong>in</strong>s of Aspergillus section Nigri <strong>in</strong> w<strong>in</strong>e<br />
grapes <strong>in</strong> Argent<strong>in</strong>a, Lett. Appl. Microbiol 37:179-184.<br />
Pitt, J. I., 1979, The Genus Penicillium and its Teleomorphic States Eupenicillium and<br />
Talaromyces, Academic Press, London.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, Blackie Academic and<br />
Professional, London.<br />
Raper, K. B., and Fennell, D. I., 1965, The Genus Aspergillus, The William and<br />
Wilk<strong>in</strong>s Co., Baltimore.<br />
Sage, L., Krivobok, S., Delbos, E., Seigle-Murandi, F., and Creppy, E. E., 2002,<br />
Fungal flora and ochratox<strong>in</strong> A production <strong>in</strong> grapes and musts from France,<br />
J. Agric. <strong>Food</strong> Chem. 50:1306-1311.<br />
Serra, R., Abrunhosa, L., Kozakiewicz, Z., and Venancio, A., 2003, Black Aspergillus<br />
species as ochratox<strong>in</strong> A producers <strong>in</strong> Portuguese w<strong>in</strong>e grapes, Int. J. <strong>Food</strong><br />
Microbiol. 88:63-68.<br />
Zimmerli, B., and Dick, R., 1996, Ochratox<strong>in</strong> A <strong>in</strong> table w<strong>in</strong>e and grape-juice: occurrence<br />
and risk assessment, <strong>Food</strong> Addit. Contam. 13:655-668.
FUNGI PRODUCING OCHRATOXIN IN<br />
DRIED FRUITS<br />
Beatriz T. Iamanaka, Marta H. Taniwaki, E. Vicente and<br />
Hilary C. Menezes *<br />
1. INTRODUCTION<br />
Ochratox<strong>in</strong> A (OA) has been shown to be a potent nephrotox<strong>in</strong> <strong>in</strong><br />
animal species and has been found <strong>in</strong> agricultural products. OA is<br />
believed to be produced <strong>in</strong> nature by three ma<strong>in</strong> species of fungi,<br />
Aspergillus ochraceus, Aspergillus carbonarius and Penicillium verrucosum,<br />
with a m<strong>in</strong>or contribution from A. niger and several species<br />
closely related to A. ochraceus (JECFA, 2001). P. verrucosum occur<br />
ma<strong>in</strong>ly <strong>in</strong> cool temperate climates, and is usually associated with cereals<br />
(Pitt, 1987; Pitt and Hock<strong>in</strong>g, 1997).<br />
Studies carried out <strong>in</strong> Europe have reported the presence of the<br />
ochratoxigenic fungi A. ochraceus, A. niger and A. carbonarius and<br />
sometimes OA <strong>in</strong> dried fruits (MAFF, 2002). A. carbonarius and<br />
A. niger were described as sources of OA <strong>in</strong> matur<strong>in</strong>g and dry<strong>in</strong>g grapes<br />
<strong>in</strong> Spa<strong>in</strong> and Australia (Abarca et al., 1994; Heenan et al., 1998).<br />
Grape juice and w<strong>in</strong>es from southern regions of Europe have been<br />
reported to conta<strong>in</strong> detectable levels of OA (Zimmerli and Dick,<br />
1996). Detectable concentrations of OA have been found <strong>in</strong> sultanas<br />
imported <strong>in</strong>to the United K<strong>in</strong>gdom: a survey of 20 samples of dried<br />
fruit found more than 80% were positive for OA (MAFF, 1997;<br />
MacDonald et al., 1999).<br />
*Beatriz T. Iamanaka, Marta H. Taniwaki and E. Vicente: <strong>Food</strong> Technology<br />
Institute, ITAL C.P 139 CEP13.073-001 Camp<strong>in</strong>as-SP, Brazil; Hilary C. Menezes:<br />
<strong>Food</strong> Eng<strong>in</strong>eer<strong>in</strong>g Faculty-Unicamp, Camp<strong>in</strong>as-SP, Brazil. Correspondence to:<br />
mtaniwak@ital.sp.gov.br<br />
181
182 Beatriz T. Iamanaka et al.<br />
The aim of this work was to <strong>in</strong>vestigate the <strong>in</strong>cidence of toxigenic<br />
fungi and OA <strong>in</strong> dried fruits from different countries of orig<strong>in</strong> sold on<br />
the Brazilian market.<br />
2. MATERIALS AND METHODS<br />
2.1. Sampl<strong>in</strong>g<br />
A total of 119 samples (500 g each) of dried fruits were purchased<br />
<strong>in</strong> Camp<strong>in</strong>as and São Paulo markets <strong>in</strong> Brazil <strong>in</strong> 2002-2003, compris<strong>in</strong>g<br />
black sultanas (24), white sultanas (19), apricots (14), figs (19),<br />
dates (22) and plums(21). The dried fruit samples orig<strong>in</strong>ated from<br />
Turkey, Spa<strong>in</strong>, Mexico, Tunisia, USA, Argent<strong>in</strong>a and Chile.<br />
2.2. Mycological Analyses<br />
Larger fruit (apricots, figs, dates and plums) were cut aseptically <strong>in</strong>to<br />
small pieces, whereas smaller fruit (sultanas) were analysed whole. Whole<br />
fruit or fruit pieces were surface dis<strong>in</strong>fected with 0.4% chlor<strong>in</strong>e solution<br />
for 1 m<strong>in</strong>. Fifty sultanas or fruit pieces were plated onto Dichloran 18%<br />
Glycerol agar (DG18; Pitt and Hock<strong>in</strong>g, 1997). The plates were <strong>in</strong>cubated<br />
at 25˚C for 5-7 days. Colonies with the appearance of A. niger, A.<br />
carbonarius and A. ochraceus were isolated onto Malt Extract agar (Pitt<br />
and Hock<strong>in</strong>g, 1997) and identified accord<strong>in</strong>g to Klich and Pitt (1988).<br />
The percentage <strong>in</strong>fection of the fruit or pieces was calculated.<br />
2.3. Ochratox<strong>in</strong> A Production<br />
Ochratox<strong>in</strong> A production from each isolate was analysed qualitatively<br />
us<strong>in</strong>g the agar plug technique of Filtenborg et al. (1983) or<br />
extracted with chloroform as described below. The isolates were <strong>in</strong>oculated<br />
onto Yeast Extract (0.1%) Sucrose (15%) agar and <strong>in</strong>cubated at<br />
25˚C for 7 days. For the agar plug technique, a small plug was cut from<br />
the colony us<strong>in</strong>g a cork borer and tested by TLC as described by<br />
Filtenborg et al. (1983). If isolates were found to be negative for OA<br />
production, the whole colony was extracted with chloroform. The<br />
whole colony from the Petri dish was placed <strong>in</strong> chloroform <strong>in</strong> a<br />
Stomacher and homogenised for 3 m<strong>in</strong>, filtered and concentrated <strong>in</strong> a<br />
water bath at 60˚C to near dryness then dried under a stream of nitrogen.<br />
The residue was resuspended <strong>in</strong> chloroform and spotted onto<br />
TLC plates which were developed <strong>in</strong> toluene: ethyl acetate: formic acid
Fungi Produc<strong>in</strong>g Ochratox<strong>in</strong> <strong>in</strong> Dried Fruits 183<br />
(5:4:1) and visualized under UV light at 365nm. An OA standard<br />
(Sigma Chemicals, St Louis, USA) was used for comparison.<br />
2.4. Analysis of Ochratox<strong>in</strong> A from Fruit Samples<br />
The fruit samples were analysed for OA us<strong>in</strong>g the Ochratest<br />
HPLC Procedure for Currants and Rais<strong>in</strong>s (Vicam, 1999). The<br />
method was validated for this study. Samples were extracted with a<br />
solution of methanol: sodium bicarbonate, 1% (70:30) <strong>in</strong> a blender at<br />
high speed for 1 m<strong>in</strong>. The extracts were filtered onto qualitative paper<br />
and an aliquot diluted with phosphate buffered sal<strong>in</strong>e conta<strong>in</strong><strong>in</strong>g<br />
0.01% Tween 20. This solution was filtered through a microfibre filter<br />
and an aliquot applied to an immunoaff<strong>in</strong>ity column (Vicam,<br />
Watertown MA, USA) conta<strong>in</strong><strong>in</strong>g a monoclonal antibody specific for<br />
OA. The column was washed with phosphate buffered sal<strong>in</strong>e with<br />
0.01% Tween 20, followed by purified water. The OA was eluted with<br />
HPLC methanol and the eluate was added to purified water, mixed<br />
and <strong>in</strong>jected <strong>in</strong> the HPLC with a fluorescence detector. The mobile<br />
phase was acetonitrile:water:acetic acid (99:99:2) and the flow rate was<br />
0.8 ml/m<strong>in</strong>. The HPLC equipment was a Shimadzu LC-10VP system<br />
(Shimadzu, Japan) set at 333 nm excitation and 477 nm emission. The<br />
HPLC was fitted with a Shimadzu CLC G-ODS(4) (4 × 10 mm) guard<br />
column and Shimadzu Shimpack CLC-ODS (4.6 × 250 mm) column.<br />
2.5. Confirmation of Ochratox<strong>in</strong> A<br />
Ochratox<strong>in</strong> A was confirmed by methyl ester formation (Pittet<br />
et al., 1996). Aliquots (200 µl) of both the sample and standard were<br />
evaporated under a stream of nitrogen and the residue redissolved <strong>in</strong><br />
300 µl of boron trifluoride-methanol complex (20% solution <strong>in</strong><br />
methanol). The solution was heated at 80˚C for 10 m<strong>in</strong> and allowed to<br />
cool to room temperature. The solution was evaporated and taken up<br />
<strong>in</strong> mobile phase (1 ml) and <strong>in</strong>jected <strong>in</strong>to the HPLC. A positive confirmation<br />
of identity was provided by the disappearance of the OA peak<br />
at retention time 16.4 m<strong>in</strong> and the appearance of a new peak (OA<br />
methyl ester) at a retention time of 41.0 m<strong>in</strong>.<br />
2.6. Water Activity<br />
The water activity (a w ) was measured us<strong>in</strong>g an Aqualab T3 device<br />
(Decagon, Pullman, WA, USA), at a constant temperature of 25˚C.<br />
The samples were analysed <strong>in</strong> triplicate.
184 Beatriz T. Iamanaka et al.<br />
3. RESULTS AND DISCUSSION<br />
The water activities of the samples exam<strong>in</strong>ed are summarised <strong>in</strong><br />
Table 1. Samples of all the fruit except plums and apricots were below<br />
0.75 a w and microbiologically stable. Some samples of apricots and<br />
plums were higher than 0.75 a w , but conta<strong>in</strong>ed preservatives and were<br />
also shelf stable.<br />
Table 2 shows the mean and range of percentage <strong>in</strong>fection by<br />
A. niger plus A. carbonarius and A. ochraceus <strong>in</strong> dried fruits. The predom<strong>in</strong>ance<br />
of black Aspergilli can be expla<strong>in</strong>ed by their black spores<br />
which possess resistance to ultraviolet light. The high sugar concentration<br />
and low water activity <strong>in</strong> dried fruits also assist the development<br />
of these fungi because they are xerophilic.<br />
Table 3 shows the results of the concentrations of ochratox<strong>in</strong> A <strong>in</strong><br />
dried fruits. The average level of contam<strong>in</strong>ation by OA <strong>in</strong> dried fruits<br />
was low except for one sample each of black sultanas and figs<br />
with more than 30 and 20 µg/kg OA and mean values of 4.73 and<br />
4.10 µg/kg respectively. OA was detected at levels rang<strong>in</strong>g from 0.13 to<br />
5.0 µg/kg <strong>in</strong> most samples (88.2%). Although date samples were<br />
contam<strong>in</strong>ated with a high level of toxigenic species of black Aspergilli<br />
Table 1. Average and range of water activity (a w ) <strong>in</strong> dried fruits<br />
a w<br />
Dried Fruits Mean Range<br />
Black sultanas 0.629 0.527–0.765<br />
White sultanas 0.567 0.473–0.638<br />
Dates 0.629 0.549–0.712<br />
Plums 0.796 0.712–0.863<br />
Apricots 0.694 0.638–0.782<br />
Figs 0.682 0.638–0.751<br />
Table 2. Percentage <strong>in</strong>fection of dried fruits by A. niger plus A. carbonarius and<br />
A. ochraceus<br />
% Infection<br />
No. A. niger + A. carbonarius A. ochraceus<br />
Dried fruits Samples Mean Range Mean Range<br />
Black sultanas 24 22 0 – 90 0.8 0 –18<br />
White sultanas 19 0.5 0 – 8 - -<br />
Dates 22 8.6 0 – 86 1.1 0 –24<br />
Plums 21 8.0 0 – 60 0.5 0 –10<br />
Apricots 14 - - - -<br />
Figs 19 4.5 0 – 38 - -
Fungi Produc<strong>in</strong>g Ochratox<strong>in</strong> <strong>in</strong> Dried Fruits 185<br />
Table 3. Ochratox<strong>in</strong> A levels <strong>in</strong> dried fruits<br />
Ochratox<strong>in</strong> Black White<br />
A (µg/kg) sultanas sultanas Dates Plums Apricots Figs<br />
30.0 1 - - - -<br />
Mean 4.73 0.52
186 Beatriz T. Iamanaka et al.<br />
Figure 1 shows the total numbers of black Aspergillus species<br />
isolated from each type of sample and the numbers of isolates<br />
produc<strong>in</strong>g OA. Of 264 isolates from black sultanas, only 6.1% were<br />
toxigenic, whereas <strong>in</strong> dates 68.4% of black Aspergilli were toxigenic.<br />
Plums and figs yielded 84 and 43 isolates, of which 28 (33.3%)<br />
and 11 (25.6%) were toxigenic, respectively. Studies on dried fruits<br />
have shown that these products are commonly contam<strong>in</strong>ated with<br />
black Aspergilli such as A. carbonarius, A. niger and related species<br />
(K<strong>in</strong>g et al., 1981; Abarca et al., 2003). Apricots were not contam<strong>in</strong>ated<br />
with ochratoxigenic fungi because the use of high levels of<br />
sulphur dioxide to preserve colour renders dried apricots essentially<br />
sterile.<br />
Abarca et al. (2003) isolated black Aspergilli from 98% of dried fruit<br />
samples (currants, rais<strong>in</strong>s and sultanas). They found that 96.7% of<br />
A. carbonarius and 0.6% of A niger isolates produced OA, <strong>in</strong>dicat<strong>in</strong>g<br />
that among black Aspergilli, A. carbonarius was the most probable<br />
source of this tox<strong>in</strong> <strong>in</strong> these substrates <strong>in</strong> Spa<strong>in</strong>. However, Da Rocha<br />
Rosa et al. (2002) found a higher percentage of A. niger produc<strong>in</strong>g OA<br />
<strong>in</strong> grapes from Argent<strong>in</strong>a (17%) and Brazil (30%). Only A. carbonarius<br />
was found <strong>in</strong> Brazilian grapes and only eight isolates (25%) were<br />
able to produce this tox<strong>in</strong>.<br />
In the present study, the high <strong>in</strong>cidence of toxigenic black Aspergilli<br />
<strong>in</strong> dates, plums and figs is of concern, but only a few isolates were<br />
identified as A. carbonarius. Compared with studies that have been<br />
carried out <strong>in</strong> several parts of the world (Heenan et al., 1998; Da<br />
Rocha Rosa et al., 2002; Abarca et al., 2003; Battilani et al., 2003), the<br />
results presented here differ significantly because they show a higher<br />
percentage of black Aspergilli other than A. carbonarius produc<strong>in</strong>g<br />
OA.<br />
Figure 2 shows the numbers of A. ochraceus found <strong>in</strong> dried fruits.<br />
A. ochraceus contam<strong>in</strong>ation was not high, only 27 isolates were found<br />
<strong>in</strong> black sultanas, dates and plums, however 52% of the isolates were<br />
toxigenic. Most of isolates from black sultanas and plums produce<br />
OA; 80% and 100%, respectively. From dates, only one of 12 isolates<br />
was toxigenic. A. ochraceus was not found <strong>in</strong> white sultanas, apricots<br />
or figs.<br />
In general, the presence of OA <strong>in</strong> dried fruits was related to contam<strong>in</strong>ation<br />
by black Aspergilli and the major <strong>in</strong>cidence occurred <strong>in</strong><br />
black sultanas, figs and dates.
Fungi Produc<strong>in</strong>g Ochratox<strong>in</strong> <strong>in</strong> Dried Fruits 187<br />
No. isolates<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
black<br />
sultanas<br />
white<br />
sultanas<br />
4. REFERENCES<br />
dates plums apricots figs<br />
A. ochraceus toxigenics<br />
Figure 2. Number of isolates of Aspergillus ochraceus from dried fruits and production<br />
of ochratox<strong>in</strong> A<br />
Abarca, M. L., Bragulat, M. R., Castella, G., and Cabanes, F. J., 1994, Ochratox<strong>in</strong> A<br />
production by stra<strong>in</strong>s of Aspergillus niger var. niger, Appl. Environ. Microbiol.<br />
60:2650-2652.<br />
Abarca, M. L., Accensi, F., Bragulat, M. R., Castella, G., and Cabanes, F. J., 2003,<br />
Aspergillus carbonarius as the ma<strong>in</strong> source of ochratox<strong>in</strong> A contam<strong>in</strong>ation <strong>in</strong> dried<br />
v<strong>in</strong>e fruits from the Spanish market, J. <strong>Food</strong> Prot. 66:504-506.<br />
Battilani, P., Pietri, A., Bertuzzi, T., Languasco, L., Giorni, P. and Kozakiewicz, Z.,<br />
2003, Occurrence of ochratox<strong>in</strong> A-produc<strong>in</strong>g fungi <strong>in</strong> grapes grown <strong>in</strong> Italy,<br />
J. <strong>Food</strong> Prot. 66:633-636.<br />
Da Rocha Rosa, C. A., Palacios, V., Comb<strong>in</strong>a, M., Fraga, M. E., De Oliveira Rekson,<br />
A., Magnoli, C. E., and Dalcero, A. M., 2002, Potential ochratox<strong>in</strong> A producers<br />
from w<strong>in</strong>e grapes <strong>in</strong> Argent<strong>in</strong>a and Brazil, <strong>Food</strong> Addit. Contam. 19:408-414.<br />
Filtenborg, O., Frisvad, J. C., and Svendensen, J. A., 1983, Simple screen<strong>in</strong>g method<br />
for molds produc<strong>in</strong>g <strong>in</strong>tracellular mycotox<strong>in</strong>s <strong>in</strong> pure cultures, Appl. Environ.<br />
Microbiol. 45:581-585.<br />
Heenan, C. N., Shaw, K. J. and Pitt, J. I., 1998, Ochratox<strong>in</strong> A production by<br />
Aspergillus carbonarius and A. niger isolates and detection us<strong>in</strong>g coconut cream<br />
agar, J. <strong>Food</strong> Mycol. 1:67-72.<br />
JECFA (Jo<strong>in</strong>t FAO/WHO Expert Committee on <strong>Food</strong> Additives), 2001, Ochratox<strong>in</strong><br />
A, <strong>in</strong>: Safety Evaluation of Certa<strong>in</strong> Mycotox<strong>in</strong>s <strong>in</strong> <strong>Food</strong>. Prepared by the Fifty-sixth<br />
meet<strong>in</strong>g of the JECFA. FAO <strong>Food</strong> and Nutrition Paper 74, <strong>Food</strong> and Agriculture<br />
Organization of the United Nations, Rome, Italy.<br />
K<strong>in</strong>g, A. D., Hock<strong>in</strong>g, A. D., and Pitt, J. I., 1981, The mycoflora of some Australian<br />
foods, <strong>Food</strong> Technol. Aust. 33:55-60.
188 Beatriz T. Iamanaka et al.<br />
Klich, M. A., and Pitt, J. I., 1988, A Laboratory Guide to Common Aspergillus Species<br />
and their Teleomorphs, CSIRO Division of <strong>Food</strong> Science and Technology, Sydney,<br />
Australia.<br />
MacDonald, S., Wilson, P., Barnes, K., Damant, A., Massey, R., Mortby, E., and<br />
Shepherd, M. J., 1999, Ochratox<strong>in</strong> A <strong>in</strong> dried v<strong>in</strong>e fruit: method development and<br />
survey, <strong>Food</strong> Addit. Contam. 6:253-260.<br />
MAFF (M<strong>in</strong>istry of Agriculture, Fisheries and <strong>Food</strong>), 1997, Survey of aflatox<strong>in</strong>s and<br />
ochratox<strong>in</strong> A <strong>in</strong> cereals and retail products, <strong>Food</strong> Surveillance Information Sheet<br />
130, MAFF, UK.<br />
MAFF (M<strong>in</strong>istry of Agriculture, Fisheries and <strong>Food</strong>), 2002, Survey of nuts, nut products<br />
and dried tree fruits for mycotox<strong>in</strong>, http://www.food.gov.uk/multimedia/<br />
pdfs/21nuts.pdf <strong>Food</strong> Surveillance Information, MAFF, UK.<br />
Pitt J. I., 1987, Penicillium viridicatum, Penicillium verrucosum and production of<br />
ochratox<strong>in</strong> A. Appl. Environ. Microbiol. 53,:266-269.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, Blackie Academic and<br />
Professional, London.<br />
Pittet, A., Tornare, D., Huggett, A., and Viani, R., 1996, Liquid chromatographic<br />
determ<strong>in</strong>ation of ochratox<strong>in</strong> A <strong>in</strong> pure and adulterated soluble coffee us<strong>in</strong>g an<br />
immunoaff<strong>in</strong>ity column cleanup procedure, J. Agric. <strong>Food</strong> Chem. 44:3564-3569.<br />
VICAM, 1999, Ochratest Instruction Manual, VICAM Pty Ltd, Watertown, MA.<br />
CD-Rom.<br />
Zimmerli, B., and Dick, R., 1996, Ochratox<strong>in</strong> A <strong>in</strong> table w<strong>in</strong>e and grape juice: occurrence<br />
and risk assessment. <strong>Food</strong> Addit. Contam. 13:655-668.
AN UPDATE ON OCHRATOXIGENIC FUNGI<br />
AND OCHRATOXIN A IN COFFEE<br />
Marta H. Taniwaki *<br />
1. INTRODUCTION<br />
The occurrence of ochratox<strong>in</strong> A (OA) <strong>in</strong> raw coffee was first<br />
reported by Levi et al. (1974). Initial data suggested that almost complete<br />
destruction of mycotox<strong>in</strong>s would occur dur<strong>in</strong>g the roast<strong>in</strong>g<br />
process. However, occurrence of ochratox<strong>in</strong> A <strong>in</strong> market samples of<br />
roasted coffee, as well as <strong>in</strong> coffee beverage, was reported after an<br />
improved detection method. Consequently, the European<br />
Commission’s Scientific Committee for <strong>Food</strong> (SCF) considered that<br />
there was real potential for OA contam<strong>in</strong>ation of coffee. S<strong>in</strong>ce then,<br />
surveys on the presence of OA <strong>in</strong> coffee have been undertaken <strong>in</strong> several<br />
countries. These surveys looked first at raw coffee imported <strong>in</strong>to<br />
Europe from different orig<strong>in</strong>s, then on roasted and soluble coffee produced<br />
and sold <strong>in</strong> Europe (MAFF, 1996; Patel et al., 1997; van der<br />
Stegen et al., 1997).<br />
Some countries have stated limits of ochratox<strong>in</strong> A for raw coffee:<br />
Italy 8 µg/kg; F<strong>in</strong>land 10 µg/kg; Greece 20 µg/kg; or for coffee products:<br />
Switzerland 5 µg/kg. Pressure from European authorities has<br />
prompted coffee importers, and the coffee-produc<strong>in</strong>g countries, to<br />
beg<strong>in</strong> surveys for the presence of OA <strong>in</strong> coffee products, and as well as<br />
<strong>in</strong>vestigation of the fate of OA dur<strong>in</strong>g the production, handl<strong>in</strong>g and<br />
manufacture of raw coffee.<br />
*<strong>Food</strong> Technology Institute (ITAL), Av. Brasil, 2880, Camp<strong>in</strong>as, SP, CEP 13.073-001,<br />
Brazil. Correspondence to: mtaniwak@ital.sp.gov.br<br />
189
190 Marta H. Taniwaki<br />
2. INCIDENCE OF OCHRATOXIN A IN<br />
COFFEE SAMPLES WORLDWIDE<br />
Surveys all over the world have confirmed the presence of OA <strong>in</strong><br />
commercial raw, roasted and soluble coffee (Tables 1-3). Extensive<br />
sampl<strong>in</strong>g of raw coffee from all orig<strong>in</strong>s and both types of coffee<br />
(Arabica, Robusta) has shown that OA contam<strong>in</strong>ation may be more<br />
frequent <strong>in</strong> some areas, but that no produc<strong>in</strong>g country is entirely free<br />
from contam<strong>in</strong>ation (Table 1). Similarly it has been shown that, while<br />
the <strong>in</strong>itial contam<strong>in</strong>ation may occur at farm level, OA formation may<br />
happen throughout the entire cha<strong>in</strong>, at every stage of production.<br />
What happens to OA dur<strong>in</strong>g process<strong>in</strong>g of coffee beans is not yet<br />
fully understood, but the results of surveys for OA <strong>in</strong> retail roasted<br />
and soluble coffees all over the world <strong>in</strong>dicate that coffee is not a<br />
major source of OA <strong>in</strong> the diet, with estimated <strong>in</strong>takes be<strong>in</strong>g well<br />
with<strong>in</strong> safety limits. The low levels of OA contam<strong>in</strong>ation found <strong>in</strong><br />
roasted and soluble coffee (Tables 2 and 3) support this conclusion.<br />
3. FUNGI PRODUCING OCHRATOXIN A IN<br />
COFFEE<br />
Aspergillus ochraceus has been isolated from coffee samples by several<br />
authors (Urbano et al., 2001b; Taniwaki et al., 2003; Mart<strong>in</strong>s<br />
et al., 2003; Batista et al., 2003; Suárez-Quiroz et al., 2004) and was<br />
proposed as the major cause of OA <strong>in</strong> coffee beans (Frank, 1999).<br />
However, A. carbonarius and A. niger, capable of produc<strong>in</strong>g OA, have<br />
also been isolated from coffee (Téren et al., 1997; Nakajima et al.,<br />
1997; Joosten et al., 2001; Urbano et al., 2001b; Taniwaki et al., 2003;<br />
Pardo et al., 2004; Suárez-Quiroz et al., 2004), <strong>in</strong>dicat<strong>in</strong>g that these<br />
two species are also potential sources of OA <strong>in</strong> coffee. These f<strong>in</strong>d<strong>in</strong>gs<br />
have led to the consensus viewpo<strong>in</strong>t that the three Aspergilli:<br />
A. ochraceus, A. carbonarius and A. niger are responsible for the<br />
formation of OA on coffee.<br />
In a study of fungi with the potential to produce OA <strong>in</strong> Brazilian<br />
coffee (Taniwaki et al., 2003), the major fungus responsible was found<br />
to be A. ochraceus. Of 269 isolates of this species, 75% were found to<br />
be capable of tox<strong>in</strong> production, a higher percentage than had been<br />
reported previously. A. carbonarius was also found, though much less<br />
commonly, and it also had the potential to produce OA <strong>in</strong> coffee. Few
An Update on Ochratoxigenic Fungi and Ochratox<strong>in</strong> A <strong>in</strong> Coffee 191<br />
Table 1. Incidence of ochratox<strong>in</strong> A (OA) <strong>in</strong> commercial raw coffee worldwide<br />
No.<br />
positive/ Range<br />
No. of OA Coffee<br />
Orig<strong>in</strong> samples (µg/kg) type Reference<br />
Angola 0/4 < 20 a N.S. b Levi et al. (1974)<br />
Brazil 3/7 Trace – 360 ” ”<br />
Colombia 17/139 Trace – 50 ” ”<br />
Cameroon 0/1 < 20 a ” ”<br />
Ivory Coast 1/12 Trace ” ”<br />
Uganda 1/2 Trace ” ”<br />
Unknown 7/102 Trace ” ”<br />
Brazil 10/14 0.2 – 3.7 Arabica Micco et al. (1989)<br />
Cameroon 3/3 Traces – 2.2 Robusta ”<br />
Colombia 1/2 3.3 Arabica ”<br />
Costa Rica 1/2 Traces Arabica ”<br />
Ivory Coast 1/2 1.3 Robusta ”<br />
Kenya 0/2 < 0.01 a Arabica ”<br />
Mexico 1/2 1.4 Arabica ”<br />
Zaire 2/2 8.4 – 15.0 Robusta ”<br />
Brazil 3/5 2.0 – 7.4 N.S. b Studer-Rhor et al.<br />
(1995)<br />
Colombia 3/5 1.2 – 9.8 ” ”<br />
Central America 0/1 < 0.5 a N.S. b ”<br />
Costa Rica 0/1 < 0.5 a ” ”<br />
Guatemala 0/1 < 0.5 a ” ”<br />
Ivory Coast 2/2 9.9 – 56.0 ” ”<br />
Kenya 0/3 < 0.5 a ” ”<br />
New Gu<strong>in</strong>ea 0/1 < 0.5 a ” ”<br />
Tanzania 1/1 2.2 ” ”<br />
Zaire 1/1 17.3 ” ”<br />
Unknown 2/4 2.2 – 11.8<br />
America, Africa, 31/153 0.2 – 9.0 Arabica MAFF, 1996<br />
Papua New<br />
Gu<strong>in</strong>ea<br />
America, Africa, 55/75 0.2 – 27.3 Robusta ”<br />
Asia<br />
Yemen 7/10 0.7 – 17.4 Arabica Nakajima et al.<br />
(1997)<br />
Tanzania 5/9 0.1 – 7.2 Arabica ”<br />
Indonesia 2/9 0.2 – 1.0 Robusta ”<br />
Ethiopia 0/1 < 0.1 a Arabica ”<br />
Central America 0/6 < 0.1 a Arabica ”<br />
South America 0/12 < 0.1 a Arabica ”<br />
East Africa 42/33 0.2 – 62.0 N.S. b Heilmann<br />
et al. (1999)<br />
West Africa 9/9 0.3 – 5.0 N.S. b Heilmann<br />
et al. (1999)
192 Marta H. Taniwaki<br />
Table 1. Incidence of ochratox<strong>in</strong> A (OA) <strong>in</strong> commercial raw coffee worldwide—cont’d<br />
No.<br />
positive/ Range<br />
No. of OA Coffee<br />
Orig<strong>in</strong> samples (µg/kg) type Reference<br />
Asia 20/29 0.2 – 4.9 N.S. b ”<br />
Central America 6/15 0.2 – 0.8 ” ”<br />
South America 5/17 0.2 – 1.0 ” ”<br />
South America 9/19 0.1 – 4.9 N.S. b Trucksess<br />
et al. (1999)<br />
Africa 76/84 0.5 – 48.0 N.S. b Romani et al.<br />
(2000)<br />
Lat<strong>in</strong> America 19/60 0.1 – 7.7 ” ”<br />
Asia 11/18 0.2 – 4.9 ” ”<br />
Brazil 17/37 0.2 – 6.2 Arabica Gollücke<br />
et al. (2001)<br />
Brazil 27/132 0.7 – 47.8 Arabica Leoni et al.<br />
(2000)<br />
Brazil 5/40 0.6 – 4.4 Arabica Batista et al.<br />
(2003)<br />
Brazil 20/60 0.2 – 7.3 Arabica Mart<strong>in</strong>s et al.<br />
(2003)<br />
Africa 12/12 2.4 – 23.3 Robusta Pardo et al.<br />
(2004)<br />
America 31/31 1.3 – 27.7 Arabica ”<br />
Asia 14/14 1.6 – 31.5 Arabica and ”<br />
Robusta<br />
a Corresponds to the detection limit of the method; b Not Specified.<br />
Table 2. Incidence of ochratox<strong>in</strong> A <strong>in</strong> commercial roasted coffee worldwide<br />
No. positive/ Range of<br />
Retail country No. samples AO (µg/kg) Reference<br />
Japan 5/68 3.2 – 17.0 Tsubouchi et al. (1988)<br />
United K<strong>in</strong>gdom 17/20 0.2 – 2.1 Patel et al. (1997)<br />
Europe ?/484 < 0.5 a – 8.2 Van der Stegen et al. (1997)<br />
Denmark 11/11 0.1 – 3.2 Jorgensen (1998)<br />
Spa<strong>in</strong> 29/29 0.22 – 5.64 Burdespal & Legarda (1998)<br />
United States 9/13 0.1 – 1.2 Trucksess et al. (1999)<br />
Brazil 23/34 0.3 – 6.5 Leoni et al. (2000)<br />
Brazil 41/47 0.99 – 5.87 Prado et al. (2000)<br />
Germany 22/67 0.3 – 3.3 Wolff (2000)<br />
Germany 273/490 0.21 – 12.1 Otteneder & Majerus (2001)<br />
Canada 42/71 0.1 – 2.3 Lombaert et al. (2002)<br />
Hungary 22/38 0.17 – 1.3 Fazekas et al. (2002)<br />
a Corresponds to the detection limit of the method.
An Update on Ochratoxigenic Fungi and Ochratox<strong>in</strong> A <strong>in</strong> Coffee 193<br />
Table 3. Incidence of ochratox<strong>in</strong> A <strong>in</strong> commercial soluble coffee worldwide<br />
No. positive/ Range of<br />
Retail country No. samples OA (µg/kg) Reference<br />
Australia 7/22 0.2 – 4.0 Pittet et al. (1996)<br />
United States 3/6 1.5 – 2.1 ”<br />
Germany 5/9 0.3 – 2.2 ”<br />
United K<strong>in</strong>gdom 64/80 0.1 – 8.0 Patel at al. (1997)<br />
Europe ?/149 < 0.5 a – 27.2 Van der Stegen et al. (1997)<br />
Spa<strong>in</strong> 9/9 0.19 – 1.08 Burdaspal & Legarda (1998)<br />
Brazil 8/10 0.31 – 1.78 Prado et al. (2000)<br />
Brazil 16/16 0.5 – 5.1 Leoni et al. (2000)<br />
Germany 23/52 0.3 – 9.5 Wolff (2000)<br />
Germany 12/41 0.28 – 4.8 Otteneder & Majerus (2001)<br />
Canada 20/30 0.1 – 3.1 Lombaert et al. (2002)<br />
a Corresponds to the detection limit of the method.<br />
cherries on the coffee trees were <strong>in</strong>fected with these species, <strong>in</strong>dicat<strong>in</strong>g<br />
that <strong>in</strong>fection mostly occurred after harvest, and the fungal sources<br />
were likely to be soil, equipment and dry<strong>in</strong>g yard surfaces. Variability<br />
<strong>in</strong> <strong>in</strong>fection rates of these toxigenic species was reflected <strong>in</strong> a wide<br />
range of ochratox<strong>in</strong> A levels <strong>in</strong> samples from the dry<strong>in</strong>g yard and storage.<br />
A. niger was more common than A. ochraceus or A. carbonarius,<br />
but only 3% of isolates were capable of produc<strong>in</strong>g OA, so this species<br />
is probably a relatively unimportant source of OA <strong>in</strong> coffee.<br />
4. COFFEE PRODUCTION<br />
The formation of OA dur<strong>in</strong>g coffee production can be assessed <strong>in</strong><br />
three stages: 1) pre-harvest; 2) post-harvest to storage and transportation<br />
of raw coffee to the process<strong>in</strong>g plant, and 3) raw coffee process<strong>in</strong>g<br />
to roasted and ground coffee and soluble coffee.<br />
4.1. Pre-harvest<br />
The issue of time of <strong>in</strong>vasion of coffee by toxigenic fungi is of<br />
great importance <strong>in</strong> understand<strong>in</strong>g the problem of OA <strong>in</strong> coffee and <strong>in</strong><br />
develop<strong>in</strong>g control strategies. Experience with other crops has shown<br />
that if <strong>in</strong>vasion occurs pre-harvest, control will be much more difficult<br />
than if it occurs post-harvest, i.e. dur<strong>in</strong>g dry<strong>in</strong>g and storage. Post-harvest<br />
problems can be expected to relate to unfavourable climates for<br />
dry<strong>in</strong>g, poor dry<strong>in</strong>g practice or quality control, or <strong>in</strong>adequate storage
194 Marta H. Taniwaki<br />
conditions. The major risk factors and process<strong>in</strong>g steps that can lead<br />
to contam<strong>in</strong>ation of raw coffee with ochratox<strong>in</strong> A have been reviewed<br />
(Bucheli and Taniwaki, 2002).<br />
The suggestion by Mantle (2000) that OA <strong>in</strong> coffee beans may result<br />
from uptake of OA <strong>in</strong> soil by the roots of the coffee tree and then<br />
translocated is conjectural. Taniwaki et al. (2003) found an average of<br />
A. ochraceus <strong>in</strong>fection of less than 0.6% of fruit on the tree. The highest<br />
percentage of A. ochraceus <strong>in</strong>fection <strong>in</strong> coffee fruit sampled from<br />
trees was 4%, but the figure <strong>in</strong>creased to 16% <strong>in</strong> fruit harvested from<br />
the ground, and to 35% <strong>in</strong> fruit dur<strong>in</strong>g dry<strong>in</strong>g and storage. No evidence<br />
has been found that either A. ochraceus or A. carbonarius<br />
<strong>in</strong>vades coffee beans before harvest or has an association with the coffee<br />
tree. These facts <strong>in</strong>dicate that <strong>in</strong>fection mostly occurs after harvest,<br />
and the fungal sources are likely to be soil, equipment and dry<strong>in</strong>g yard<br />
surfaces.<br />
4.2. Post-harvest, storage and transport<br />
Cherries conta<strong>in</strong> sufficient amounts of water to support mould<br />
growth and OA formation on the outer part of the cherries dur<strong>in</strong>g the<br />
<strong>in</strong>itial 3-5 days of dry<strong>in</strong>g. Sun dry<strong>in</strong>g of coffee cherries if done <strong>in</strong>correctly<br />
can potentially result <strong>in</strong> OA contam<strong>in</strong>ation. In general, dry<strong>in</strong>g<br />
of fruits appears to be a particularly risky step for the natural occurrence<br />
of black Aspergilli. This may be l<strong>in</strong>ked to the resistance of the<br />
black spores of this species to UV irradiation. Dry<strong>in</strong>g is also the most<br />
favourable time for development of A. ochraceus, the ma<strong>in</strong> problem<br />
be<strong>in</strong>g the time it takes for the berries to dry below a critical water<br />
activity (a w ) of about 0.80. Berries should spend no more that 4 days<br />
between 0.97 to 0.80 a w . Palacios-Cabrera et al. (2004) showed that A.<br />
ochraceus produced little OA (0.15 µg/kg) <strong>in</strong> coffee beans at a w of 0.80<br />
and temperature of 25:C, but at 0.86 and 0.90 a w the production was<br />
2500 and over 7000 µg/kg, respectively.<br />
4.2.1. Dry<strong>in</strong>g<br />
The formation of OA dur<strong>in</strong>g coffee dry<strong>in</strong>g was studied <strong>in</strong> Thailand<br />
by Bucheli et al. (2000), who showed that the tox<strong>in</strong> was normally produced<br />
dur<strong>in</strong>g the sun dry<strong>in</strong>g of coffee; overripe cherries were more<br />
susceptible than green ones. They also noted that dur<strong>in</strong>g sun dry<strong>in</strong>g,<br />
OA was formed <strong>in</strong> the coffee cherry pericarp (pulp and parchment),<br />
the part of the cherry that is removed as husk <strong>in</strong> the dehull<strong>in</strong>g process.<br />
The results obta<strong>in</strong>ed by Bucheli et al. (2000) demonstrated that the <strong>in</strong>i-
An Update on Ochratoxigenic Fungi and Ochratox<strong>in</strong> A <strong>in</strong> Coffee 195<br />
tial raw material quality, weather conditions dur<strong>in</strong>g dry<strong>in</strong>g, dry<strong>in</strong>g<br />
management, presence of OA produc<strong>in</strong>g fungi, and local farm conditions,<br />
undoubtedly played a more important role <strong>in</strong> OA contam<strong>in</strong>ation<br />
<strong>in</strong> raw coffee than the dry<strong>in</strong>g methodology used, on bamboo<br />
tables, bare ground or concrete. Taniwaki et al. (2003) agreed with<br />
Bucheli et al. (2000) that if dry<strong>in</strong>g is rapid and effective, OA will not<br />
be produced. Good sun dry<strong>in</strong>g or a comb<strong>in</strong>ation of sun dry<strong>in</strong>g and<br />
mechanical dehydration provide effective control.<br />
4.2.2. Dehull<strong>in</strong>g<br />
Dried coffee cherries are subjected to a dehull<strong>in</strong>g process to separate<br />
the raw coffee beans from the husks. This is often a rather dusty<br />
procedure and it is possible that part of the OA conta<strong>in</strong>ed <strong>in</strong> the husks<br />
will be transferred as dust particles or husk fragments to the raw coffee.<br />
After dehull<strong>in</strong>g, husks can be highly contam<strong>in</strong>ated with OA.<br />
Bucheli et al. (2000) studied Robusta coffee dry<strong>in</strong>g <strong>in</strong> Thailand over<br />
three seasons. They found that sun dry<strong>in</strong>g of cherries consistently<br />
resulted <strong>in</strong> OA formation <strong>in</strong> the pulp and husks. The coffee beans, on<br />
the other hand, had only about 1% of the OA found <strong>in</strong> husks. OA contam<strong>in</strong>ation<br />
of raw coffee depended on cherry maturation, with overripe<br />
cherries be<strong>in</strong>g the most susceptible. Inclusion of defective berries<br />
and husks was found to be the most important source of OA contam<strong>in</strong>ation.<br />
The ma<strong>in</strong> fungal source of OA <strong>in</strong> this case was A. carbonarius.<br />
The largest proportion (more than 90%) of OA contam<strong>in</strong>ation <strong>in</strong><br />
coffee was usually concentrated <strong>in</strong> the husks. Clean<strong>in</strong>g, grad<strong>in</strong>g and<br />
hygienic stor<strong>in</strong>g of raw coffee is, therefore, of paramount importance.<br />
An <strong>in</strong>direct confirmation of the importance of husk removal to<br />
reduce OA contam<strong>in</strong>ation is the observation that husk addition, a<br />
fraudulent practice sometimes encountered <strong>in</strong> the manufacture of soluble<br />
coffee, may lead to the presence of relatively high levels of OA<br />
contam<strong>in</strong>ation <strong>in</strong> adulterated soluble coffee (Pittet et al., 1996). More<br />
recently, Suárez-Quiroz et al. (2004) reported that tox<strong>in</strong> was not<br />
totally elim<strong>in</strong>ated after hull<strong>in</strong>g coffee conta<strong>in</strong><strong>in</strong>g OA. The content of<br />
the tox<strong>in</strong> <strong>in</strong> coffee beans, parchment or husk was similar. However,<br />
these authors analysed only seven samples and the contam<strong>in</strong>ation<br />
level was between a trace and 0.3 µg/kg.<br />
4.2.3. Wet process<strong>in</strong>g<br />
Bucheli and Taniwaki (2002), review<strong>in</strong>g the impact of wet process<strong>in</strong>g<br />
on the presence of OA <strong>in</strong> coffee, noted that there is <strong>in</strong>direct evidence
196 Marta H. Taniwaki<br />
that the depulp<strong>in</strong>g process must reduce significantly the risk of OA<br />
contam<strong>in</strong>ation, as the fruit pulp is an excellent substrate for the<br />
growth of OA produc<strong>in</strong>g fungi. Suárez-Quiroz et al. (2004) evaluated<br />
the effect on growth of fungi produc<strong>in</strong>g OA of three methods of coffee<br />
process<strong>in</strong>g (wet, mechanical and dry) at different stages from harvest<br />
to storage. These authors concluded that for A. ochraceus there<br />
was no great difference between the three processes, but the dry<br />
method seemed to promote the presence of A. niger. This may be due<br />
to the black spores which give protection from sunlight and UV light,<br />
provid<strong>in</strong>g a competitive advantage <strong>in</strong> such habitats. Black Aspergilli<br />
have been frequently isolated from sun dried products, such as dried<br />
v<strong>in</strong>e fruits, dried fish and spices (Pitt and Hock<strong>in</strong>g, 1997).<br />
Whether the wet or dry process is preferred, some measures must be<br />
taken to avoid contam<strong>in</strong>ation, by hav<strong>in</strong>g a good <strong>in</strong>itial quality of harvested<br />
coffee, and well controlled process<strong>in</strong>g conditions.<br />
4.2.4. Storage<br />
Bucheli et al. (1998) reported on the impact of storage on mould<br />
growth on <strong>in</strong>dustrial green Robusta coffee and consequent OA formation.<br />
Under the storage conditions tested (bag storage, and silo storage<br />
under air-condition<strong>in</strong>g, aeration and non-aeration), neither<br />
growth and presence of OA produc<strong>in</strong>g fungi, nor consistent OA production,<br />
was observed. On average, an 18-fold decrease of fungal<br />
counts was found. This storage study demonstrated that safe storage<br />
of green Robusta coffee under humid tropical conditions can be<br />
achieved, even over a ra<strong>in</strong>y period of several months, without f<strong>in</strong>d<strong>in</strong>g<br />
OA formation and bean damage dur<strong>in</strong>g storage. However, the <strong>in</strong>itial<br />
a w of the beans stored <strong>in</strong> bags was 0.72 and did not exceed 0.75 even<br />
<strong>in</strong> the ra<strong>in</strong>y season. Improper storage and transportation do not<br />
appear to be a major route for OA contam<strong>in</strong>ation of coffee, unless coffee<br />
is remoistened.<br />
4.2.5. Transportation<br />
Condensation can occur dur<strong>in</strong>g transportation of raw coffee to the<br />
consum<strong>in</strong>g countries and lead to mould growth. Blanc et al. (2001)<br />
reported on transportation of raw coffee <strong>in</strong> bulk or bags <strong>in</strong> shipp<strong>in</strong>g<br />
conta<strong>in</strong>ers and showed that condensation sometimes occurred at the<br />
top of the conta<strong>in</strong>er, for example dur<strong>in</strong>g w<strong>in</strong>ter <strong>in</strong> a European harbour.<br />
The development of areas with high moisture can favour mould<br />
growth and potentially OA formation.
An Update on Ochratoxigenic Fungi and Ochratox<strong>in</strong> A <strong>in</strong> Coffee 197<br />
Storage and transportation trials have shown that risk of condensation<br />
and wett<strong>in</strong>g of coffee occur ma<strong>in</strong>ly dur<strong>in</strong>g transport overland to<br />
the harbour for shipp<strong>in</strong>g and/or arrival at the dest<strong>in</strong>ation.<br />
4.3. Effect of roast<strong>in</strong>g on ochratox<strong>in</strong> levels<br />
The roast<strong>in</strong>g process subjects raw coffee to temperatures of 180-<br />
250:C for 5 to 15 m<strong>in</strong>. Dur<strong>in</strong>g roast<strong>in</strong>g, chemical changes occur with<br />
the development of aromas and the formation of dark coloured compounds,<br />
while physical changes <strong>in</strong>clude loss of water and dry material,<br />
ma<strong>in</strong>ly CO 2 and other volatiles (Clarke, 1987).<br />
Conflict<strong>in</strong>g data with respect to the <strong>in</strong>fluence of roast<strong>in</strong>g (Table 4),<br />
gr<strong>in</strong>d<strong>in</strong>g and beverage preparation on the residual levels of OA are<br />
found <strong>in</strong> the literature. Blanc et al. (1998) <strong>in</strong>vestigated the behaviour<br />
of OA dur<strong>in</strong>g roast<strong>in</strong>g and the production of soluble coffee. In this<br />
study, a small proportion of the OA was elim<strong>in</strong>ated dur<strong>in</strong>g the <strong>in</strong>itial<br />
clean<strong>in</strong>g process, due to the discard<strong>in</strong>g of defective and black beans,<br />
but the most significant reduction occurred dur<strong>in</strong>g the roast<strong>in</strong>g<br />
process. The ground roasted coffee conta<strong>in</strong>ed only 16% of the orig<strong>in</strong>al<br />
Table 4. Effect of roast<strong>in</strong>g on ochratox<strong>in</strong> A reduction<br />
No Roast<strong>in</strong>g %<br />
samples Tox<strong>in</strong> orig<strong>in</strong> condition reduction References<br />
4 Inoculationa 200˚C/10-20 m<strong>in</strong> 0 –12 Tsubouchi<br />
et al. (1988)<br />
2 Naturalb 5 – 6 m<strong>in</strong>/dark roast<strong>in</strong>g 90 – 100 Micco et al.<br />
(1989)<br />
3 Naturalb 252˚C/100-190 seg 14 – 62 Studer-Rohr<br />
et al. (1995)<br />
2 Inoculationa 252˚C/100-190 seg 2 – 28 ”<br />
6 Naturalb 223˚C / 14 m<strong>in</strong> 84 Blanc et al.<br />
(1998)<br />
3 Inoculationa 200:C/10 m<strong>in</strong> (medium 22.5 Urbano et al.<br />
roast<strong>in</strong>g) (2001a)<br />
3 ” 200:C/15 m<strong>in</strong> (medium<br />
roast<strong>in</strong>g)<br />
48.1 ”<br />
3 ” 210:C/10 m<strong>in</strong> (medium<br />
dark)<br />
39.2 ”<br />
3 ” 210:C/15 m<strong>in</strong> (medium<br />
dark)<br />
65.6 ”<br />
3 ” 220:C/10 m<strong>in</strong> (dark) 88.4 ”<br />
3 ” 220:C/15 m<strong>in</strong> (dark) 93.9 ”<br />
a b Coffee beans <strong>in</strong>oculated with toxigenic spores of Aspergillus ochraceus; Naturally<br />
contam<strong>in</strong>ated beans.
198 Marta H. Taniwaki<br />
OA present <strong>in</strong> the raw coffee. Clean<strong>in</strong>g and thermal degradation were<br />
the most important factors <strong>in</strong> the elim<strong>in</strong>ation of OA.<br />
Heilmann et al. (1999), studied OA reduction <strong>in</strong> raw coffee beans<br />
roasted <strong>in</strong>dustrially, and showed that levels of OA were significantly<br />
reduced, especially <strong>in</strong> coffee decaffe<strong>in</strong>ated by solvent extraction. Leoni<br />
et al. (2000) studied 34 samples of ground roasted coffee, 14 <strong>in</strong>stant<br />
coffee and 2 decaffe<strong>in</strong>ated coffee. They found an average of 2.2 µg/kg<br />
of OA <strong>in</strong> the <strong>in</strong>stant coffee, and <strong>in</strong> 23 of the ground roasted samples,<br />
values between 0.3 and 6.5 µg/kg of OA were found. When coffee<br />
beverage was prepared from the ground, roasted samples, the OA<br />
content of the resultant coffee was 74 to 86% of that <strong>in</strong> the orig<strong>in</strong>al<br />
ground samples.<br />
Urbano et al. (2001a) analysed 18 samples of coffee <strong>in</strong>oculated with<br />
a stra<strong>in</strong> of Aspergillus ochraceus that produced OA. The samples were<br />
subjected to temperatures of 200˚, 210˚ and 220˚C for 10 to 15 m<strong>in</strong> as<br />
shown <strong>in</strong> Table 4. The level of OA destruction varied from 22% to 94%<br />
depend<strong>in</strong>g on the time and temperature comb<strong>in</strong>ation. In practice,<br />
however, a treatment of 220˚C for 10 to 15 m<strong>in</strong> may not produce a<br />
sensorially acceptable beverage.<br />
The data <strong>in</strong> the literature show evidence that the roast<strong>in</strong>g process is<br />
efficient <strong>in</strong> reduc<strong>in</strong>g OA, but there is a lack of more conclusive<br />
research on the effects of the stages of roast<strong>in</strong>g, gr<strong>in</strong>d<strong>in</strong>g and beverage<br />
preparation on the stability of the tox<strong>in</strong>. The effect on OA destruction<br />
of coffee production processes such as roast<strong>in</strong>g, dr<strong>in</strong>k<br />
preparation, soluble coffee and decaffe<strong>in</strong>ation have been reviewed<br />
elsewhere (Gollucke et al., 2004). It is important that to ma<strong>in</strong>ta<strong>in</strong> good<br />
quality coffee, it is advisable to avoid coffee with a high OA content.<br />
Even though roast<strong>in</strong>g or other processes may reduce this tox<strong>in</strong>, the<br />
quality of the coffee can be affected.<br />
5. OCHRATOXIN A CONSUMPTION FROM<br />
COFFEE<br />
A great deal of <strong>in</strong>terest has been focused on the possible role of<br />
coffee <strong>in</strong> ochratox<strong>in</strong> A consumption. JECFA (2001) has set a<br />
Provisional Tolerable Weekly Intake for OA of 100 ng/kg bw/week<br />
which corresponds to a Provisional Tolerable Daily Intake (PTDI) of<br />
14 ng/kg bw/day. From a survey of coffee dr<strong>in</strong>kers <strong>in</strong> the United<br />
K<strong>in</strong>gdom and the data shown <strong>in</strong> Table 2, Patel et al. (1997) developed<br />
an Estimated Weekly Intake of OA from soluble coffee. Based on
An Update on Ochratoxigenic Fungi and Ochratox<strong>in</strong> A <strong>in</strong> Coffee 199<br />
a consumption of 4.5 g of soluble coffee per day, the average coffee<br />
dr<strong>in</strong>ker <strong>in</strong>gested 0.4 ng/kg body weight/week, while the heavy consumer<br />
(97.5% percentile) consumed nearly 20 g soluble coffee per day<br />
to give an OA <strong>in</strong>take of 1.9 ng/kg body weight/week. Those figures<br />
translate <strong>in</strong>to 3.5 ng and 17 ng <strong>in</strong>take of OA per day and 0.4% or 2%<br />
of the PTDI respectively (Patel et al., 1997). Compar<strong>in</strong>g these data<br />
with the data obta<strong>in</strong>ed by Leoni et al. (2000) <strong>in</strong> Brazil, the average of<br />
OA concentration <strong>in</strong> roasted coffee was 0.9 µg/kg (Table 2). Ground<br />
and roasted coffee is the type of coffee most used by Brazilians and<br />
many other coffee produc<strong>in</strong>g countries. Accord<strong>in</strong>g to Leoni et al.<br />
(2000), the average Brazilian adult dr<strong>in</strong>ks five cups of coffee per day<br />
and this would correspond to 30 g of roast and ground coffee to brew<br />
five cups of the beverage (each 60 ml) prepared accord<strong>in</strong>g to common<br />
household procedures. The probable daily <strong>in</strong>take of OA by a 70 kg<br />
adult would be 0.4 ng/kg bw/day and this falls far below the JECFA<br />
PTDI. Exam<strong>in</strong><strong>in</strong>g a worst case situation of a heavy coffee dr<strong>in</strong>ker who<br />
may dr<strong>in</strong>k up to 33 cups of coffee a day (Camargo et al. 1999) this<br />
would mean an <strong>in</strong>gestion of 2.5 ng/kg bw/day which is still well below<br />
JECFA PTDI. These results <strong>in</strong>dicate that coffee is not a major dietary<br />
source of ochratox<strong>in</strong> A <strong>in</strong> the UK or Brazil and the situation should<br />
not be very different <strong>in</strong> other coffee dr<strong>in</strong>k<strong>in</strong>g countries.<br />
6. CONCLUSION<br />
As the evidence shows that ochratox<strong>in</strong> A is formed <strong>in</strong> raw coffee<br />
beans after harvest, OA contam<strong>in</strong>ation can be m<strong>in</strong>imized by follow<strong>in</strong>g<br />
good agricultural practice and post-harvest handl<strong>in</strong>g consist<strong>in</strong>g of<br />
appropriate techniques for dry<strong>in</strong>g, grad<strong>in</strong>g, transportation and storage<br />
of raw coffee. Moreover, better quality raw material, appropriate<br />
dehull<strong>in</strong>g procedures and reduction of defects us<strong>in</strong>g colour sort<strong>in</strong>g<br />
can substantially reduce the concentration of OA <strong>in</strong> raw coffee. These<br />
procedures are well established. In June 2002, the European coffee<br />
associations and bodies published the Code of Practice<br />
“Enhancement of coffee quality through prevention of mould formation”<br />
(www.ecf-coffee.org). The objective of this code of practice is to<br />
assist operators to apply Good Agricultural Practice, Good Practices<br />
<strong>in</strong> Transport and Storage and Good Manufactur<strong>in</strong>g Practices prevent<strong>in</strong>g<br />
OA contam<strong>in</strong>ation and formation throughout the coffee<br />
cha<strong>in</strong>. Preventive measures taken by all participants <strong>in</strong> the cha<strong>in</strong> from<br />
tree to cup are the best way to prevent OA contam<strong>in</strong>ation <strong>in</strong> coffee.
200 Marta H. Taniwaki<br />
7. REFERENCES<br />
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Lombaert, G. A., Pellaers, P., Chettiar, M., Lavalce, D., Scott, P. M., and Lau, B. P.<br />
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Phytochemistry 53:377-378<br />
Mart<strong>in</strong>s, M. L., Mart<strong>in</strong>s, H. M., and Gimeno, A., 2003, Incidence of microflora and<br />
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by ochratox<strong>in</strong> A of green and roasted coffee beans, <strong>Food</strong> Addit. Contam.<br />
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determ<strong>in</strong>ation of ochratox<strong>in</strong> A <strong>in</strong> pure and adulterated soluble coffee us<strong>in</strong>g an<br />
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na cidade de Belo Horizonte, MG, Ciência e Tecnologia de Alimentos<br />
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MYCOBIOTA, MYCOTOXIGENIC FUNGI,<br />
AND CITRININ PRODUCTION IN BLACK<br />
OLIVES<br />
Dilek Heperkan, Burcak E. Meric, Gülc<strong>in</strong> Sismanoglu,<br />
Gözde Dalkiliç and Funda K. Güler *<br />
1. INTRODUCTION<br />
Olives have been reported to be a poor substrate for the production<br />
of aflatox<strong>in</strong>s (Mahjoub and Bullerman, 1987; Eltem, 1996;<br />
Leontopoulos et al., 2003). However Yassa et al., (1994) isolated A.<br />
flavus and A. parasiticus from black table olives <strong>in</strong> Egypt, from which<br />
n<strong>in</strong>e stra<strong>in</strong>s of A. flavus and five stra<strong>in</strong>s of A. parasiticus were found<br />
to produce aflatox<strong>in</strong> B 1 on olive paste. Toussa<strong>in</strong>t (1997) and<br />
Daradimos et al. (2000) found aflatox<strong>in</strong> B 1 <strong>in</strong> olive oil samples at levels<br />
of 5-10 µg/kg and 3-46 ng/kg respectively.<br />
Mahjoub and Bullerman (1987) found that mix<strong>in</strong>g olive paste with<br />
yeast extract sucrose agar caused a moderate <strong>in</strong>crease of aflatox<strong>in</strong> B 1<br />
production compared with yeast extract sucrose medium alone, and<br />
that heat and sodium hydroxide treatments stimulated growth and<br />
aflatox<strong>in</strong> production <strong>in</strong> olives. However, Leontopoulos et al. (2003)<br />
found higher levels of aflatox<strong>in</strong> produced <strong>in</strong> olives sanitized with<br />
chlor<strong>in</strong>e than <strong>in</strong> those sterilized by autoclav<strong>in</strong>g.<br />
* Dilek Heperkan, Burcak E. Meric, Gülc<strong>in</strong> Sismanoglu, Gözde Dalkiliç, and Funda<br />
K. Güler, Istanbul Technical University, Dept. of <strong>Food</strong> Eng<strong>in</strong>eer<strong>in</strong>g Istanbul, Turkey,<br />
34469 Maslak. Correspondence to: heperkan @itu.edu.tr<br />
203
204 Dilek Heperkan et al.<br />
Daradimos et al. (2000) highlighted the importance of methods for<br />
aflatox<strong>in</strong> analysis. Apply<strong>in</strong>g two different methods for determ<strong>in</strong>ation<br />
of aflatox<strong>in</strong> B 1 <strong>in</strong> olive oil, with one method they found 72% of the<br />
samples were contam<strong>in</strong>ated with aflatox<strong>in</strong> B 1 , whereas no aflatox<strong>in</strong> B 1<br />
was detected with the other method.<br />
Mycotox<strong>in</strong>s such as ochratox<strong>in</strong> and patul<strong>in</strong> (Gourama and<br />
Bullerman, 1987) were not produced by A. ochraceus, A. petrakii<br />
and P. expansum isolated from olives. However citr<strong>in</strong><strong>in</strong> production<br />
by Penicillium citr<strong>in</strong>um has been reported <strong>in</strong> olives (Oral and<br />
Heperkan, 1999).<br />
Citr<strong>in</strong><strong>in</strong> contam<strong>in</strong>ation of foods is important because citr<strong>in</strong><strong>in</strong> is<br />
nephrotoxic (Krogh et al., 1973; Frank, 1992) and genotoxic<br />
(Föllmann, et al., 1998) and enhances the effect of ochratox<strong>in</strong> A <strong>in</strong><br />
<strong>in</strong>duction of renal tumors <strong>in</strong> animals such as rats and mice (Pfohl-<br />
Leszkowicz et al., 2002). The LD 50 of citr<strong>in</strong><strong>in</strong> varies from 20 mg/kg<br />
b.w. by subcutaneous <strong>in</strong>jection <strong>in</strong> the rabbit to 112 mg/kg b.w. <strong>in</strong> mice<br />
after <strong>in</strong>traperitoneal adm<strong>in</strong>istration (Pfohl-Leszkowicz et al., 2002).<br />
Citr<strong>in</strong><strong>in</strong> is produced by some Penicillium, Aspergillus and Monascus<br />
species. Pitt (2002) <strong>in</strong>dicated that production of citr<strong>in</strong><strong>in</strong> has been<br />
reported from at least 22 Penicillium species, but the true number of<br />
producers was less than five. Citr<strong>in</strong><strong>in</strong> produc<strong>in</strong>g stra<strong>in</strong>s <strong>in</strong>clude P. citr<strong>in</strong>um,<br />
P. verrucosum (Frisvad and Thrane, 2002; Pitt and Hock<strong>in</strong>g,<br />
1997; Pitt, 2002), P. expansum (V<strong>in</strong>as et al., 1993) A. terreus (Frisvad<br />
and Thrane, 2002), Monascus ruber and M. purpureus (Blanc et al.,<br />
1995; Hajjaj et al., 1999; Xu et al., 1999).<br />
Penicillium and Aspergillus commonly occur on black olives<br />
(Fernandez et al., 1997; Sah<strong>in</strong> et al., 1999). Olives are grown ma<strong>in</strong>ly <strong>in</strong><br />
Spa<strong>in</strong>, Italy, Greece and Turkey and are used to produce olive oil or<br />
consumed directly. Fermented olives are an important product worldwide:<br />
table olive production and total olive production are approximately<br />
350,000 and 1,150,000 tonnes respectively per year <strong>in</strong> Turkey.<br />
Table olives comprise approximately 30% of Turkey’s total olive production.<br />
They have high economic value for the produc<strong>in</strong>g country as<br />
well as be<strong>in</strong>g an important source of nutrition.<br />
The process, by which olives are produced varies from country to<br />
country and table olives often take their name from the country of orig<strong>in</strong>,<br />
such as Turkish style, Greek style and Californian style, etc.<br />
Dur<strong>in</strong>g conventional olive production, the surface of the br<strong>in</strong>e may be<br />
covered with a thick layer of mould. Mould growth may also be visible<br />
on one end of black olives sold <strong>in</strong> transparent packages <strong>in</strong> the market.<br />
Mould growth can cause soften<strong>in</strong>g of the olive tissue, a mouldy<br />
taste and appearance and thus reduce the acceptable quality of olives.
Mycobiota and Citr<strong>in</strong><strong>in</strong> <strong>in</strong> Black Olives 205<br />
Moulds may also shorten the shelf life and produce mycotox<strong>in</strong>s. P. citr<strong>in</strong>um<br />
and P. crustosum have been isolated from the surface of olives<br />
dur<strong>in</strong>g fermentation (Oral and Heperkan, 1999).<br />
This study provides <strong>in</strong>formation on the mycoflora and citr<strong>in</strong><strong>in</strong> production<br />
<strong>in</strong> black table olives obta<strong>in</strong>ed from a survey conducted <strong>in</strong><br />
Turkey.<br />
2. MATERIALS AND METHODS<br />
2.1. Samples<br />
Whole black table olives were surveyed. A total of 69 samples were<br />
randomly collected from markets <strong>in</strong> the Marmara and Aegean<br />
Regions, Turkey <strong>in</strong> 2000-2001. Each sample comprised approximately<br />
2 kg and was ma<strong>in</strong>ta<strong>in</strong>ed at 4˚C until analyzed.<br />
2.2. Mycobiota<br />
Dilution plate techniques were used to determ<strong>in</strong>e the mycobiota.<br />
For the enumeration and isolation of moulds Malt Extract Agar with<br />
chloramphenicol was used. Plates were <strong>in</strong>cubated at 25˚C for 7 days.<br />
Moulds were identified accord<strong>in</strong>g to Pitt and Hock<strong>in</strong>g (1997) and<br />
Samson et al. (1996).<br />
2.3. Citr<strong>in</strong><strong>in</strong> Analysis<br />
Citr<strong>in</strong><strong>in</strong> standards were purchased from Sigma Chemical, St<br />
Louis, MO. Pre-coated silica gel TLC plates (Merck) were used for<br />
citr<strong>in</strong><strong>in</strong> analyses. The citr<strong>in</strong><strong>in</strong> standard was dissolved <strong>in</strong> chloroform<br />
and the UV absorption was read at 322 nm. The concentration of<br />
the solution was calculated us<strong>in</strong>g the formula for aflatox<strong>in</strong>s<br />
(Trucksess, 2000). The extraction and separation method of<br />
Comerio et al. (1998) was modified. Olive samples (25 g) were<br />
blended with acetonitrile (180 ml), 4% KCl (20 ml) and 20% H 2 SO 4<br />
(2 ml) for two m<strong>in</strong> at high speed and filtered through Whatman No<br />
4 filter paper. After filtration, hexane (50 ml) was added and the mixture<br />
was shaken for 15 m<strong>in</strong> <strong>in</strong> a separat<strong>in</strong>g funnel. The oil from the<br />
olives was extracted <strong>in</strong>to the hexane (upper) phase prevent<strong>in</strong>g it from<br />
<strong>in</strong>terfer<strong>in</strong>g with the assay. Citr<strong>in</strong><strong>in</strong> partitioned <strong>in</strong>to the lower phase.<br />
The first 100 ml of the lower phase was transferred to a second
206 Dilek Heperkan et al.<br />
separat<strong>in</strong>g funnel to which chloroform (50 ml) and distilled water (25<br />
ml) were added. After extraction, the tox<strong>in</strong> conta<strong>in</strong>ed <strong>in</strong> the lower<br />
phase was collected <strong>in</strong> a beaker and was evaporated to dryness under<br />
stream of nitrogen <strong>in</strong> water bath at approximately 55˚C. The dry<br />
extract was taken up <strong>in</strong> 1 ml chloroform <strong>in</strong> a tube and the chloroform<br />
removed under a stream of nitrogen. Before extracts were<br />
spotted, TLC plates were dipped <strong>in</strong>to 10% glycolic acid solution <strong>in</strong><br />
ethanol for two m<strong>in</strong>utes and then dried for 10 m<strong>in</strong> at 110˚C.<br />
The dried tox<strong>in</strong> extracts were dissolved <strong>in</strong> chloroform (100 µl) and<br />
were spotted onto a TLC plate us<strong>in</strong>g a micropipette. The plate was<br />
developed <strong>in</strong> a tank conta<strong>in</strong><strong>in</strong>g toluene:ethyl acetate:chloroform:90%<br />
formic acid (70:50:50:20), dried and treated with ammonia vapour for<br />
10-15 sec (Mart<strong>in</strong>s et al., 2002).<br />
Citr<strong>in</strong><strong>in</strong> was detected under UV light at 366 nm and the amount<br />
determ<strong>in</strong>ed visually by compar<strong>in</strong>g the fluorescence of the sample with<br />
the citr<strong>in</strong><strong>in</strong> standard. The recovery of citr<strong>in</strong><strong>in</strong> was determ<strong>in</strong>ed by spik<strong>in</strong>g<br />
a certa<strong>in</strong> amount of citr<strong>in</strong><strong>in</strong> standard e.g 100 µl <strong>in</strong>to 25 g of olive<br />
sample. Four parallel experiments were carried out and the mean<br />
amount of citr<strong>in</strong><strong>in</strong> was determ<strong>in</strong>ed. In these experiments the<br />
calculated average recovery value was 76.3%.<br />
3. RESULTS AND DISCUSSION<br />
Of the olive samples exam<strong>in</strong>ed, 17 of 42 (40%) and 15 of 27<br />
(55.5%) from the Marmara and Aegean Regions respectively were<br />
found to be contam<strong>in</strong>ated with moulds. The mould species isolated<br />
from the black olive samples are given <strong>in</strong> Table 1.<br />
As can be seen from Table 1, almost all the isolates were Penicillium<br />
species, with P. crustosum and P. viridicatum be<strong>in</strong>g the most common.<br />
No Aspergillus species were isolated <strong>in</strong> the Marmara Region. In the<br />
Aegean Region, A. versicolor was isolated from only 2 samples; the<br />
flora was ma<strong>in</strong>ly Penicillium spp. with P. roqueforti and P. viridicatum<br />
be<strong>in</strong>g most frequently detected. The citr<strong>in</strong><strong>in</strong> content of samples is<br />
shown <strong>in</strong> Table 2.<br />
Citr<strong>in</strong><strong>in</strong> was detected <strong>in</strong> 34 of the 42 samples (81%) from the<br />
Marmara Region; the amount of citr<strong>in</strong><strong>in</strong> varied from a m<strong>in</strong>imum of<br />
75 and a maximum of 350 µg/kg. In the Aegean Region 20 of the 27<br />
(74%) samples conta<strong>in</strong>ed citr<strong>in</strong><strong>in</strong> with the highest value be<strong>in</strong>g 100<br />
µg/kg. There was a large difference between the citr<strong>in</strong><strong>in</strong> amounts <strong>in</strong><br />
the two regions, with the Marmara Region hav<strong>in</strong>g much higher levels.
Mycobiota and Citr<strong>in</strong><strong>in</strong> <strong>in</strong> Black Olives 207<br />
Table 1. Mould flora <strong>in</strong> black olives<br />
Number of positive samples<br />
Mould species Marmara Region Aegean Region<br />
Aspergillus versicolor − 2<br />
Cladosporium spp. 2 3<br />
Penicillium citr<strong>in</strong>um 2 −<br />
P. crustosum 10 2<br />
P. digitatum 2 2<br />
P. roqueforti 2 14<br />
P. viridicatum 5 4<br />
P. solitum − 2<br />
P. brevicompactum − 3<br />
Total stra<strong>in</strong>s isolated 23 32<br />
Table 2. Distribution of citr<strong>in</strong><strong>in</strong> <strong>in</strong> black olives<br />
Concentration of Number of positive samples<br />
citr<strong>in</strong><strong>in</strong> (µg/kg) Marmara Region Aegean Region<br />
208 Dilek Heperkan et al.<br />
production was assumed to have taken place dur<strong>in</strong>g the residence <strong>in</strong><br />
the concrete vats under adverse conditions. Studies on other products<br />
<strong>in</strong> Turkey also show the frequent occurrence of P. citr<strong>in</strong>um. P. citr<strong>in</strong>um<br />
was isolated from 23% of pistachio nuts (Heperkan et al., 1994),<br />
27.5% of stored corn (Özay and Heperkan, 1989) and 14.3% of animal<br />
feed (Heperkan and Alperden, 1988).<br />
Although citr<strong>in</strong><strong>in</strong> is considered a m<strong>in</strong>or mycotox<strong>in</strong> compared with<br />
aflatox<strong>in</strong>s, ochratox<strong>in</strong> A, fumonis<strong>in</strong>s, patul<strong>in</strong>, zearalenone and<br />
deoxynivalenol (Miller, 1995) it is moderately toxic: <strong>in</strong> humans kidney<br />
damage appears to be a likely result of prolonged <strong>in</strong>gestion (Pitt,<br />
2002). Citr<strong>in</strong><strong>in</strong> co-occurs with ochratox<strong>in</strong> A (Vrabcheva et al., 2000)<br />
and patul<strong>in</strong> (Mart<strong>in</strong>s et al., 2002) and is nephrotoxic and teratogenic<br />
<strong>in</strong> test animals (Abramson, 1999).<br />
4. CONCLUSIONS<br />
The results of our study and the literature survey <strong>in</strong>dicate that<br />
olives, which are widely consumed <strong>in</strong> Turkey, could be a significant<br />
source of mycotox<strong>in</strong>s <strong>in</strong> the human diet: our survey found 77% of the<br />
samples conta<strong>in</strong>ed citr<strong>in</strong><strong>in</strong>, with some relatively high amounts<br />
detected. As contam<strong>in</strong>ation occurs ma<strong>in</strong>ly dur<strong>in</strong>g production, olive<br />
production methods should be reviewed. Residence <strong>in</strong> vats should be<br />
elim<strong>in</strong>ated or mould growth prevented dur<strong>in</strong>g this stage of production.<br />
Storage and market<strong>in</strong>g stages should also be exam<strong>in</strong>ed, and<br />
procedures to prevent mould contam<strong>in</strong>ation should be developed.<br />
Although there are no limits for citr<strong>in</strong><strong>in</strong> set by either the Turkish<br />
government or the European Union, some countries may choose to set<br />
more str<strong>in</strong>gent levels based on dietary <strong>in</strong>take of their populations<br />
(Park and Troxell, 2002). The presence of mycotox<strong>in</strong>s <strong>in</strong> food can<br />
cause health problems and endanger the commercial value of the<br />
product. Therefore susta<strong>in</strong>able strategies such as Good Agricultural<br />
Practices, (GAP) and Hazard Analyses Critical Control Po<strong>in</strong>t<br />
(HACCP) systems should be established.<br />
5. REFERENCES<br />
Abramson, D., 1999, Rapid determ<strong>in</strong>ation of citr<strong>in</strong><strong>in</strong> <strong>in</strong> corn by fluorescence liquid<br />
chromatography and enzyme immunoassay, J. AOAC Int. 82:1353-1355.
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Blanc, P. J., Laussac, J. P., Le Bars, J., Le Bars, P., Loret, M. O., Pareilleux, A., Prome,<br />
D., Prome, J. C., Santerre, A. L., and Goma, G., 1995, Characterization of<br />
monascid<strong>in</strong> A from Monascus as citr<strong>in</strong><strong>in</strong>, Int. J. <strong>Food</strong> Microbiol. 27:201-213.<br />
Comerio, R., P<strong>in</strong>to, V. E. F., and Vamonde, G., 1998, Influence of water activity on<br />
Penicillium citr<strong>in</strong>um growth and k<strong>in</strong>etics of citr<strong>in</strong><strong>in</strong> accumulation <strong>in</strong> wheat, Int.<br />
J. <strong>Food</strong> Microbiol. 42:219-223.<br />
Daradimos, E., Markaki, P., and Koupparis, M., 2000, Evaluation and validation of<br />
two fluorometric HPLC methods for the determ<strong>in</strong>ation of aflatox<strong>in</strong> B 1 <strong>in</strong> olive oil,<br />
<strong>Food</strong> Addit. Contam. 17:65-63.<br />
Eltem, R., 1996, Growth and aflatox<strong>in</strong> B 1 production on olives and olive paste by<br />
moulds isolated from ‘Turkish style’ natural black olives <strong>in</strong> br<strong>in</strong>e, Int. J. <strong>Food</strong><br />
Microbiol. 32:217-223.<br />
Fernandez, G. A., Diez, M. J. F., and Adams, M. R., 1997, Table Olives: Production<br />
and Process<strong>in</strong>g, Chapman and Hall, London.<br />
Föllmann, W., Dorrenhaus, A., and Bolt, H. M., 1998, Induktion von mutationen im<br />
HPRT-Gen, DNA-reparatursynthese und schwesterchromatidaustausch durch<br />
ochratox<strong>in</strong> A und citr<strong>in</strong><strong>in</strong> <strong>in</strong> vitro, <strong>in</strong>: Proceed<strong>in</strong>gs Mycotox<strong>in</strong> Workshop, J.Wolff,<br />
T. Betsche., eds, Detmold, Germany, June 8-10.<br />
Frank, H. K., 1992, Citr<strong>in</strong><strong>in</strong>, Zeits. Ernahrungswiss. 31:164-177.<br />
Frisvad, J. C., and Thrane, U., 2002, Mycotox<strong>in</strong> production by food-borne fungi, <strong>in</strong>:<br />
Introduction to <strong>Food</strong> Borne Fungi, R. A. Samson, E. S. Hoekstra, J. C. Frisvad, and<br />
O. Filtenborg, eds, 6th edition, Centraalbureau voor Schimmelcultures, CBS,<br />
Delft, pp. 251-261.<br />
Gourama, H., and Bullerman, L. B., 1987, Mycotox<strong>in</strong> production by moulds isolated<br />
from Greek-style black olives, Int. J. <strong>Food</strong> Microbiol. 1:81-90.<br />
Hajjaj, H., Blanc, P. J., Groussac, E., Goma, G., Uribellarrea, J. I. and Loubiere, P.,<br />
1999, Improvement of red pigment citr<strong>in</strong><strong>in</strong> production ratio as a function of environmental<br />
conditions by Monascus ruber, Biotechnol. Bioeng. 64:497-501.<br />
Heperkan, D., and Alperden, I., 1988, Mycological survey of chicken feed and feed<br />
<strong>in</strong>gredients <strong>in</strong> Turkey, J. <strong>Food</strong> Prot. 51:807-810.<br />
Heperkan, D., Aran, N., and Ayfer, M., 1994, Mycoflora and aflatox<strong>in</strong> contam<strong>in</strong>ation<br />
<strong>in</strong> shelled pistachio nuts, J. Sci. <strong>Food</strong> Agric. 66:273-278.<br />
Korukluogˇlu, M., Gürbüz, O., Uylas¸er, V., Yildirim, A., and Sah<strong>in</strong>, I., 2000, Gemlik<br />
tipi zeyt<strong>in</strong>lerde mikotoks<strong>in</strong> kirlilig<strong>in</strong><strong>in</strong> arastirilmasi, Türkiye 1. Zeyt<strong>in</strong>cilik<br />
Sempozyumu. Uludagˇ Üniversitesi 6-9 Haziran, Bursa. Bildiri Kitabi, pp. 218-218.<br />
Krogh, P., Hald, B., and Pedersen, E.J., 1973, Occurrence of ochratox<strong>in</strong> A and citr<strong>in</strong><strong>in</strong><br />
<strong>in</strong> cereals associated with mycotoxic porc<strong>in</strong>e nephropathy, Acta. Path. Microbiol.<br />
Scand. Sect. B. 81:689-695.<br />
Leontopoulos, D., Siafaka, A., Markaki, P., 2003, Black olives as substrate for Aspergillus<br />
parasiticus growth and aflatox<strong>in</strong> B 1 production, <strong>Food</strong> Microbiol. 20:119-126.<br />
Mahjoub, A., and Bullerman, L. B., 1987, Effects of nutrients and <strong>in</strong>hibitors <strong>in</strong> olives<br />
on aflatoxigenic molds, J. <strong>Food</strong> Prot. 50:959-963.<br />
Mart<strong>in</strong>s, M. L., Gimeno, A., Mart<strong>in</strong>s, H. M., and Bernardo, F., 2002, Co-occurrence<br />
of patul<strong>in</strong> and citr<strong>in</strong><strong>in</strong> <strong>in</strong> Portuguese apples with rotten spots, <strong>Food</strong> Addit. Contam.<br />
19:568-574.<br />
Miller, J. D., 1995, Fungi and mycotox<strong>in</strong>s <strong>in</strong> gra<strong>in</strong>: implications for stored product<br />
research, J. Stored Prod. Res. 31:1-16.<br />
Oral, J., and Heperkan, D., 1999, Penicillic acid and citr<strong>in</strong><strong>in</strong> production <strong>in</strong> olives, <strong>in</strong>:<br />
<strong>Food</strong> Microbiology and <strong>Food</strong> Safety <strong>in</strong>to the Next Millenium. Proceed<strong>in</strong>gs of the
210 Dilek Heperkan et al.<br />
17th International ICFMH Conference. A. C. J. Tuijtelaars, R. A. Samson, F. M.<br />
Rombouts, S. Notermans, eds, Veldhoven, The Netherlands, pp. 138-140.<br />
Özay, G., and Heperkan, D., 1989, Mould and mycotox<strong>in</strong> contam<strong>in</strong>ation of stored<br />
corn <strong>in</strong> Turkey, Mycotox<strong>in</strong> Res. 5:81-89.<br />
Park, D. L., and Troxell, T. C., 2002, U.S. perspective on mycotox<strong>in</strong> regulatory issues,<br />
<strong>in</strong>: Mycotox<strong>in</strong>s and <strong>Food</strong> Safety. J. W. DeVries, M. W. Trucksess, and L. S. Jackson,<br />
eds, Kluwer Academic / Plenum Publishers, New York, pp. 277-285.<br />
Pfohl-Leszkowicz, A., Petkova-Bocharova, T., Chernozemsky, I. N., and Castegnaro,<br />
M., 2002, Balkan endemic nephropathy and associated ur<strong>in</strong>ary tract tumors: a<br />
review on aetiological causes and potential role of mycotox<strong>in</strong>s, <strong>Food</strong> Addit.<br />
Contam. 19:282-302.<br />
Pitt, J. I., 2002, Biology and ecology of toxigenic Penicillium species, <strong>in</strong>: Mycotox<strong>in</strong>s<br />
and <strong>Food</strong> Safety. J. W. DeVries, M. W. Trucksess, and L. S. Jackson., eds, Kluwer<br />
Academic / Plenum Publishers, New York, pp. 29-41.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Penicillium and related genera, <strong>in</strong>: Fungi and <strong>Food</strong><br />
Spoilage, 2nd edition, Blackie Academic and Professional, London, pp. 251-255.<br />
Samson, R. A., Hoekstra, E. S., Frisvad, J. C., and Filtenborg, O., 1996, Introduction<br />
to <strong>Food</strong> Borne Fungi. 6th edition, Centraalbureau voor Schimmelcultures, Baarn,<br />
The Netherlands.<br />
Sah<strong>in</strong>, I., Basoglu, F., Korukluoglu, M., and Göcmen, D., 1999, Salamura siyah<br />
zeyt<strong>in</strong>lerde rastlanan küfler ve mikotoks<strong>in</strong> riskleri, Kükem Dergisi 22(2):1-8.<br />
Toussa<strong>in</strong>t, G., Lafaverges, F., and Walker, E. A., 1997, The use of high pressure liquid<br />
chromatography for determ<strong>in</strong>ation of aflatox<strong>in</strong> <strong>in</strong> olive oil, Arch. Inst. Pasteur,<br />
Tunis 3-4:325-334.<br />
Trucksess, M. W., 2000, Committee on natural tox<strong>in</strong>s, General referee reports,<br />
J. AOAC Int. 83:442-448<br />
V<strong>in</strong>as, I., Dadon, J., and Sanchis, V., 1993, Citr<strong>in</strong><strong>in</strong> production capacity of Penicillium<br />
expansum stra<strong>in</strong>s from apple packag<strong>in</strong>g houses of Lerida (Spa<strong>in</strong>), Int. J. <strong>Food</strong><br />
Microbiol. 19:153-156.<br />
Vrabcheva, T., Usleber, E., Dietrich, R., and Martlauber, E., 2000, Co-ocurrence of<br />
ochratox<strong>in</strong> A and citr<strong>in</strong><strong>in</strong> <strong>in</strong> cereals from Bulgarian villages with a history of<br />
Balkan endemic nephropathy, J. Agric. <strong>Food</strong> Chem. 48: 2483-2488.<br />
Xu, Y., Li, Y., Sun, H., Lai, W., and Xu, E., 1999, Study on the production of citr<strong>in</strong><strong>in</strong><br />
<strong>in</strong> submerged cultures of Monascus spp., <strong>in</strong>: <strong>Food</strong> Microbiology and <strong>Food</strong> Safety<br />
Into the Next Millenium. Proceed<strong>in</strong>gs of the 17th International ICFMH Conference,<br />
A. C. J. Tuijtelaars, R. A. Samson, F. M. Rombouts and S. Notermans, eds,<br />
Veldhoven, The Netherlands, pp. 150.<br />
Yassa, I. A., Abdalla, E. A. M., and Aziz, S. Y., 1994, Aflatox<strong>in</strong> B 1 production by<br />
moulds isolated from black table olives, Ann. Agric. Sci. 39:525-537.
BYSSOCHLAMYS: SIGNIFICANCE OF HEAT<br />
RESISTANCE AND MYCOTOXIN<br />
PRODUCTION<br />
Jos Houbraken, Robert A. Samson and Jens C. Frisvad *<br />
1. INTRODUCTION<br />
Byssochlamys species produce ascospores that are very heat-resistant<br />
and survive heat<strong>in</strong>g above 85°C for considerable periods (Beuchat<br />
and Rice, 1981; Splittstoesser, 1987). Besides their heat resistance,<br />
Byssochlamys species are also able to grow under very low oxygen tensions<br />
(Tanawaki, 1995) and are capable of produc<strong>in</strong>g pect<strong>in</strong>olytic<br />
enzymes. The comb<strong>in</strong>ation of these three physiological characteristics<br />
make Byssochlamys species very important spoilage fungi <strong>in</strong> pasteurized<br />
and canned fruit <strong>in</strong> which they can cause great economical losses.<br />
The natural habitat of Byssochlamys is soil. Fruit that grow near the<br />
soil, or are harvested from the ground may thus become contam<strong>in</strong>ated<br />
with Byssochlamys (Olliver and Rendle, 1934; Hull, 1939).<br />
Besides caus<strong>in</strong>g spoilage of pasteurized products, some<br />
Byssochlamys species are also capable of produc<strong>in</strong>g mycotox<strong>in</strong>s,<br />
<strong>in</strong>clud<strong>in</strong>g patul<strong>in</strong>, byssotox<strong>in</strong> A and byssochlamic acid (Kramer et al.,<br />
1976; Rice, 1977). An antitumor metabolite, byssochlamysol, a steroid<br />
aga<strong>in</strong>st IGF-1 dependent cancer cells, is also produced by<br />
Byssochlamys nivea (Mori et al. 2003).<br />
*J. Houbraken and R. A. Samson: Centraalbureau voor Schimmelcultures, P.O. Box<br />
85167, 3508 AD, Ultrecht, Netherlands. J. C. Frisvad: Centre for Process<br />
Biotechnology, Biocentrum-DTU, Technical University of Denmark, 2800 Kgs.<br />
Lyngby, Denmark. Correspondence to: samson@cbs.knaw.nl<br />
211
212 Jos Houbraken et al.<br />
In the taxonomic revision of Samson (1974), three Byssochlamys<br />
species were accepted: B. fulva, B. nivea and B. zollerniae. B. verrucosa<br />
was subsequently described (Samson and Tansey, 1975). Only B. nivea<br />
and B. fulva are currently considered important food spoilage fungi or<br />
mycotox<strong>in</strong> producers.<br />
Recently we have encountered numerous food spoilage problems <strong>in</strong><br />
which species of Byssochlamys and their Paecilomyces anamorphs<br />
were <strong>in</strong>volved. To elucidate the taxonomic and ecological characteristics<br />
of these isolates <strong>in</strong> relation to heat resistance and mycotox<strong>in</strong> production<br />
we have <strong>in</strong>vestigated Byssochlamys and Paecilomyces isolates<br />
from various orig<strong>in</strong>s, <strong>in</strong>clud<strong>in</strong>g pasteurized fruit, <strong>in</strong>gredients based on<br />
fruit, and soil. Us<strong>in</strong>g a polyphasic approach, a classification of foodrelated<br />
heat resistant Byssochlamys species is presented.<br />
2. MATERIALS AND METHODS<br />
2.1. Isolates<br />
The 39 isolates of Byssochlamys and Paecilomyces studied are<br />
listed <strong>in</strong> Table 1. All isolates are ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong> the collection of the<br />
Centraalbureau voor Schimmelcultures (CBS), Utrecht, Netherlands.<br />
2.2. Media<br />
The follow<strong>in</strong>g media were used <strong>in</strong> this study: Czapek Yeast<br />
Autolysate agar (CYA), Malt Extract Agar (MEA), Potato Dextrose<br />
Agar (PDA), Yeast Extract Sucrose agar (YES), Hay <strong>in</strong>fusion agar<br />
(HAY), Cornmeal agar (CMA), Creat<strong>in</strong>e sucrose agar (CREA)<br />
(Samson et al., 2004) and Alkaloid agar (ALK) (V<strong>in</strong>okurova et al.).<br />
2.3. Macromorphological characterisation<br />
The macroscopical features used were acid production on CREA,<br />
colony diameter on MEA after 72 h. and colony diameter and degree<br />
of growth on CYA after 7 days at 30°C.<br />
2.4. Micromorphological characterisation<br />
Isolates were grown on several media for 5-70 d. Micromorphology<br />
of the anamorphic Paecilomyces states was characterised on MEA,
Byssochlamys Heat Resistance and Mycotox<strong>in</strong>s 213<br />
Table 1. Byssochlamys and Paecilomyces isolates used <strong>in</strong> this study<br />
CBS<br />
Asco-<br />
a No. Species Source, remarks about culture spores<br />
132.33 Byssochlamys fulva Bottled fruit, Type of<br />
Paecilomyces fulvus<br />
+<br />
146.48 Byssochlamys fulva Bottled fruit, Type of<br />
Byssochlamys fulva<br />
−<br />
135.62 Byssochlamys fulva Fruit juice, Type of<br />
Paecilomyces todicus<br />
+<br />
604.71 Byssochlamys fulva Unknown source −<br />
113245 Byssochlamys fulva Pasteurized fruit juice +<br />
113225 Byssochlamys fulva Multifruit juice +<br />
100.11 Byssochlamys nivea Unknown source, Type of<br />
Byssochlamys nivea<br />
+<br />
133.37 Byssochlamys nivea Milk of cow, Type of<br />
Arachniotus trisporus<br />
−<br />
606.71 Byssochlamys nivea Oat gra<strong>in</strong>, received as<br />
Byssochlamys musticola<br />
−<br />
546.75 Byssochlamys nivea Unknown source +<br />
271.95 Byssochlamys nivea Mushroom bed +<br />
102192 Byssochlamys nivea Pasteurized dr<strong>in</strong>k yoghurt +<br />
113246 Byssochlamys nivea Apple compote +<br />
373.70 Byssochlamys Wood of Laguncularia racemosa +<br />
lagunculariae (Mangue), Type of B. nivea var.<br />
lagunculariae<br />
696.95 Byssochlamys<br />
lagunculariae<br />
Pasteurized strawberries +<br />
338.51 Byssochlamys<br />
spectabilis<br />
Fruit juice −<br />
102.74 Byssochlamys Unknown source, Type of −<br />
spectabilis Paecilomyces variotii<br />
298.93 Byssochlamys<br />
spectabilis<br />
Man, breast milk of patient −<br />
101075 Byssochlamys Heat processed fruit beverage, +<br />
spectabilis Type of Talaromyces spectabilis<br />
109072 Byssochlamys<br />
spectabilis<br />
Pect<strong>in</strong>, teleomorph present +<br />
109073 Byssochlamys<br />
spectabilis<br />
Pect<strong>in</strong>, teleomorph present +<br />
110431 Byssochlamys<br />
spectabilis<br />
Rye bread −<br />
284.48 Byssochlamys Mucilage bottle with library −<br />
divaricatum paste,<br />
Type of Penicillium divaricatum <strong>in</strong>itials<br />
110428 Byssochlamys<br />
divaricatum<br />
Pect<strong>in</strong> −, <strong>in</strong>itials<br />
110429 Byssochlamys<br />
divaricatum<br />
Pect<strong>in</strong> −, <strong>in</strong>itials
214 Jos Houbraken et al.<br />
Table 1. Byssochlamys and Paecilomyces isolates used <strong>in</strong> this study—cont’d<br />
CBS<br />
Asco-<br />
a No. Species Source, remarks about culture spores<br />
110430 Byssochlamys<br />
divaricatum<br />
Pect<strong>in</strong> +<br />
604.74 Byssochlamys Nest<strong>in</strong>g material of Leipoa +<br />
verrucosa ocellata<br />
605.74 Byssochlamys Nest<strong>in</strong>g material of Leipoa +<br />
verrucosa ocellata, Type<br />
374.70 Byssochlamys Wood of Zollernia ilicifolia +<br />
zollerniae and Protium heptaphyllum, Type<br />
628.66 Paecilomyces Quebracho-tanned sheep −<br />
maximus leather, France<br />
371.70 Paecilomyces Annona squamosa, Brazil, −<br />
maximus Type of Paecilomyces maximus<br />
990.73B Paecilomyces Unknown source, Type of −<br />
maximus Monilia Formosa<br />
296.93 Paecilomyces<br />
maximus<br />
Man, bone marrow of patient −<br />
297.93 Paecilomyces<br />
maximus<br />
Man, blood of patient −<br />
323.34 Paecilomyces Unknown source, Type of −<br />
dactylethromorphus Paecilomyces mandshuricus<br />
var. saturatus<br />
223.52 Paecilomyces<br />
dactylethromorphus<br />
Leather −<br />
251.55 Paecilomyces Acetic acid, Type of −<br />
dactylethromorphus Paecilomyces dactylethromorphus<br />
990.73A Paecilomyces Unknown source, Type of −<br />
dactylethromorphus Penicillium v<strong>in</strong>iferum<br />
492.84 Paecilomyces<br />
dactylethromorphus<br />
Lepidium sativum −<br />
aCBS is the culture collection of the Centraalbureau voor Schimmelcultures, Utrecht,<br />
Netherlands<br />
HAY and YES agars (Samson et al., 2004). The latter was used for the<br />
determ<strong>in</strong>ation of the presence of chlamydospores. For the analyses of<br />
the features of the teleomorphic Byssochlamys state, OA and PDA<br />
(Samson et al., 2004) were used. The microscopical features recorded<br />
<strong>in</strong>cluded shape and size of conidia, size and ornamentation of<br />
ascospores and presence and ornamentation of chlamydospores.<br />
Ornamentation of the surface of the conidia, chlamydospores and<br />
ascospores was determ<strong>in</strong>ed by light microscopy after prolonged<br />
<strong>in</strong>cubation up to 70 days.
Byssochlamys Heat Resistance and Mycotox<strong>in</strong>s 215<br />
2.5. Multivariate analyses<br />
A matrix consist<strong>in</strong>g of 39 objects (fungal isolates) and 10 variables<br />
(macro- and microscopical features) was constructed. Cluster analysis<br />
by unweighted pair-group method, arithmetic average (UMPGA) was<br />
performed on the data matrix us<strong>in</strong>g BIOLOMICS software<br />
(Bioaware S.A., Hannut, Belgium).<br />
2.6. Secondary metabolite analysis<br />
Isolates studied (see Table 1) were three po<strong>in</strong>t <strong>in</strong>oculated on MEA,<br />
YES, PDA, OA, CYA and ALK agars. All isolates were analysed for<br />
secondary metabolites after two weeks growth at 30°C. The cultures<br />
were extracted accord<strong>in</strong>g to the method of Smedsgaard (1997) and<br />
analysed by HPLC with diode array detection (Frisvad and Thrane,<br />
1993). The metabolites found were compared with a spectral UV<br />
library made from authentic standards run under the same conditions,<br />
and retention <strong>in</strong>dices were compared with those of standards. The<br />
maximal similarity was a match of 1000.<br />
2.7. Heat resistance<br />
The stra<strong>in</strong> Talaromyces spectabilis CBS 109073 (now considered to<br />
be a Byssochlamys species, see below) was <strong>in</strong>oculated at three po<strong>in</strong>ts<br />
on CMA and <strong>in</strong>cubated for 45 days at 30°C. After <strong>in</strong>cubation, the<br />
parts of the colony where ascospores were produced were comb<strong>in</strong>ed<br />
and transferred to 10 mM ACES buffer (pH 6.8, N-[2-acetamido]-<br />
2-am<strong>in</strong>oethane-sulfonic acid; Sigma) supplemented with 0.05% Tween<br />
80. The <strong>in</strong>tact asci were ruptured by suction through a 0.9 mm hypodermic<br />
needle with a syr<strong>in</strong>ge and by agitation with glass beads. The<br />
suspension was sonicated briefly (3 times for 30 s) and filtered through<br />
sterile glass wool. The ascospores together with conidia and other fungal<br />
fragments were centrifuged at 1,100×g (5 m<strong>in</strong>) and washed three<br />
times <strong>in</strong> buffer.<br />
D values <strong>in</strong> ACES buffer were determ<strong>in</strong>ed at 85°C <strong>in</strong> duplicate. The<br />
spore suspension was pre-treated by heat<strong>in</strong>g at 65°C for 10 m<strong>in</strong> to<br />
elim<strong>in</strong>ate conidia, chlamydospores and hyphae. The suspension was<br />
then heated at 85°C at various times (see Figure 2). After heat<strong>in</strong>g, the<br />
suspension was cooled, serially diluted <strong>in</strong> sterile water, then spread<br />
plated onto CYA and <strong>in</strong>cubated for 5-10 days at 30°C. Colonies were<br />
counted and D 85 values calculated.
216 Jos Houbraken et al.<br />
3. RESULTS<br />
3.1. Morphological analyses<br />
Paecilomyces variotii sensu lato and anamorphs of Byssochlamys<br />
species share several micromorphological characteristics, <strong>in</strong>clud<strong>in</strong>g<br />
phialides with cyl<strong>in</strong>drical bases taper<strong>in</strong>g abruptly <strong>in</strong>to long cyl<strong>in</strong>drical<br />
necks. The conidia are produced <strong>in</strong> long divergent cha<strong>in</strong>s<br />
(Samson, 1974). There are characters that are constant at the species<br />
level but dist<strong>in</strong>ct between species. Microscopical features such as the<br />
shape of the conidia, the sizes of the conidia and ascospores and the<br />
presence of chlamydospores can be used to group species belong<strong>in</strong>g<br />
to Byssochlamys and Paecilomyces. Acid production on CREA,<br />
colony diameters and degree of growth on CYA are useful characters<br />
too. Table 2 summarises the results of the micro- and macroscopical<br />
analyses.<br />
The classification of the Byssochlamys and Paecilomyces taxa based<br />
on phenotypical characters is also supported by a molecular taxonomic<br />
study of this complex (partial β-tubul<strong>in</strong> gene sequenc<strong>in</strong>g)<br />
(Samson et al., submitted).<br />
Figure 1 shows that n<strong>in</strong>e clades could be dist<strong>in</strong>guished among the<br />
isolates studied (Table 1). Clades 1, 2 4 and 5 separate the four known<br />
species of Byssochlamys: B. verrucosa, B. zollerniae, B. fulva and<br />
B. nivea. Clade 6 <strong>in</strong>cludes the ex-type culture of B. nivea var. lagunculariae<br />
(CBS 373.70) and CBS 696.95, isolated from strawberries.<br />
Clades 3, 7, 8 and 9 are isolates have been classified <strong>in</strong> Paecilomyces<br />
variotii complex, now seen to <strong>in</strong>clude several taxa. Clade 8 <strong>in</strong>cludes<br />
the ex-type cultures of Paec. variotii and Talaromyces spectabilis. Reexam<strong>in</strong>ation<br />
of the ex-type culture of T. spectabilis shows that it logically<br />
belongs <strong>in</strong> Byssochlamys and the formal comb<strong>in</strong>ation<br />
Byssochlamys spectabilis (Udagawa & Suzuki) Samson et al. is proposed<br />
(Samson et al., submitted). Stra<strong>in</strong>s which produce ascospores<br />
are rare and if ascospores are formed, they often develop only after<br />
prolonged <strong>in</strong>cubation. In this clade many stra<strong>in</strong>s isolated from<br />
dr<strong>in</strong>k<strong>in</strong>g yoghurt and pect<strong>in</strong> are accommodated.<br />
Clade 3 conta<strong>in</strong>s the ex-type culture of Penicillium divaricatum. Pen.<br />
divaricatum Thom 1910 was considered to be a synonym of Paec. variotii<br />
by Thom (1930). In one isolate, CBS 110430, we have observed<br />
ascospore production of the Byssochlamys type after prolonged<br />
<strong>in</strong>cubation (70 d). We have therefore erected the new name<br />
Byssochlamys divaricata Samson et al. for Pen. divaricatum (Samson<br />
et al., submitted). The stra<strong>in</strong>s exam<strong>in</strong>ed were isolated from pect<strong>in</strong> and
Byssochlamys Heat Resistance and Mycotox<strong>in</strong>s 217<br />
Table 2. Macro- and microscopical features of Byssochlamys and Paecilomyces isolates<br />
Ascospore size Colony<br />
Conidial size Chlamydo- (µm) and diameter Degree<br />
Species (µm) and shape sporesa ornamentation (mm) b of growthc Acidd B. divaricatum 3.2-4.6 × 1.6-2.5; − (+) 5.3-7.0 × 3.8-4.9, 10-17 Moderate −<br />
ellipsoidal to smooth<br />
cyl<strong>in</strong>drical with<br />
truncate ends<br />
B. fulva 3.7-7.5 × 1.4-2.5; cyl<strong>in</strong>drical − (+) 5.3-7.1 × 3.3-4.3, (50), >80 Good +<br />
with truncate ends smooth<br />
B. lagunculariae 2.7-4.5 × 2.2-3.3; globose +, smooth 3.8-5.0 × 3.0-3.9, 45-55 Good −<br />
with flattened base smooth<br />
B. nivea 3.0-4.7 × 2.3-4.0; globose to +, smooth to 4.1-5.5 × 2.9-3.9, (8) 28-50 Weak − (+)<br />
ellipsoidal with flattened base f<strong>in</strong>ely smooth<br />
roughened<br />
B. spectabilis 3.3-6.1 × 1.5-4.4; mostly +, smooth to 5.2-6.8 × 3.5-4.5, 25-40 (56) Good −<br />
ellipsoidal and ellipsoidal f<strong>in</strong>ely almost smooth, sl.<br />
with truncated ends roughened roughened<br />
B. verrucosa 6.3-13.1 × 1.6-4.7; − 6.6-8.4 × 4.0-6.1, 25-40 Good −<br />
cyl<strong>in</strong>drical with truncate ends rough<br />
B. zollerniae 2.5-4.0 × 1.5-3.0; globose to +, warted 3.0-4.5 × 2.5-3.0, 30-35 Weak −<br />
ellipsoidal, apiculate smooth<br />
P. dactylethro- 2.3-7.0 × 1.7-3.4; mostly +, smooth No ascospores 22-55 Good −<br />
morphus cyl<strong>in</strong>drical and ellipsoidal detected<br />
without truncated ends<br />
P. maximus 3.0-10 × 1.8-3.5; ellipsoidal +, smooth No ascospores 18 − >80 Good +<br />
to cyl<strong>in</strong>drical with truncate and often detected<br />
ends pigmented<br />
a +, chlamydospores present; −, chlamydospores absent, (+) chlamydospores produced by some isolates after prolonged <strong>in</strong>cubation (40 days); b<br />
Colony diameter on CYA, 72 h, 30°C; c Degree of growth on CYA, 7 d, 30°C; d Acid production <strong>in</strong> CREA, 7 d, 30°C
218 Jos Houbraken et al.<br />
0.175 0.572 0.429 0.286 0.143 0<br />
CBS 605.74 B.venucosa<br />
CBS 604.74 B.venucosa<br />
CBS 374.70 B.zollemiae<br />
CBS 284.48 B.divaricatum<br />
CBS 110430 B.divaricatum<br />
CBS 110428 B.divaricatum<br />
CBS 110429 B.divaricatum<br />
CBS 604.71 B.fulva<br />
CBS 113225 B.fulva<br />
CBS 132.33 B.fulva<br />
CBS 146.48 B.fulva<br />
CBS 113245 B.fulva<br />
CBS 135.62 B.fulva<br />
CBS 271.95 B.nivea<br />
CBS 113246 B.nivea<br />
CBS 606.71 B.nivea<br />
CBS 102192 B.nivea<br />
CBS 546.75 B.nivea<br />
CBS 133.37 B.nivea<br />
CBS 100.11 B.nivea<br />
CBS 696.95 B.lagunculariae<br />
CBS 373.70 B.lagunculariae<br />
CBS 990.73A P.dactylethromorplus<br />
CBS 323.34 P.dactylethromorplus<br />
CBS 251.55 P.dactylethromorplus<br />
CBS 492.84 P.dactylethromorplus<br />
CBS 223.52 P.dactylethromorplus<br />
CBS 102.74 B.spectabilis<br />
CBS 298.73 B.spectabilis<br />
CBS 109073 B.spectabilis<br />
CBS 338.51 B.spectabilis<br />
CBS 110431 B.spectabilis<br />
CBS 109072 B.spectabilis<br />
CBS 101075 B.spectabilis<br />
CBS 990.73B P.maximus<br />
CBS 296.93 P.maximus<br />
CBS 297.93 P.maximus<br />
CBS 628.66 P.maximus<br />
CBS 371.70 P.maximus<br />
Figure 1. An UPGMA dendrogram based upon micro- and macromorphological<br />
characteristics of Paecilomyces variotii and Byssochlamys isolates.<br />
fruit concentrates, and the ex-type culture came from a mucilage bottle<br />
with library paste. Clade 7 <strong>in</strong>cludes the ex-type cultures of Paec.<br />
dactylethromorphus Batista & H. Maia and Paec. mandshuricus var.<br />
saturatus Nakazawa et al. while the ex-type culture Paec. maximus C.<br />
1<br />
2<br />
3<br />
4<br />
5<br />
6<br />
7<br />
8<br />
9
Byssochlamys Heat Resistance and Mycotox<strong>in</strong>s 219<br />
Ram is accommodated <strong>in</strong> Clade 9. These three taxa are now considered<br />
to be synonyms of Paec. variotii. Both clades conta<strong>in</strong> strictly<br />
conidial isolates and no ascospores have been observed.<br />
3.2. Mycotox<strong>in</strong> analysis<br />
Species <strong>in</strong> Byssochlamys and related Paecilomyces species can also<br />
be dist<strong>in</strong>guished by differences <strong>in</strong> secondary metabolites. The results<br />
of the analyses are summarized <strong>in</strong> Table 3. B. spectabilis produced the<br />
mycotox<strong>in</strong> viriditox<strong>in</strong>. Some isolates of B. nivea and P. dactylethromorphus<br />
produced patul<strong>in</strong>, however, many did not. Mycophenolic<br />
acid was produced by B. nivea and B. lagunculariae, whereas emod<strong>in</strong><br />
was produced by B. divaricatum. Byssotox<strong>in</strong> A has been reported to be<br />
produced by isolates of B. fulva (Kramer, 1976) but because the structure<br />
of byssotox<strong>in</strong> A was not elucidated, isolates were not screened for<br />
the presence of this mycotox<strong>in</strong>.<br />
3.3. Heat resistance<br />
Paecilomyces and Byssochlamys isolates <strong>in</strong> the CBS collection were<br />
re-identified as described above. The results of the identification were<br />
related to the orig<strong>in</strong> of the isolates. In Table 4 the number of stra<strong>in</strong>s<br />
isolated from heat-treated products or samples is correlated with the<br />
total number of isolates. The Table shows that species with a teleomorph<br />
are more often found <strong>in</strong> heat treated products, with the exception<br />
of B. verrucosa and B. zollerniae which do not occur <strong>in</strong> foods. The<br />
presence of P. variotii sensu stricto (the anamorph of B. spectabilis) <strong>in</strong><br />
heat treated products could be expla<strong>in</strong>ed by the production of heat<br />
resistant ascospores.<br />
Table 3. Mycotox<strong>in</strong> production by Byssochlamys and Paecilomyces species a<br />
Species Known mycotox<strong>in</strong>s<br />
Byssochlamys fulva Byssochlamic acid<br />
Byssochlamys nivea Patul<strong>in</strong>, mycophenolic acid, byssochlamic acid<br />
Byssochlamys lagunculariae Mycophenolic acid, byssochlamic acid<br />
Byssochlamys spectabilis Viriditox<strong>in</strong><br />
Byssochlamys divaricatum Emod<strong>in</strong><br />
Byssochlamys verrucosa Byssochlamic acid<br />
Byssochlamys zollerniae No known mycotox<strong>in</strong>s detected<br />
Paecilomyces maximus No known mycotox<strong>in</strong>s detected<br />
Paecilomyces dactylethromorphus Patul<strong>in</strong><br />
a Many other metabolites are produced but not listed here
220 Jos Houbraken et al.<br />
Table 4. Overview of isolates <strong>in</strong> CBS collection correlated with orig<strong>in</strong>a No.<br />
isolates from Percentage from<br />
No. isolates heat-treated heat-treated<br />
Species <strong>in</strong>vestigated products products<br />
Byssochlamys fulva 5 5 100<br />
Byssochlamys nivea 5 3 60<br />
Byssochlamys languculariae 2 1 50<br />
Byssochlamys spectabilis 17 6 35<br />
Byssochlamys divaricatum 4 3 75<br />
Byssochlamys verrucosa 2 0 0<br />
Byssochlamys zollerniae 1 0 0<br />
Paecilomyces maximus 6 0 0<br />
Paecilomyces<br />
dactylethromorphus<br />
5 0 0<br />
a CBS is the culture collection of the Centraalbureau voor Schimmelcultures, Utrecht,<br />
Netherlands. Isolates of unknown orig<strong>in</strong> are excluded<br />
Two laboratory experiments were conducted on 45 day-old<br />
ascospores of B. spectabilis <strong>in</strong> ACES buffer. Conidia, chlamydospores<br />
and other fungal fragments were <strong>in</strong>activated as described above<br />
(Section 2.7.). Figure 2 shows one of the two thermal death rate<br />
curves. Between 0 and 8 m<strong>in</strong>utes of the heat-treatment, activation of<br />
the ascospores occurs, then a l<strong>in</strong>ear correlation exists between time<br />
and the logarithm of surviv<strong>in</strong>g ascospores. Regression analyses on the<br />
best fit resulted <strong>in</strong> two equations:<br />
and<br />
log CFU = −0.0170 * T (m<strong>in</strong>) + 5.9779 (r 0.972, p
Byssochlamys Heat Resistance and Mycotox<strong>in</strong>s 221<br />
Log CFU<br />
7.00<br />
6.00<br />
5.00<br />
4.00<br />
0 20 40 60 80 100 120 140<br />
Heat<strong>in</strong>g time (m<strong>in</strong>)<br />
Figure 2. Thermal death curve of Byssochlamys spectabilis CBS 109073 at 85°C.<br />
lagunculariae can be considered as a separate taxon, <strong>in</strong> addition to the<br />
known taxa, B. fulva, B. nivea, B. verrucosa and B. zollerniae. With<strong>in</strong><br />
the species complex, Byssochlamys teleomorphs are observed <strong>in</strong> two<br />
other taxa, B. spectabilis and B. divaricata, which are clearly different<br />
from the other members of the genus Byssochlamys.<br />
Accord<strong>in</strong>g to the literature, Paec. variotii sensu lato plays a role <strong>in</strong><br />
mycotoxicoses described <strong>in</strong> several types of animals. In 1916,<br />
Turresson reported that rabbits died after <strong>in</strong>gestion of conidia and<br />
mycelium of Pen. divaricatum (= B. divaricata). B. divaricata produces<br />
the mycotox<strong>in</strong> emod<strong>in</strong>, a genotoxic and diarrhoeagenic<br />
anthraqu<strong>in</strong>one (Müller et al., 1996), which could be the cause of this<br />
described mycotoxicosis. Our <strong>in</strong>vestigation showed that byssochlamic<br />
acid is formed by B. nivea, B. fulva, B. lagunculariae and B. verrucosa.<br />
Byssochlamic acid was shown by Raistrick and Smith (1933) to be<br />
toxic to mice, and it is weakly hepatotoxic to gu<strong>in</strong>ea pigs (Gedek,<br />
1971).<br />
Patul<strong>in</strong> was reported to be produced by B. nivea (Karrow and<br />
Foster, 1944; Kis et al., 1969) and B. fulva (Rice et al., 1977). However,<br />
we could not detect patul<strong>in</strong> production by any of the <strong>in</strong>vestigated<br />
B. fulva isolates. Patul<strong>in</strong> production by stra<strong>in</strong>s of B. nivea was confirmed<br />
and its production by Paec. dactylethromorphus is described. Patul<strong>in</strong>
222 Jos Houbraken et al.<br />
was not produced by any of the stra<strong>in</strong>s of B. lagunculariae exam<strong>in</strong>ed<br />
<strong>in</strong> this study. Mycophenolic acid was produced by stra<strong>in</strong>s of B. nivea<br />
and B. lagunculariae. This metabolite is an antibiotic, with antitumour,<br />
anti-psoriasis and immunosuppressive features (Bentley,<br />
2000) and may be of relevance for secondary mycotoxicosis (bacterial<br />
<strong>in</strong>fections caused by <strong>in</strong>take of an immunosuppressive mycotox<strong>in</strong>).<br />
Paec. variotii sensu lato is a rather common fungus <strong>in</strong> the air, <strong>in</strong> soil<br />
(subtropical and tropical climates), <strong>in</strong> compost (Knösel and Rész,<br />
1973) and on wood (Ram, 1968). It is also common <strong>in</strong> foods such as<br />
rye bread, margar<strong>in</strong>e, peanuts and peanut cake (Joffe, 1969; K<strong>in</strong>g<br />
et al., 1981), cereals (Pelhate, 1968) and heat treated fruit juices<br />
(S. Udagawa, A. D. Hock<strong>in</strong>g , unpublished data).<br />
From our study it can be concluded that the D 85 value of B. spectabilis<br />
<strong>in</strong> ACES buffer was between 47 and 75 m<strong>in</strong>utes. Compar<strong>in</strong>g these<br />
results with other data, it seems that the ascospores of this species are<br />
one of the most heat resistant fungal ascospores. As this species is also<br />
capable of produc<strong>in</strong>g viriditox<strong>in</strong>, it is an important spoilage fungus <strong>in</strong><br />
pasteurized food and feed.<br />
Paec. maximus commonly occurs <strong>in</strong> subtropical and tropical soils<br />
and Paec. dactylethromorphus is isolated from products such as acetic<br />
acid, leather and wood. Both species form chlamydospores, but we<br />
have never detected them from heat-treated samples. This <strong>in</strong>dicates<br />
that ascospores, not thick walled chlamydospores, are the survival<br />
structures.<br />
B. divaricata has also been isolated from heat-treated samples.<br />
This fungus does not form chlamydospores and therefore the<br />
mode of heat-survival is probably due to ascospores. B. divaricata and<br />
B. spectabilis make ascomata <strong>in</strong> culture only sparsely (and only after<br />
prolonged <strong>in</strong>cubation at 30°C), nevertheless these structures should<br />
be present <strong>in</strong> nature. Soil (Udagawa et al., 1994) could be its natural<br />
habitat but also wood (Cartwright, 1937; Ram, 1968) should not be<br />
excluded.<br />
5. ACKNOWLEDGEMENTS<br />
The authors thank Joost Eleveld and Erik Dekker for their data on<br />
heat <strong>in</strong>activation of various structures of Byssochlamys spectabilis.<br />
Jan Dijksterhuis k<strong>in</strong>dly assisted with the statistical analyses of the<br />
thermal death curves. Jens C. Frisvad acknowledges the Danish<br />
Technical Research Council and Centre for Advanced <strong>Food</strong> Studies<br />
(LMC) for f<strong>in</strong>ancial support.
Byssochlamys Heat Resistance and Mycotox<strong>in</strong>s 223<br />
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Gesundheit des Menschen, Materia Med. Normark 23:130-141.<br />
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Ann. Appl. Biol. 26:800-822.<br />
Joffe, A. Z., 1969, The mycoflora of fresh and stored groundnuts kernels <strong>in</strong> Israel,<br />
Mycopathol. Mycol. Appl. 39:255-264.<br />
Karrow, E. O., and Foster, J. W., 1944, An antibiotic substance from species of<br />
Gymnoascus and Penicillium, Science, N.Y. 99:265-266.<br />
K<strong>in</strong>g, A. D., Hock<strong>in</strong>g, A. D. and Pitt, J. I., 1981, The mycoflora of some Australian<br />
foods, <strong>Food</strong> Technol. Aust. 33:55-60.<br />
Kis, Z., Furger, P., and Sigg, H. P., 1969, Isolation of pyrenophorol, Experientia<br />
25:123-124.<br />
Knösel, D., and Rézs, A., 1973, Pilze ays Müllkompost. Enzymatischer Abbau von<br />
Pekt<strong>in</strong> und Zellulose durch wärmeliebende Spezies, Städtehygiene 6, 6 pp.<br />
Kramer, R. K., Davis, N. D., and Diener, U. L., 1976, Byssotox<strong>in</strong> A, a secondary<br />
metabolite of Byssochlamys fulva, Appl. Environ. Microbiol. 31:249-253.<br />
Mori, T., Sh<strong>in</strong>-ya, K., Takatori, K., Aihara, M., and Hayakawa, Y., 2003,<br />
Byssochlamysol, a new antitumor steroid aga<strong>in</strong>st IGF-1-dependent cells from<br />
Byssochlamys nivea. II. Physico-chemical properties and structure elucidation,<br />
J Antibiot. (Tokyo) 56(1):6-8.<br />
Müller, S. O., Eckert, I., Lutz, W. K., and Stopper, H., 1996, Genotoxicity of the laxative<br />
drug components emod<strong>in</strong>, aloe-emod<strong>in</strong> and danthron <strong>in</strong> mammalian cells:<br />
topoisomerase II mediated? Mutation Res. 371:165-173.<br />
Olliver, M., and Rendle, T., 1934, A new problem <strong>in</strong> food preservation. Studies on<br />
Byssochlamys fulva and its effect on the tissues of processed fruit, J. Soc. Chem.<br />
Ind. London 53:66-172.<br />
Pelhate, J., 1968, Inventaire de la mycoflore des blés de conservation, Bull. Trimest.<br />
Soc. Mycol. Fr. 84:127-143.<br />
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XXXV. The metabolic products of Byssochlamys fulva Oliiver & Smith, Biochem.<br />
J. 27:1814-1819,<br />
Ram, C., 1968, Timber-attack<strong>in</strong>g fungi from the state of Maranhao, Brazil. Some new<br />
species of Paecilomyces and its perfect state Byssochlamys Westl., VIII. Nova<br />
Hedwigia 16:305-314<br />
Rice S. L., Beuchat, L. R., and Worth<strong>in</strong>gton, R. E., 1977, Patul<strong>in</strong> production by<br />
Byssochlamys spp. <strong>in</strong> fruit juices, Appl. Environ. Microbiol. 34:791-796.<br />
Samson, R. A., 1974, Paecilomyces and some allied hyphomycetes, Stud. Mycol.<br />
Baarn 6. Centraalbureau voor Schimmelcultures, Baarn, Netherlands
224 Jos Houbraken et al.<br />
Samson, R. A., and Tansey M. R., 1975, Byssochlamys verrucosa sp. nov., Trans. Br.<br />
Mycol. Soc. 65:512-514.<br />
Samson, R. A., Hoekstra, E. S. and Frisvad, J. C., eds, 2004, Introduction to <strong>Food</strong>- and<br />
Airborne Fungi, 7th edition, Centraalbureau voor Schimmelcultures, Utrecht,<br />
Netherlands.<br />
Samson, R. A., Houbraken, J., Kuijpers, A., and Frisvad, J. C., 2005, Revision of<br />
Paecilomyces with its teleomorph Byssochlamys and its mycotox<strong>in</strong> production,<br />
Mycol. Res. (submitted).<br />
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fungal metabolite production <strong>in</strong> cultures, J. Chromatog. A, 760:264-270.<br />
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fungus Penicillium roquefortii Thom 1906., Appl. Biochem. Microbiol. 37:184-187.
EFFECT OF WATER ACTIVITY AND<br />
TEMPERATURE ON PRODUCTION OF<br />
AFLATOXIN AND CYCLOPIAZONIC ACID<br />
BY ASPERGILLUS FLAVUS IN PEANUTS<br />
Graciela Vaamonde, Andrea Patriarca and Virg<strong>in</strong>ia E.<br />
Fernández P<strong>in</strong>to *<br />
1. INTRODUCTION<br />
It is well known that some isolates of Aspergillus flavus are able to<br />
produce cyclopiazonic acid (CPA) <strong>in</strong> addition to aflatox<strong>in</strong>s (Luk et al.,<br />
1977; Gallagher et al., 1978). CPA produc<strong>in</strong>g stra<strong>in</strong>s of A. flavus are frequently<br />
isolated from substrates such as peanuts (Blaney et al., 1989;<br />
Vaamonde et al., 2003) and maize (Resnik et al., 1996), <strong>in</strong>dicat<strong>in</strong>g that<br />
this tox<strong>in</strong> could be a common metabolite and thus is likely to be present<br />
<strong>in</strong> some aflatox<strong>in</strong> contam<strong>in</strong>ated foods. Natural co-occurrence of both<br />
tox<strong>in</strong>s has been detected <strong>in</strong> peanuts (Urano et al., 1992; Fernández P<strong>in</strong>to<br />
et al., 2001) and it has been hypothesized that the presence of both tox<strong>in</strong>s<br />
<strong>in</strong> food and feeds may result <strong>in</strong> additive or synergistic effects.<br />
CPA is toxic to poultry and may have contributed to the outbreak<br />
of the classic “Turkey X” disease which killed about 100,000 turkey<br />
poults <strong>in</strong> England <strong>in</strong> 1960 (Cole, 1986). Some disease outbreaks of<br />
unknown aetiology observed <strong>in</strong> chickens <strong>in</strong> Argent<strong>in</strong>a could also<br />
be attributed to the presence of CPA <strong>in</strong> peanut meal used as a raw<br />
material <strong>in</strong> poultry feeds, as stra<strong>in</strong>s of A. flavus capable of produc<strong>in</strong>g<br />
*Laboratorio de Microbiología de Alimentos, Departamento de Química Orgánica,<br />
Area Bromatología, Facultad de Ciencias Exactas y Naturales, Universidad de<br />
Buenos Aires, Ciudad Universitaria, Pabellón II, 3˚ Piso, 1428, Buenos Aires,<br />
Argent<strong>in</strong>a. Correspondence to: vaamonde@qo.fcen.uba.ar<br />
225
226 Graciela Vaamonde et al.<br />
high levels of CPA are frequently isolated from peanuts grown <strong>in</strong> this<br />
country (Vaamonde et al., 2003). CPA accumulates <strong>in</strong> the muscle of<br />
chickens follow<strong>in</strong>g oral dos<strong>in</strong>g (Norred et al., 1988) so that a potential<br />
for contam<strong>in</strong>ation of meat and meat products exists.<br />
In view of the health hazards for animals and humans caused by the<br />
co-occurrence of aflatox<strong>in</strong>s and CPA, the production of these tox<strong>in</strong>s<br />
<strong>in</strong> agricultural commodities must be controlled. Water activity (a w )<br />
and temperature are the most important environmental factors prevent<strong>in</strong>g<br />
fungal growth and mycotox<strong>in</strong> biosynthesis. Conditions for<br />
mycotox<strong>in</strong> production are generally more restrictive than those for<br />
growth and can differ between mycotox<strong>in</strong>s produced by the same fungal<br />
species, as well as between fungi produc<strong>in</strong>g the same mycotox<strong>in</strong><br />
(Frisvad and Samson, 1991). Interactions between factors such as a w ,<br />
temperature and time are also important and should be taken<br />
<strong>in</strong>to account <strong>in</strong> the design of experiments to study their effects on<br />
mycotox<strong>in</strong> production (Gqaleni et al., 1997).<br />
The effects of temperature and a w on the production of aflatox<strong>in</strong>s<br />
by A. flavus have been widely studied (ICMSF, 1996) but very little is<br />
known about the effect of these factors on CPA production. Besides,<br />
most published studies on mycotox<strong>in</strong> formation have been concerned<br />
with s<strong>in</strong>gle mycotox<strong>in</strong>s. Few studies have exam<strong>in</strong>ed how environmental<br />
factors can affect the simultaneous production of these two tox<strong>in</strong>s<br />
on agar culture media (Gqaleni et al., 1997) and natural substrates<br />
(Gqaleni et al.,1996b).<br />
In the present work an experiment with a full factorial design was<br />
used to study the effects of, and <strong>in</strong>teractions among, temperature, a w<br />
and <strong>in</strong>cubation period on co-production of AF and CPA on peanuts<br />
<strong>in</strong>oculated with a cocktail of A. flavus stra<strong>in</strong>s. Peanuts were sterilized<br />
by gamma irradiation to keep, as closely as possible, to natural conditions<br />
for mycotox<strong>in</strong> production. In this way, it was hoped that a<br />
different fungal response to environmental conditions <strong>in</strong> relation to<br />
biosynthesis of both secondary metabolites could be observed.<br />
2. MATERIALS AND METHODS<br />
2.1. Substrate<br />
Samples of peanuts (Arachis hypogaea cv Runner) from the<br />
peanut grow<strong>in</strong>g area <strong>in</strong> the prov<strong>in</strong>ce of Córdoba, Argent<strong>in</strong>a, were
Aflatox<strong>in</strong> and CPA Production <strong>in</strong> Peanuts 227<br />
used. Samples were analysed for aflatox<strong>in</strong>s and CPA and shown to<br />
be free of both tox<strong>in</strong>s. Peanut kernels were distributed <strong>in</strong> plastic<br />
bags <strong>in</strong> portions of 2.5 kg of material and were treated by gamma<br />
irradiation (Cuero et al., 1986) to kill contam<strong>in</strong>ant microorganisms.<br />
Prelim<strong>in</strong>ary experiments showed that a dosage rate of 6 kGy produced<br />
kernels free of viable microorganisms without adversely<br />
affect<strong>in</strong>g seed germ<strong>in</strong>ation. Irradiation was carried out at the<br />
Comisión Nacional de Energía Atómica, Buenos Aires, Argent<strong>in</strong>a,<br />
us<strong>in</strong>g a 60 Co source.<br />
Water activity was adjusted by add<strong>in</strong>g sterile water accord<strong>in</strong>g to<br />
data from the water sorption isotherms of peanuts (Karon and<br />
Hillery, 1949). The material was mixed and left to equilibrate at 7°C<br />
until measurements of a w <strong>in</strong> three consecutives days showed variation<br />
228 Graciela Vaamonde et al.<br />
the correspond<strong>in</strong>g a w . Each experiment was carried out with three<br />
replicates.<br />
2.4. Mycotox<strong>in</strong> Analysis<br />
Three flasks were removed weekly from each set of conditions<br />
and analysed for AFB 1 and CPA. Analysis of AFB 1 was carried out<br />
accord<strong>in</strong>g to the BF method (AOAC, 1995). Peanut kernels (25 g)<br />
were extracted with methanol-water (125 ml; 55+45), hexane (50 ml)<br />
and NaCl (1 g) <strong>in</strong> a high-speed blender (1 m<strong>in</strong>). The extract was filtered<br />
through Whatman No 4 filter paper and centrifuged at 2000<br />
rpm (5 m<strong>in</strong>). Aliquots of the aqueous phase (25 ml) were transferred<br />
to a separatory funnel and extracted with chloroform (25 ml) by<br />
shak<strong>in</strong>g for 1 m<strong>in</strong>. The chloroform extract was filtered through<br />
anhydrous Na 2 SO 4 and evaporated to dryness. AFB 1 was detected by<br />
TLC on silica gel G60 plates us<strong>in</strong>g chloroform:acetone (90:10) as a<br />
develop<strong>in</strong>g solvent. AFB 1 concentrations were determ<strong>in</strong>ed by visual<br />
comparison of fluorescence under UV light (366 nm) with standard<br />
solutions (Sigma Chemical, St. Louis, MO, USA) dissolved <strong>in</strong><br />
benzene:acetonitrile 98:2.<br />
CPA was analysed by the method of Fernández P<strong>in</strong>to et al. (2001).<br />
Peanuts (25 g) were mixed with methanol:2% sodium hydrogen carbonate<br />
mixture (7:3; 100 ml). The mixture was blended at high speed<br />
for 3 m<strong>in</strong>, centrifuged (1500 rpm) for 15 m<strong>in</strong> and then filtered. Filtrate<br />
(50 ml) was defatted twice with hexane (50 ml) by shak<strong>in</strong>g <strong>in</strong> a wrist<br />
action shaker (3 m<strong>in</strong>). KCl solution (10%; 25 ml) was added to the<br />
sample layer and acidified to pH 2 with 6N HCl. The solution was<br />
transferred quantitatively to a separatory funnel, extracted twice with<br />
chloroform (25 ml), and filtered over anhydrous Na 2 SO 4 . This extract<br />
was evaporated to dryness and redissolved <strong>in</strong> chloroform (200 µl).<br />
CPA was detected by TLC separation on silica gel G60 plates. TLC<br />
plates were immersed completely <strong>in</strong> oxalic acid solution (2% wt/wt) <strong>in</strong><br />
methanol for 10 m<strong>in</strong>, heated at 100°C for 1 h and cooled. Extracts<br />
(2 µl) were applied to the plates with standard solution every fourth<br />
track. The plates were developed <strong>in</strong> ethyl acetate:2-propanol: ammonium<br />
hydroxide (40:30:20). After development, the plates were dried<br />
5 m<strong>in</strong> at 50°C to drive off ammonia and then sprayed with Erlich’s<br />
reagent [4-dimethylam<strong>in</strong>obenzaldehyde (1 g) <strong>in</strong> ethanol (75 ml) and<br />
concentrated HCl (25 ml)]. Blue spots were analysed after 10 m<strong>in</strong> by<br />
visual comparison with a standard CPA solution (Sigma Chemical<br />
Co., St. Louis, MO, USA). The detection limit of the method was<br />
50 µg/kg.
Aflatox<strong>in</strong> and CPA Production <strong>in</strong> Peanuts 229<br />
3. RESULTS AND DISCUSSION<br />
Figure 1 shows the <strong>in</strong>fluence of water activity and temperature on<br />
CPA accumulation over a 28-day period. CPA production was <strong>in</strong>hibited<br />
at the lowest a w studied (0.86) at all temperatures. At higher a w<br />
levels, CPA was detected after a lag period of between 7 and 14 d. The<br />
amount of CPA produced was determ<strong>in</strong>ed by the complex <strong>in</strong>teraction<br />
of a w , temperature and <strong>in</strong>cubation time. Measurements performed<br />
only at a fixed time could lead to mis<strong>in</strong>terpretation of the results. For<br />
example, from the curve at a w 0.92 (Figure 1) different conclusions<br />
could be drawn regard<strong>in</strong>g the <strong>in</strong>fluence of temperature on CPA production<br />
by consider<strong>in</strong>g the accumulation after 14 or 28 d. Tak<strong>in</strong>g <strong>in</strong>to<br />
account the whole <strong>in</strong>cubation period at the various a w levels, CPA<br />
reached a maximal accumulation (4469.2 µg/kg) after 28 d at 0.94 a w<br />
and 25°C, but also a considerable production (2690 ppb) was detected<br />
at the lowest temperature studied (20°C) after 21 d at relatively high<br />
ppb<br />
ppb<br />
4000<br />
3000<br />
2000<br />
1000<br />
0<br />
a w = 0.86<br />
0 7 14 21 28 35<br />
days<br />
0 0<br />
0 7 14 21 28 35 0 7 14 21 28 35<br />
days<br />
days<br />
30�C 25�C 20�C<br />
ppb<br />
4000<br />
3000<br />
2000<br />
1000<br />
4000 aw = 0.92 aw = 0.94<br />
4000<br />
3000 3000<br />
2000 2000<br />
1000 1000<br />
ppb<br />
0<br />
a w = 0.88<br />
0 7 14 21 28 35<br />
days<br />
Figure 1. Interaction between a w and temperature and their effect on cyclopiazonic<br />
acid production by A. flavus on peanuts over a 28 day period
230 Graciela Vaamonde et al.<br />
a w . These results are <strong>in</strong> concordance with those obta<strong>in</strong>ed by Gqaleni<br />
et al. (1996a) who reported that A. flavus produced higher levels of<br />
CPA at 20°C than at 30°C on maize gra<strong>in</strong>s held at constant a w . The<br />
same effect was observed by Sosa et al. (2000) who found maximum<br />
CPA production by Penicillium commune at 20°C and 0.90 a w <strong>in</strong> a<br />
medium based on meat extract. Accord<strong>in</strong>g to Gqaleni et al (1996b)<br />
CPA production proceeded <strong>in</strong> a similar pattern for A. flavus and<br />
P. commune with a comb<strong>in</strong>ation of high a w and low temperature favour<strong>in</strong>g<br />
high CPA production and low a w and high temperature support<strong>in</strong>g<br />
least CPA production.<br />
AFB 1 was produced over the whole a w range (Figure 2). AFB 1 concentrations<br />
were low after 7 d at all temperatures, but as time progressed,<br />
the maximal accumulation was observed at the lowest a w<br />
(0.86) with temperatures of 25°C and 30°C, temperatures reported as<br />
favourable for aflatox<strong>in</strong> production by other authors (ICMSF, 1996).<br />
A comb<strong>in</strong>ed effect of a w and temperature was observed as AFB 1 production<br />
was <strong>in</strong>hibited at low a w (0.86) and low temperature (20°C). At<br />
ppb<br />
ppb<br />
5000 5000<br />
aw = 0.86<br />
4000 4000<br />
3000 3000<br />
2000 2000<br />
1000 1000<br />
0 0<br />
0 7 14 21 28 35<br />
days<br />
5000 5000<br />
a w = 0.88<br />
aw = 0.92 aw = 0.94<br />
4000 4000<br />
3000 3000<br />
2000 2000<br />
1000 1000<br />
0<br />
0 7 14 21 28 35<br />
days<br />
30�C 25�C 20�C<br />
ppb<br />
ppb<br />
0<br />
0 7 14 21 28 35<br />
days<br />
0 7 14 21 28 35<br />
days<br />
Figure 2. Interaction between a w and temperature and their effect on aflatox<strong>in</strong> B 1 production<br />
by A. flavus on peanuts over a 28 day period
Aflatox<strong>in</strong> and CPA Production <strong>in</strong> Peanuts 231<br />
higher a w , AFB 1 production taken over the whole <strong>in</strong>cubation period<br />
was lower than at low a w . The lowest accumulation was detected at<br />
0.94 a w . At this a w level the fungus grew vigorously at 25°C and 30°C<br />
but very low quantities of AFB 1 were detected <strong>in</strong> such conditions,<br />
while at 20°C slower growth was observed but a considerable amount<br />
of AFB 1 (1375 ppb) was produced after 28 d.<br />
As mycotox<strong>in</strong>s are fungal secondary metabolites, production need<br />
not to be correlated with the growth of the fungus and factors such as<br />
<strong>in</strong>duction, end product <strong>in</strong>hibition, catabolite repression and phosphate<br />
regulation can <strong>in</strong>fluence production (Tuomi et al., 2000).<br />
However, the results obta<strong>in</strong>ed for AFB 1 accumulation were unexpected<br />
and contradict data from previous studies which reported that<br />
aflatox<strong>in</strong> production <strong>in</strong>creases with <strong>in</strong>creas<strong>in</strong>g a w (Diener and Davis,<br />
1970; Montani et al., 1988; Gqaleni et al., 1996b).<br />
Differences could be due to several factors. Gqaleni et al. (1996a),<br />
us<strong>in</strong>g several A. flavus stra<strong>in</strong>s known as co-producers of aflatox<strong>in</strong>s<br />
and CPA, reported that the pattern of co-production of these tox<strong>in</strong>s<br />
by a particular isolate can vary widely depend<strong>in</strong>g on the type of cultural<br />
system. When they used yeast extract sucrose agar or a liquid<br />
medium, aflatox<strong>in</strong>s were produced <strong>in</strong> very low quantities over a period<br />
of 21 d of <strong>in</strong>cubation at 30°C despite the high a w of these substrates.<br />
When they used autoclaved maize gra<strong>in</strong>s (a w 0.992) higher concentrations<br />
of both tox<strong>in</strong>s were detected and the ratios between concentrations<br />
of aflatox<strong>in</strong>s and CPA were 16.6:1, 5.7:1 and 1.6:1 after 7, 15<br />
and 21 d respectively (Gqaleni et al., 1996a). The heat sterilised maize<br />
gra<strong>in</strong>s used by Gqaleni et al. (1996a; 1996b) differed from the cultural<br />
system used <strong>in</strong> the present work, where liv<strong>in</strong>g seeds were used. In this<br />
case, the substrate conditioned at higher a w levels might perhaps have<br />
had some defence mechanism, e.g. synthesis of <strong>in</strong>hibitors of aflatox<strong>in</strong><br />
biosynthesis. The fungus might respond by <strong>in</strong>creas<strong>in</strong>g production of<br />
CPA s<strong>in</strong>ce the biosynthetic pathways of the two tox<strong>in</strong>s are different.<br />
CPA is derived from tryptophan (Holzapfel, 1971) whereas aflatox<strong>in</strong>s<br />
are decaketides (Smith and Moss, 1985). On the other hand, reduced<br />
metabolic activity of viable kernels associated with decreased a w could<br />
<strong>in</strong>crease susceptibility to A. flavus growth and aflatox<strong>in</strong> production,<br />
an effect observed <strong>in</strong> the field with decreased pod moisture content<br />
under drought stress (Mehan et al., 1991).<br />
Other factors such as the peanut variety, the size of <strong>in</strong>oculum and<br />
oxygen availability could also be responsible for the unexpected results<br />
obta<strong>in</strong>ed <strong>in</strong> the present work. The possibility of competitive growth of<br />
microorganisms that could have survived the dis<strong>in</strong>fection treatment<br />
should be also considered s<strong>in</strong>ce the radiation dose employed (6 kGy)
232 Graciela Vaamonde et al.<br />
was relatively low when compared with that applied <strong>in</strong> other studies<br />
(Marín et al., 1999), although microbial contam<strong>in</strong>ation was not visually<br />
detected. Further experiments are currently <strong>in</strong> progress <strong>in</strong> order to<br />
clarify the possible <strong>in</strong>fluence of some of the above mentioned factors,<br />
e.g. to perform mycotox<strong>in</strong> accumulation curves us<strong>in</strong>g A. flavus and<br />
A. parasiticus stra<strong>in</strong>s capable of produc<strong>in</strong>g only aflatox<strong>in</strong>s with other<br />
peanut varieties and different <strong>in</strong>oculum levels.<br />
It is evident that accumulation of two (or more) tox<strong>in</strong>s produced<br />
simultaneously by a fungus is affected by numerous factors, as well as<br />
their <strong>in</strong>teractions, as the result of a very complex relationship between<br />
the microorganism, the substrate and the environment. The <strong>in</strong>fluence<br />
of each of these factors on the production of each tox<strong>in</strong> may be different.<br />
In fact, variation of the relative amounts of the tox<strong>in</strong>s produced<br />
with chang<strong>in</strong>g a w and temperature has been observed for other<br />
fungal species produc<strong>in</strong>g several tox<strong>in</strong>s (Magan et al., 1984; Wagener<br />
et al., 1980). Biosynthesis of different mycotox<strong>in</strong>s produced by<br />
Alternaria spp. is favoured by different temperatures. Production of<br />
tenuazonic acid, a tetramic acid like CPA, occurred at 20°C (Young<br />
et al., 1980), and was affected <strong>in</strong> a very different way by environmental<br />
factors compared with other Alternaria tox<strong>in</strong>s such as alternariol<br />
and alternariol monomethyl ether (Magan et al.,1984).<br />
The conditions for maximum production of aflatox<strong>in</strong>s and CPA by<br />
A. flavus are different, accord<strong>in</strong>g to results of the present work and<br />
those obta<strong>in</strong>ed by Gqaleni et al. (1996a; 1996b). Table 1 illustrates the<br />
relative concentrations of both tox<strong>in</strong>s <strong>in</strong> some of the conditions used<br />
<strong>in</strong> our study. It can be po<strong>in</strong>ted out that these conditions are different<br />
from those <strong>in</strong> most published studies on mycotox<strong>in</strong> formation because<br />
a) a cocktail of native stra<strong>in</strong>s that co-produce aflatox<strong>in</strong>s and CPA was<br />
used; b) the substrate was not autoclaved and; c) the study covered<br />
sufficient time to observe the evolution of the <strong>in</strong>teraction between the<br />
Table 1. Mean levels of cyclopiazonic acid (CPA) and aflatox<strong>in</strong> B 1 (AFB 1 ) produced<br />
by Aspergillus flavus <strong>in</strong> peanuts under conditions of controlled temperature, a w and<br />
time<br />
Water Temperature Time CPA AFB 1<br />
activity (°C) (days) (µg/kg) (µg/kg)<br />
0.94 25 28 4469.2 a 118.7<br />
0.94 25 14 109.8 14.8<br />
0.92 25 21 550.2 1972.8<br />
0.88 25 28 2205.7 3411.7<br />
0.86 30 28 134.5 4450.0<br />
0.86 20 28 ND b 0.4<br />
a Optimal conditions for CPA production; b Limit<strong>in</strong>g conditions for CPA production
Aflatox<strong>in</strong> and CPA Production <strong>in</strong> Peanuts 233<br />
fungus and the substrate under different environmental conditions. It<br />
can be seen that at the po<strong>in</strong>ts at which one of the tox<strong>in</strong>s reaches the<br />
highest concentration the other is accumulated <strong>in</strong> m<strong>in</strong>imal quantities.<br />
While concentrations of both tox<strong>in</strong>s <strong>in</strong> extreme conditions are quite<br />
different, at <strong>in</strong>termediate po<strong>in</strong>ts the relative concentrations are more<br />
similar as can be observed <strong>in</strong> Table 1 for 0.88 a w ,25°C and 28 d.<br />
The ability to respond <strong>in</strong> a different way to environmental factors <strong>in</strong><br />
relation to the biosynthesis of toxic secondary metabolites may aid<br />
survival of the fungus <strong>in</strong> a particular ecological niche allow<strong>in</strong>g it to<br />
colonize the substrate more efficiently than other competitors.<br />
4. REFERENCES<br />
AOAC (Association of Official Analytical Chemists), 1995, Official Methods of<br />
Analysis of the Association of Official Analytical Chemists, 16th edition, Arl<strong>in</strong>gton,<br />
USA, p. 970.45.<br />
Blaney, B. J., Kelly, M. A., Tyler, A. L., and Connole, M. D., 1989, Aflatox<strong>in</strong> and<br />
cyclopiazonic acid production by Queensland isolates of Aspergillus flavus and<br />
Aspergillus parasiticus, Aust. J. Agric. Res. 40:395-400.<br />
Cole, R. J., 1986, Etiology of turkey “X” disease <strong>in</strong> retrospect: a case for the <strong>in</strong>volvement<br />
of cyclopiazonic acid, Mycotox<strong>in</strong> Res. 2:3-7.<br />
Cuero, R. G., Smith, J. E., and Lacey, J., 1986, The <strong>in</strong>fluence of gamma irradiation<br />
and sodium hypochlorite sterilization on maize seed microflora and germ<strong>in</strong>ation,<br />
<strong>Food</strong> Microbiol. 3:107-113.<br />
Diener, U. L., and Davis, N. D., 1970, Limit<strong>in</strong>g temperature and relative humidity for<br />
aflatox<strong>in</strong> production by Aspergillus flavus <strong>in</strong> stored peanuts, J. Am. Oil Chem. Soc.<br />
47:347-351.<br />
Fernández P<strong>in</strong>to, V., Patriarca, A., Locani, O., and Vaamonde, G., 2001, Natural cooccurrence<br />
of aflatox<strong>in</strong> and cyclopiazonic acid <strong>in</strong> peanuts grown <strong>in</strong> Argent<strong>in</strong>a,<br />
<strong>Food</strong> Addit. Contam. 18:1017-1020.<br />
Frisvad, J. C., and Samson, R. A., 1991, Filamentous fungi <strong>in</strong> foods and feeds: ecology,<br />
spoilage and mycotox<strong>in</strong> production, <strong>in</strong>: Handbook of Applied <strong>Mycology</strong>:<br />
<strong>Food</strong>s and Feeds, D. K. Arora, K. G. Mukerji and E. H. Marth, eds, Marcel<br />
Dekker, Inc., New York, N.Y., pp. 31-68.<br />
Gallagher, R. T., Richard, J. L., Stahr, H. M., and Cole, R. J., 1978, Cyclopiazonic<br />
acid production by aflatoxigenic and non-aflatoxigenic stra<strong>in</strong>s of Aspergillus<br />
flavus, Mycopathologia, 66:31-36.<br />
Gqaleni, N., Smith, J. E., and Lacey, J., 1996a, Co-production of aflatox<strong>in</strong>s and<br />
cyclopiazonic acid <strong>in</strong> isolates of Aspergillus flavus, <strong>Food</strong> Addit. Contam. 13:<br />
677-685<br />
Gqaleni, N., Smith, J. E., Lacey, J., and Gett<strong>in</strong>by, G., 1996b, The production of<br />
cyclopiazonic acid by Penicillium commune and cyclopiazonic acid and aflatox<strong>in</strong>s<br />
by Aspergillus flavus as affected by water activity and temperature on maize gra<strong>in</strong>s,<br />
Mycopathologia, 136:103-108.<br />
Gqaleni, N., Smith, J. E., Lacey, J., and Gett<strong>in</strong>by, G., 1997, Effects of temperature,<br />
water activity and <strong>in</strong>cubation time on production of aflatox<strong>in</strong>s and cyclopiazonic
234 Graciela Vaamonde et al.<br />
acid by an isolate of Aspergillus flavus <strong>in</strong> surface agar culture, Appl. Environ.<br />
Microbiol. 63:1048-1053.<br />
Holzapfel, C. W., 1971, On the biosynthesis of cyclopiazonic acid, Phytochemistry,<br />
10:351-358.<br />
ICMSF (International Commission on Microbiological Specifications for <strong>Food</strong>s),<br />
1996, Microorganisms <strong>in</strong> <strong>Food</strong>s 5. Characteristics of Microbial Pathogens, Blackie<br />
Academic and Professional, London, pp. 347-381.<br />
Karon, M. L., and Hillery, B. E., 1949, Hygroscopic equilibrium of peanuts. J. Am.<br />
Oil Chem. Soc. 26:16. [89]<br />
Luk, K. C., Kobbe, B., and Townsend, J. M., 1977, Production of cyclopiazonic acid<br />
by Aspergillus flavus L<strong>in</strong>k, Appl. Environ. Microbiol. 33:211-212.<br />
Magan, N., Cayley, G. R., and Lacey, J., 1984, Effect of water activity and temperature<br />
on mycotox<strong>in</strong> production by Alternaria alternata <strong>in</strong> culture and on wheat<br />
gra<strong>in</strong>, Appl. Environ. Microbiol. 47:1113-1117.<br />
Marín, S., Sanchis, V., Sanz, D., Castel, I., Ramos, A. J., Canela, R., and Magan, N.,<br />
1999, Control of growth and fumonis<strong>in</strong> B 1 production by Fusarium verticillioides<br />
and Fusarium proliferatum isolates <strong>in</strong> moist maize with propionate preservatives,<br />
<strong>Food</strong> Addit.Contam. 16: 555-563.<br />
Mehan, V. K., Mc Donald, D., Haravu, L. J., and Jayanthi, S., 1991, The groundnut<br />
aflatox<strong>in</strong> problem. Review and Literature Database, ICRISAT, International<br />
Crops Research Institute for the Semi-Arid Tropics, Patancheru, Andra Pradesh<br />
502 324, India, p. 59.<br />
Montani, M. L., Vaamonde, G., Resnik, S. L., and Buera, P., 1988, Water activity<br />
<strong>in</strong>fluence on aflatox<strong>in</strong> accumulation <strong>in</strong> corn, Int. J. <strong>Food</strong> Microbiol. 6:349-353.<br />
Norred, W. P., Porter, J. K., Dorner, J.W., and Cole, R. J., 1988, Occurrence of the<br />
mycotox<strong>in</strong>, cyclopiazonic acid, <strong>in</strong> meat after oral adm<strong>in</strong>istration to chickens,<br />
J. Agric. <strong>Food</strong> Chem. 36:113-116.<br />
Resnik, S. L., González, H. H. L., Pac<strong>in</strong>, A. M., Viora, M., Caballero, G. M., and<br />
Gros, E. G., 1996, Cyclopiazonic acid and aflatox<strong>in</strong>s production by Aspergillus<br />
flavus isolated from Argent<strong>in</strong>ian corn, Mycotox<strong>in</strong> Res. 12:61-66.<br />
Sosa, M. J., Córdoba, J. J., Díaz, C., Rodríguez, M., Bermúdez, E., Asensio, M. A.,<br />
and Núñez, F., 2002, Production of cyclopiazonic acid by Penicillium commune<br />
isolated from dry-cured ham on a meat extract-based substrate, J. <strong>Food</strong> Prot.<br />
65:988-992.<br />
Tuomi, T., Reijula, K., Johnsson, T., Hemm<strong>in</strong>ki, K., H<strong>in</strong>tikka, E., L<strong>in</strong>droos, O.,<br />
Kalso, S., Koukila-Kähkölä, P., Mussalo-Rauhamaa, H., and Haahtela, T., 2000,<br />
Mycotox<strong>in</strong>s <strong>in</strong> crude build<strong>in</strong>g materials from water-damaged build<strong>in</strong>gs, Appl.<br />
Environ. Microbiol. 66:1899-1904.<br />
Urano, T., Trucksess, M. W., Beaver, R. W., Wilson, D. M., Dorner, J. W., and Dowell,<br />
F. E., 1992, Co-occurrence of cyclopiazonic acid and aflatox<strong>in</strong>s <strong>in</strong> corn and<br />
peanuts, J. AOAC Int. 75:838-841.<br />
Vaamonde, G., Patriarca, A., Fernández P<strong>in</strong>to, V., Comerio, R., and Degrossi, C.,<br />
2003, Variability of aflatox<strong>in</strong> and cyclopiazonic acid production by Aspergillus section<br />
flavi from different substrates <strong>in</strong> Argent<strong>in</strong>a, Int. J. <strong>Food</strong> Microbiol. 88:79-84.<br />
Wagener, R. E., Davies, N. D. and Diener, U. L., 1980, Penitrem A and roquefort<strong>in</strong>e<br />
production by Penicillium commune, Appl. Environ. Microbiol. 39:882-887.<br />
Young, A. B. , Davis, N. D., and Diener, U. L., 1980, The effect of temperature and<br />
moisture on tenuazonic acid production by Alternaria tenuissima, Phytopathology<br />
70:607-609.
Aflatox<strong>in</strong> and CPA Production <strong>in</strong> Peanuts 235<br />
Editors’ note<br />
As the authors po<strong>in</strong>t out, the results of this work are unexpected,<br />
i.e. higher mycotox<strong>in</strong> production at lower water activities. Of the<br />
explanations suggested by the authors, the most likely reason is limitation<br />
of oxygen dur<strong>in</strong>g growth of the fungi, due to the use of<br />
Erlenmeyer flasks as experimental vessels. Oxygen limitation dur<strong>in</strong>g<br />
growth of fungi <strong>in</strong> narrow necked flasks such as Erlenmeyers has long<br />
been known. Fungal growth under optimal conditions is characterised<br />
by very high oxygen consumption. The wide mouthed Fernbach flask<br />
(illustrated <strong>in</strong> Raper and Thom, Manual of the Penicillia, Williams<br />
and Wilk<strong>in</strong>s, Baltimore, 1949, p. 91) was found to be superior for<br />
growth of fungi. Even with the use of wide mouthed flasks, care needs<br />
to be taken to use th<strong>in</strong> cotton wool closures, to permit the maximum<br />
possible diffusion of oxygen.<br />
The view po<strong>in</strong>t that oxygen limitation produced these unusual<br />
results is supported by the data: at lower water activities where growth<br />
is slower, oxygen consumption is much lower, and sufficient for normal<br />
growth and mycotox<strong>in</strong> production.
Section 4.<br />
Control of fungi and mycotox<strong>in</strong>s <strong>in</strong><br />
foods<br />
Inactivation of fruit spoilage yeasts and moulds us<strong>in</strong>g high pressure process<strong>in</strong>g<br />
Ailsa D. Hock<strong>in</strong>g, Mariam Begum and C<strong>in</strong>dy M. Stewart<br />
Activation of ascospores by novel food preservation techniques<br />
Jan Dijksterhuis and Robert A. Samson<br />
Mixtures of natural and synthetic antifungal agents<br />
Aurelio López-Malo, Enrique Palou, Reyna León-Cruz, and Stella M.<br />
Alzamora<br />
Probabilistic modell<strong>in</strong>g of Aspergillus growth<br />
Enrique Palou and Aurelio López-Malo<br />
Antifungal activity of sourdough bread cultures<br />
Lloyd B. Bullerman, Marketa Giesova, Yousef Hassan, Dwayne Deibert and<br />
Doj<strong>in</strong> Ryu<br />
Prevention of ochratox<strong>in</strong> A <strong>in</strong> cereals <strong>in</strong> Europe<br />
Monica Olsen, Nils Jonsson, Naresh Magan, John Banks, Corrado Fanelli,<br />
Aldo Rizzo, Auli Haikara, Alan Dobson, Jens Frisvad, Stephen Holmes, Juhani<br />
Olkku, Sven-Johan Persson and Thomas Börjesson
INACTIVATION OF FRUIT SPOILAGE<br />
YEASTS AND MOULDS USING HIGH<br />
PRESSURE PROCESSING<br />
Ailsa D. Hock<strong>in</strong>g, Mariam Begum * and C<strong>in</strong>dy M. Stewart †<br />
1. INTRODUCTION<br />
Process<strong>in</strong>g of foods us<strong>in</strong>g ultra high pressures offers an alternative<br />
non-thermal treatment for the production of high quality processed<br />
food products which ma<strong>in</strong>ta<strong>in</strong> many of the organoleptic qualities of<br />
fresh foods (Stewart and Cole, 2001). High pressure process<strong>in</strong>g (HPP)<br />
is particularly useful for acid foods such as fruit pieces, purees and<br />
juices as although it does not <strong>in</strong>activate bacterial spores, it provides a<br />
pasteurisation process. Many studies on the application of high<br />
pressure treatments have focused on the destruction of pathogenic<br />
bacteria, with less attention be<strong>in</strong>g paid to spoilage microorganisms,<br />
particularly yeasts and moulds.<br />
This work described here was undertaken as part of a larger project<br />
to develop a high pressure processed shelf-stable pear product<br />
(Gamage et al., 2004). The project encompassed enzyme (polyphenyloxidase)<br />
<strong>in</strong>activation and shelf life studies, as well as microbiological<br />
stability. As yeasts and moulds, <strong>in</strong>clud<strong>in</strong>g heat resistant moulds, are<br />
the most common spoilage microorganisms <strong>in</strong> processed fruit<br />
products, representative species of these fungi were chosen for the<br />
microbiological <strong>in</strong>vestigations.<br />
* A. D. Hock<strong>in</strong>g and M. Begum, <strong>Food</strong> Science Australia, CSIRO, P.O. Box 52, North<br />
Ryde, NSW Australia 1670. Correspondence to ailsa.hock<strong>in</strong>g@csiro.au<br />
† C. Stewart, National Center for <strong>Food</strong> Safety & Technology, 6502 S. Archer Rd,<br />
Summit-Argo, IL 60501, USA<br />
239
240 Ailsa D. Hock<strong>in</strong>g et al.<br />
2. MATERIALS AND METHODS<br />
2.1. Yeast and mould cultures<br />
A s<strong>in</strong>gle stra<strong>in</strong> of each of two yeasts and four filamentous fungi<br />
was chosen for <strong>in</strong>clusion <strong>in</strong> this study. The species/stra<strong>in</strong>s exam<strong>in</strong>ed<br />
were Saccharomyces cerevisiae FRR 1813, beer fermentation stra<strong>in</strong>;<br />
Pichia anomala FRR 5220 isolated from ferment<strong>in</strong>g vanilla-blueberry<br />
yoghurt; Penicillium expansum FRR 1536 isolated from mouldy pears;<br />
Fusarium oxysporum FRR 5610, isolated from spoiled UHT treated<br />
fruit juice; Byssochlamys fulva FRR 3792 from heat treated strawberry<br />
puree; and Neosartorya fischeri FRR 4595 from heat treated strawberries.<br />
FRR is the acronym of the fungal culture collection of <strong>Food</strong><br />
Science Australia, CSIRO, North Ryde, NSW. The two yeast species<br />
were selected because of their propensity for spoilage of fruit products<br />
and their ability to form ascospores, which may be more resistant to<br />
high pressure treatment than vegetative cells. P. expansum was<br />
<strong>in</strong>cluded because of its significance <strong>in</strong> post harvest spoilage of apple<br />
and pears and the consequent possibility of high numbers of spores<br />
on pears before process<strong>in</strong>g. F. oxysporum has recently been caus<strong>in</strong>g<br />
spoilage problems <strong>in</strong> UHT processed juice products, <strong>in</strong>dicat<strong>in</strong>g that it<br />
may be heat resistant and, therefore, possibly pressure resistant also.<br />
The two heat resistant moulds were <strong>in</strong>cluded because it was considered<br />
important to be able to control these species if a shelf stable fruit<br />
product were to be developed.<br />
2.2. Preparation of cell suspensions<br />
The two yeasts were grown on Malt Extract agar (MEA) at 25°C<br />
for 10 days. P. expansum was grown on Czapek Yeast Extract agar<br />
(CYA) at 25°C for 7 days. F. oxysporum was grown on Tap Water Agar<br />
with carnation leaf pieces at 25°C for 14 days under a light bank provid<strong>in</strong>g<br />
a 12 hour photoperiod to <strong>in</strong>duce formation of chlamydoconidia.<br />
The formulae for these media are from Pitt and Hock<strong>in</strong>g (1997).<br />
The heat resistant moulds B. fulva and N. fischeri were grown on Malt<br />
Extract Agar (MEA) at 30°C for 14 days. Ascospore production <strong>in</strong> the<br />
yeasts and the heat resistant fungi was confirmed by microscopy. Cells<br />
from culture plates were suspended <strong>in</strong> sucrose solution (20° Brix)<br />
adjusted to pH 4.2 with citric acid, to yield ca 10 4 ascospores/ml for<br />
heat resistant moulds, and 10 7 cfu/ml for other microorganisms.<br />
Suspensions of cells from the two yeast species conta<strong>in</strong>ed 20-25%<br />
ascospores. Duplicate suspensions were prepared.
High Pressure Inactivation of Fungi 241<br />
2.3. High pressure treatment<br />
Cell suspensions (1.2 ml) were transferred to sterile high pressure<br />
process<strong>in</strong>g vials (1.5 ml). High pressure treatments were applied us<strong>in</strong>g<br />
a U-111 High Pressure Multi-vessel Apparatus (High Pressure<br />
Research Centre, Polish Academy of Sciences, Warsaw, Poland), compris<strong>in</strong>g<br />
five separate vessels which can be pressurised simultaneously,<br />
but depressurised <strong>in</strong>dividually. The equipment also has the capacity<br />
for temperature control, but these experiments were performed at<br />
ambient temperature (20°C).<br />
For the two yeasts and P. expansum and F. oxysporum, the unit was<br />
pressurised to 400 MPa, with pressure applied for 15, 30, 45, 60 and<br />
120 sec. This pressure treatment regimen was selected to allow the<br />
acquisition of data that would provide an <strong>in</strong>activation response curve.<br />
Treatment at the pressure <strong>in</strong>tended for the pear product (600 MPa)<br />
would have resulted <strong>in</strong> <strong>in</strong>activation times that were too short to measure<br />
with the equipment available.<br />
To assess the effect of a proposed blanch<strong>in</strong>g process for the fruit<br />
product, cell suspensions of the two heat resistant moulds, B. fulva<br />
and N. fischeri, were heated at 95°C for five m<strong>in</strong>utes before they were<br />
subjected to high pressure treatment. Cell suspensions (approx. 10 ml)<br />
were filled <strong>in</strong>to sterile plastic vials which were then immersed <strong>in</strong> a<br />
water bath at 95°C. A thermocouple attached to a data logger was<br />
placed <strong>in</strong> a vial conta<strong>in</strong><strong>in</strong>g a similar volume of suspend<strong>in</strong>g fluid (20<br />
°Brix sucrose, pH 4.2). The 5 m<strong>in</strong> blanch<strong>in</strong>g period was taken from the<br />
time the control vial reached 95°C.<br />
For the two heat resistant moulds, the pressure treatment was 600<br />
MPa applied at the same time <strong>in</strong>tervals as used for the yeasts and heat<br />
sensitive moulds, us<strong>in</strong>g a 2 l high pressure process<strong>in</strong>g unit (Flow<br />
International Corporation, USA). Spore suspensions (approximately<br />
4 ml) were aseptically filled <strong>in</strong>to the bulb of sterile disposable plastic<br />
Pasteur pipettes (5 ml) (Copan Italia S.P.A., Italy), and the end heat<br />
sealed. To protect the high pressure unit from contam<strong>in</strong>ation <strong>in</strong> the<br />
event of sample tube leakage, the sealed tubes were immersed <strong>in</strong> peroxyacetic<br />
acid (250 ppm) (Proxitane Sanitiser, Solvay Interox,<br />
Australia) with<strong>in</strong> high barrier vacuum bags (Cryovac Australia Pty<br />
Ltd, Australia), which were heat sealed. The pressure fluid was water<br />
and pressurization was carried out at ambient temperature (18-20°C).<br />
Come up times to the designated pressure (600 MPa) were less than 10<br />
sec, and depressurization required less than 5 sec. The pressure run<br />
was repeated us<strong>in</strong>g separate cell suspensions of each species to provide<br />
duplicate data.
242 Ailsa D. Hock<strong>in</strong>g et al.<br />
2.4. Assessment of cell survival<br />
Initial and surviv<strong>in</strong>g populations were enumerated <strong>in</strong> duplicate<br />
us<strong>in</strong>g the spread plat<strong>in</strong>g technique on appropriate growth media,<br />
be<strong>in</strong>g MEA for the yeasts and heat resistant moulds, and CYA for<br />
P. expansum and F. oxysporum, with <strong>in</strong>cubation at the same temperatures<br />
used to grow the cultures <strong>in</strong>itially. The limit of detection for the<br />
method was 10 cfu/ml.<br />
3. RESULTS<br />
3.1. Inactivation of yeasts<br />
After 60 sec pressurisation at 400 MPa, a 3 to 4 log 10 reduction was<br />
achieved for both S. cerevisiae and P. anomala, with a 4-5 log 10 reduction<br />
after 120 sec at 400 MPa (Figure 1). The <strong>in</strong>activation curve for<br />
S. cerevisiae showed a quite l<strong>in</strong>ear response between zero and 60 sec at<br />
400 MPa for the first <strong>in</strong>activation run, dropp<strong>in</strong>g from an <strong>in</strong>itial level of<br />
9 × 10 7 cfu/ml to 3.3 × 10 3 cfu/ml after 60 sec. The duplicate run yielded<br />
a curve more similar to that of P. anomala, dropp<strong>in</strong>g rapidly from<br />
an <strong>in</strong>itial level of 1.4 × 10 7 to 4.9 × 10 4 after 30 sec, then tail<strong>in</strong>g off to<br />
2.1 ×10 4 after 60 sec, and 1.9 × 10 3 cfu/ml after 120 sec (Figure 1a).<br />
P. anomala exhibited a relatively sharp drop <strong>in</strong> viable cells after 15 sec<br />
HPP treatment, from 1-2 × 10 8 to 1-2 × 10 5 cfu/ml, with a steady,<br />
Survivors (cfu/ml)<br />
1.00E+08<br />
1.00E+07<br />
1.00E+06<br />
1.00E+05<br />
1.00E+04<br />
1.00E+03<br />
1.00E+02<br />
1.00E+01<br />
Saccharomyces cerevisiae<br />
1.00E+00<br />
0 15 30 45 60 75 90 105 120 135<br />
(a)<br />
Time (seconds)<br />
Survivors ( cfu/ml)<br />
1.00E+09<br />
1.00E+08<br />
1.00E+07<br />
1.00E+06<br />
1.00E+05<br />
1.00E+04<br />
1.00E+03<br />
1.00E+02<br />
1.00E+01<br />
Pichia anomala<br />
1.00E+00<br />
0 15 30 45 60 75 90 105 120<br />
(b)<br />
Time (seconds)<br />
Figure 1. Effect of pressure treatment of 400 MPa at 25°C on (a) Saccharomyces cerevisiae<br />
and (b) Pichia anomala suspensions of mixed vegetative cells and ascospores.<br />
Cells were suspended <strong>in</strong> 20 °Brix sucrose solution, pH 4.2 (acidified with citric acid).<br />
Graphs show replicates 1 (◆) and 2 (■) for each pressure run.
High Pressure Inactivation of Fungi 243<br />
gradual decl<strong>in</strong>e with <strong>in</strong>creas<strong>in</strong>g treatment times to 1-2 × 10 4 cfu/ml<br />
after 60 sec, and 10 3 after 120 sec (Figure 1b).<br />
3.2. Inactivation of heat sensitive moulds<br />
Both P. expansum and F. oxysporum were pressure treated at<br />
400 MPa. P. expansum was quite sensitive to pressure treatment.<br />
A 3-4 log 10 reduction was achieved after 15-30 sec (Figure 2). After<br />
60 sec, the reduction was >5 log 10 and by 120 sec, no survivors were<br />
detected (limit of detection 10 cfu/ml). F. oxysporum conidia and<br />
chlamydoconidia were even more sensitive to pressure treatment. A<br />
suspension of 2.5 × 10 7 mixed microconidia and chlamydoconidia was<br />
reduced to 1.4 × 10 2 cfu/ml after 15 sec at 400 MPa, and after 30 sec,<br />
no survivors were detected (results not shown).<br />
3.3. Inactivation of heat resistant moulds<br />
As the ascospores of heat resistant moulds are known also to be<br />
pressure resistant (Butz et al., 1996; Reyns et al., 2003; Voldrich et al.,<br />
2004), both B. fulva and N. fischeri spore suspensions were pressure<br />
treated at 600 MPa. Spore suspensions were divided <strong>in</strong>to two portions,<br />
and one of each was blanched at 95°C for 5 m<strong>in</strong>. Both blanched and<br />
unblanched spore suspensions were then pressure treated simultaneously<br />
to ensure that they were exposed to exactly the same conditions.<br />
B. fulva ascospores were more pressure sensitive than those of<br />
N. fischeri (Figure 3). Unblanched ascospore suspensions of B. fulva,<br />
Survivors (cfu/ml)<br />
1.00E+08<br />
1.00E+07<br />
1.00E+06<br />
1.00E+05<br />
1.00E+04<br />
1.00E+03<br />
1.00E+02<br />
1.00E+01<br />
1.00E+00<br />
Penicillium expansum<br />
0 15 30 45 60 75 90 105 120 135<br />
Time (seconds)<br />
Figure 2. Effect of pressure treatment of 400 MPa at 25°C on Penicillium expansum<br />
conidia. Cells were suspended <strong>in</strong> 20 °Brix sucrose solution, pH 4.2 (acidified with citric<br />
acid). Graphs show replicates 1 (◆) and 2 (■) for each pressure run.
244 Ailsa D. Hock<strong>in</strong>g et al.<br />
Survivors (cfu/ml)<br />
(a)<br />
Survivors (cfu/ml)<br />
(c)<br />
1.00E+04<br />
1.00E+03<br />
1.00E+02<br />
1.00E+01<br />
1.00E+00<br />
1.00E+05<br />
1.00E+04<br />
1.00E+03<br />
1.00E+02<br />
1.00E+01<br />
1.00E+00<br />
B. fulva (unblanched)<br />
1.00E+04<br />
1.00E+03<br />
1.00E+02<br />
1.00E+01<br />
B. fulva (blanched)<br />
1.00E+00<br />
0 15 30 45 60 120 0 0 15 30 45 60 120<br />
0<br />
Time (seconds) (b)<br />
Time (seconds)<br />
N. fischeri (unblanched)<br />
showed a 1.5 log 10 reduction after 15 sec at 600 MPa (Figure 3a). The<br />
decrease with longer pressure treatments was more gradual, with only<br />
2 log 10 reduction after 60 sec and 2.5 log 10 reduction after 120 sec.<br />
Blanch<strong>in</strong>g at 95°C for 5 m<strong>in</strong> resulted <strong>in</strong> a reduction of 1-2 log 10<br />
(Figure 3b). Pressure treatment of the blanched ascospores resulted <strong>in</strong><br />
a 2 log 10 reduction after 60 sec, and after 120 sec at 600 MPa, there<br />
were less than 10 1 ascospores/ml.<br />
Unblanched ascospores of N. fischeri showed a slight reduction (less<br />
than 1 log 10 ) after 15 sec at 600 MPa (Figure 3c). Longer treatments<br />
(up to 120 sec) appeared to have no further effect on ascospore viability,<br />
with barely 1 log 10 reduction after 120 sec treatment. In the first<br />
experiment, numbers were reduced from 1.50 × 10 4 to 1.53 × 10 3 after<br />
120 sec at 600 MPa, while <strong>in</strong> the second experiment, <strong>in</strong>itial numbers of<br />
7.0 × 10 3 were reduced only to 2.0 × 10 3 .<br />
Blanch<strong>in</strong>g at 95°C caused activation of N. fischeri ascospores<br />
(Figure 3d) with viable numbers ris<strong>in</strong>g by 0.5 log 10 or slightly more.<br />
Subsequent high pressure treatment at 600 MPa resulted <strong>in</strong> a slight<br />
decrease (less than 0.5 log 10 ), but longer treatment times to 60 sec had little<br />
further effect. After 120 sec at 600 MPa, f<strong>in</strong>al numbers were reduced<br />
by slightly less than one log 10 from the <strong>in</strong>itial, unblanched levels.<br />
Survivors (cfu/ml)<br />
Survivors (cfu/ml)<br />
1.00E+05<br />
1.00E+04<br />
1.00E+03<br />
1.00E+02<br />
1.00E+01<br />
N. fischeri (blanched)<br />
15 30 45 60 120<br />
1.00E+00<br />
0 0 15 30 45 60 120<br />
Time (seconds)<br />
(d)<br />
Time (seconds)<br />
Figure 3. Effect of pressure treatment of 600 MPa at 25°C on Byssochlamys fulva (a,<br />
b) and Neosartorya fischeri (c, d) ascospores. Cells were suspended <strong>in</strong> 20 °Brix sucrose<br />
solution, pH 4.2 (acidified with citric acid). Blanched samples (b, d) were heated to<br />
95°C for 5 m<strong>in</strong> before pressure treatment. Graphs show replicates 1 (◆) and 2 (■) for<br />
each pressure run.
High Pressure Inactivation of Fungi 245<br />
4. DISCUSSION<br />
Inactivation of yeasts by ultra high pressure has been reported previously<br />
(Ogawa et al., 1990; Palou et al., 1997; Parish, 1998; Zook<br />
et al., 1999; Basak et al., 2002). Most studies have <strong>in</strong>vestigated the<br />
effects of HPP on the common spoilage yeasts Saccharomyces<br />
cerevisiae and Zygosaccharomyces bailii. The responses of Pichia<br />
anomala to HPP treatment have not previously been reported. As<br />
observed <strong>in</strong> this study, other workers have shown that treatment at 400<br />
MPa or higher will result <strong>in</strong> <strong>in</strong>activation of yeasts provided that the<br />
pressure is applied for several m<strong>in</strong>utes. However, the time required for<br />
<strong>in</strong>activation also depends on the composition of the menstruum <strong>in</strong><br />
which the yeasts are suspended. Yeasts are less susceptible to pressure<br />
treatment when suspended <strong>in</strong> juice concentrate than <strong>in</strong> s<strong>in</strong>gle strength<br />
juice (Ogawa et al., 1990; Basak et al., 2002). Although ascospores of<br />
yeasts are more resistant to pressure treatment than vegetative cells,<br />
they are relatively quickly <strong>in</strong>activated at 500 MPa, with D values of<br />
less than 0.2 m<strong>in</strong> <strong>in</strong> s<strong>in</strong>gle strength juice (Zook et al., 1999).<br />
The conidia of Penicillium expansum were more sensitive to pressure<br />
treatment than yeast cells. Our study showed a 6 log 10 reduction <strong>in</strong><br />
viability of P. expansum conidia pressure treated at 400 MPa for 60 sec.<br />
A suspension of microconidia and chlamydoconidia of F. oxysporum<br />
was even more sensitive to pressure treatment than P. expansum. These<br />
results are <strong>in</strong> agreement with results reported by Ogawa et al. (1990)<br />
who found that treatment at 400 MPa at room temperature resulted <strong>in</strong><br />
at least 4 log 10 reduction <strong>in</strong> viability of conidia of Aspergillus awamori<br />
and sporangiospores of Mucor plumbeus. Voldrich et al. (2004) also<br />
reported that conidia of Talaromyces avellaneus exhibited comparable<br />
pressure sensitivity to vegetative cells of other yeast and mould species.<br />
Ascospores of the two heat resistant mould species were relatively<br />
resistant to the pressure treatments received, as observed by other workers<br />
(Butz et al., 1996; Reyns et al., 2003; Voldrich et al., 2004).<br />
Blanch<strong>in</strong>g at 95°C for 5 m<strong>in</strong> did not affect the pressure resistance, and<br />
<strong>in</strong> fact, ascospores of N. fischeri became more resistant to pressure after<br />
this mild heat treatment. The ascospores used <strong>in</strong> these studies were relatively<br />
young (from 14 day old cultures), and it could be expected that<br />
older ascospores would be more pressure resistant, as heat resistance <strong>in</strong><br />
N. fischeri ascospores <strong>in</strong>creases with age (Conner at al., 1987).<br />
The results of this study <strong>in</strong>dicate that high pressure process<strong>in</strong>g (600<br />
MPa for several m<strong>in</strong>utes) should be sufficient to <strong>in</strong>activate vegetative<br />
cells of yeasts and moulds and also the ascospores of yeasts, provid<strong>in</strong>g<br />
a process that is equivalent to pasteurisation. However, treatment
246 Ailsa D. Hock<strong>in</strong>g et al.<br />
with high pressure alone is <strong>in</strong>sufficient to <strong>in</strong>activate even relatively<br />
young ascospores of heat resistant moulds, <strong>in</strong>dicat<strong>in</strong>g that comb<strong>in</strong>ed<br />
treatments (for example heat and pressure) will be needed to control<br />
the outgrowth of these fungi dur<strong>in</strong>g storage of HPP fruit products at<br />
ambient temperature.<br />
5. REFERENCES<br />
Basak, S., Ramaswamy, H. S., and Piette, J. P. G., 2002, High pressure destruction k<strong>in</strong>etics<br />
of Leuconostoc mesenteroides and Saccharomyces cerevisiae <strong>in</strong> s<strong>in</strong>gle strength and<br />
concentrated orange juice, Innov. <strong>Food</strong> Sci. Emerg<strong>in</strong>g Technol. 3:223-231.<br />
Butz., P., Funtenberger, S., Haberditzl, T., and Tauscher, B., 1996, High pressure <strong>in</strong>activation<br />
of Byssochlamys nivea ascospores and other heat resistant moulds,<br />
Lebensm.-Wiss. u.-Technol. 29:404-410.<br />
Conner, D. E., Beuchat, L. R., and Chang, C. J., 1987, Age-related changes <strong>in</strong> ultrastructure<br />
and chemical composition associated with changes <strong>in</strong> heat resistance of<br />
Neosartorya fischeri ascospores, Trans. Br. Mycol. Soc. 89:539-550.<br />
Gamage, T.V., Hock<strong>in</strong>g, A., Begum, M., Stewart, C.M., Vu, T., Ng, S., Sellahewa, J.,<br />
and Versteeg, C., 2004, Quality attributes of high pressure processed pears,<br />
Proceed<strong>in</strong>gs of the 9th International Congress on Eng<strong>in</strong>eer<strong>in</strong>g and <strong>Food</strong>,<br />
Montpellier, France, March 2004, pp. 325-330.<br />
Ogawa, H., Fukuhisa, K., Kubo, Y., and Fukomoto, H., 1990, Pressure <strong>in</strong>activation<br />
of yeasts, molds, and pect<strong>in</strong>esterase <strong>in</strong> Satsuma mandar<strong>in</strong> juice: effects of juice<br />
concentration, pH, and organic acids, and comparison with heat sanitation, Agric.<br />
Biol. Chem. 54:1219-1225.<br />
Palou, E., López-Malo, A., Barbosa-Cánovas, G. V., Welti-Chanes, J., and Swanson,<br />
B. G., 1997, K<strong>in</strong>etic analysis of Zygosaccharomyces bailii <strong>in</strong>activation by high<br />
hydrostatic pressure, Lebensm.-Wiss. u.-Technol. 30:703-708.<br />
Parish, M. E., 1998, High pressure <strong>in</strong>activation of Saccharomyces cerevisiae, endogenous<br />
microflora and pect<strong>in</strong>methylesterase <strong>in</strong> orange juice, J. <strong>Food</strong> Safety 18:57-65.<br />
Pitt, J. I. and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, 2nd edition, Blackie<br />
Academic & Professional, London.<br />
Reyns, K. M. F. A., Veberke, E. A. V., and Michiels, C. W., 2003, Activation and <strong>in</strong>activation<br />
of Talaromyces macrosporus ascospores by high hydrostatic pressure,<br />
J. <strong>Food</strong> Prot. 66:1035-1042.<br />
Stewart, C. M., and Cole, M. B., 2001, Preservation by the application of nonthermal<br />
process<strong>in</strong>g, <strong>in</strong>: Spoilage of Processed <strong>Food</strong>s: Causes and Diagnosis, C. J. Moir,<br />
C. Andrew-Kabilafkas, G. Arnold, B. M. Cox, A. D. Hock<strong>in</strong>g and I. Jenson, eds,<br />
AIFST (Inc.), Sydney, NSW Australia, pp. 53-61.<br />
Voldrich, M., Dobiás, J., Tichá, L., Cerovsky, M., and Krátká, J., 2004, Resistance of<br />
vegetative cells and ascospores of heat resistant mould Talaromyces avellaneus to<br />
the high pressure treatment <strong>in</strong> apple juice, J. <strong>Food</strong> Eng. 61:541-543.<br />
Zook, C. D., Parish, M.E., Braddock, R. J., and Balaban, M. O., 1999, High pressure<br />
<strong>in</strong>activation k<strong>in</strong>etics of Saccharomyces cerevisiae ascospores <strong>in</strong> orange and apple<br />
juice, J. <strong>Food</strong> Sci. 64:533-535.
ACTIVATION OF ASCOSPORES BY NOVEL<br />
FOOD PRESERVATION TECHNIQUES<br />
Jan Dijksterhuis and Robert A. Samson *<br />
1. INTRODUCTION<br />
Most fungal survival structures can be regarded as heat resistant to<br />
some extent: sclerotia, conidia and ascospores can survive temperatures<br />
between 55 and 95°C. Byssochlamys, Neosartorya and<br />
Talaromyces are the most well known heat-resistant fungal genera.<br />
Ascospores of these fungi are the most resilient eukaryotic structures<br />
currently known. A decimal reduction time of 1.5-11 m<strong>in</strong> at 90°C has<br />
been reported for some species (Scholte et al., 2004). Recently,<br />
Panagou et al. (2002) reported moderate heat resistance (D 75 4.9-7.8<br />
m<strong>in</strong>) <strong>in</strong> ascospores of Monascus ruber isolated from br<strong>in</strong>e of a commercial<br />
thermally processed can of green olives. There appeared to be<br />
a complex <strong>in</strong>teraction between pH and salt content of the heat<strong>in</strong>g<br />
menstruum and decimal reduction time for this fungus.<br />
Dur<strong>in</strong>g recent work undertaken <strong>in</strong> our laboratory, moderate heat<br />
resistance has been observed for Talaromyces stipitatus and T. helicus<br />
(J. Dijksterhuis, unpublished results), and studies <strong>in</strong>volv<strong>in</strong>g ascospores<br />
of Byssochlamys spectabilis have resulted <strong>in</strong> a calculated decimal<br />
reduction time at 85°C of 47-75 m<strong>in</strong> (J. Houbraken, unpublished<br />
results). Table 1 shows a compilation of heat resistance data for many<br />
known heat resistant species and their D values <strong>in</strong> various heat<strong>in</strong>g<br />
menstrua (modified from Scholte et al., 2004).<br />
* Jan Dijksterhuis and Robert Samson, Department of Applied Research and<br />
Services, Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre,<br />
Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands. Correspondence to dijksterhuis<br />
@cbs.knaw.nl<br />
247
248 Jan Dijksterhuis and Robert A. Samson<br />
Table 1. Heat-resistance of ascospores at different temperatures and medium composition<br />
Fungal species D-value (m<strong>in</strong>) Medium Reference<br />
Byssochlamys fulva 86°C, 13-14 Grape Juice Michener and K<strong>in</strong>g<br />
(1974)<br />
90°C, 4-36 Buffer pH 3.6 Bayne and Michener<br />
(3 log 10 and 5.0, (1979)<br />
reduction) 16 °Brix<br />
90°C, 8.1 Tomato juice Kotzekidou (1997)<br />
Byssochlamys nivea 85°C, 1.3-4.5 Buffer pH 3.5 Casella et al. (1990)<br />
88°C, 8-9 sec R<strong>in</strong>ger solution Engel and Teuber<br />
(1991)<br />
90°C, 1.5 Tomato juice Kotzekidou (1997)<br />
Byssochlamys 85°C, 47-75 Buffer, pH 6.8 Authors’ unpublished<br />
spectabilis data<br />
Eurotium 70°C, 1.1-4.6 Grape Juice, Splittstoesser et al.<br />
herbariorum 65 °Brix (1989)<br />
Eurotium chevalieri 70°C, 17.2 Plum extract Pitt and Christian<br />
(pH 3.8, (1970)<br />
80°C, 3.3 20 °Brix,<br />
a w 0.98)<br />
Monascus ruber 80°C, 1.7-2.0 Buffers (pH Panagou et al. (2002)<br />
3,0 ; pH 7,0)<br />
80°C, 0.9-1.0 Br<strong>in</strong>e<br />
Neosartorya fischeri 85°C, 13.2 Apple Juice Conner and Beuchat<br />
(1987b)<br />
85°C, 10.1 Grape Juice Conner and Beuchat<br />
(1987b)<br />
85°C, 10-60 In ACES-buffer, Authors’ unpublished<br />
10 mM, pH 6.8 data<br />
CBS 133.64<br />
85°C, 10.4 Buffer pH 7.0 Conner and Beuchat<br />
(1987b)<br />
85°C, 35.3 Buffer pH 7.0 Rajashekhara et el.<br />
(1996)<br />
88°C, 1.4 Apple Juice Scott and Bernard<br />
(1987)<br />
88°C, 4.2-16.2 Heated fruit Beuchat (1986)<br />
fill<strong>in</strong>gs<br />
90°C, 4.4-6.6 Tomato Juice Kotzekidou (1997)<br />
91°C, < 2 Heated fruit Beuchat (1986)<br />
fill<strong>in</strong>gs<br />
Neosartorya 95°C, 20 sec Authors’ unpublished<br />
pseudofischeri data<br />
Talaromyces flavus 85°C, 39 Buffer pH 5.0, K<strong>in</strong>g (1997)<br />
(macrosporus) glucose,<br />
16 °Brix<br />
85°C, 20-26 Buffer pH 5.0, K<strong>in</strong>g and Halbrook<br />
glucose (1987)
Activation of Ascospores by Novel <strong>Food</strong> Preservation Techniques 249<br />
Table 1. Heat-resistance of ascospores at different temperatures and medium composition<br />
Fungal species D-value (m<strong>in</strong>) Medium Reference<br />
85°C, 30-100 ACES-buffer, Dijksterhuis and<br />
10 mM, pH 6.8 Teunissen (2004)<br />
88°C, 7.8 Apple Juice Scott and Bernard<br />
(1987)<br />
88°C, 7.1-22.3 Heated fruit Beuchat (1986)<br />
fill<strong>in</strong>gs<br />
90°C, 2-8 Buffer pH 5.0, K<strong>in</strong>g and Halbrook<br />
glucose (1987)<br />
90°C, 6.2 Buffer pH 5.0, K<strong>in</strong>g (1997)<br />
glucose<br />
90°C, 6.0 Buffer pH 5.0, K<strong>in</strong>g (1997)<br />
glucose<br />
Slug flow heat<br />
exchanger<br />
90°C, 2.7-4.1 Organic acids K<strong>in</strong>g and Whitehand<br />
(1990)<br />
Talaromyces flavus 90°C, 2.5-11.1 Sugar 0-60 °Brix) K<strong>in</strong>g and Whitehand<br />
(1990)<br />
90°C, 5.2-7.1 pH 3.6-6.6 K<strong>in</strong>g and Whitehand<br />
(1990)<br />
91°C, 2.1-11.7 Heated fruit Beuchat (1986)<br />
fill<strong>in</strong>gs<br />
T. trachyspermus 85°C, 45 sec Authors’ unpublished<br />
data<br />
T. helicus 70°C, approx. 20 ”<br />
T. stipitatus 72°C, approx. 85<br />
Xeromyces bisporus 82°C, 2.3 Pitt and Hock<strong>in</strong>g<br />
(1982)<br />
2. HEAT-RESISTANT ASCOSPORES OF<br />
TALAROMYCES MACROSPORUS<br />
Germ<strong>in</strong>ation of heat-resistant ascospores of different species is<br />
activated and also synchronised by a heat treatment (see for a survey,<br />
Dijksterhuis and Samson, 2002). Germ<strong>in</strong>ation of these spores has<br />
been studied <strong>in</strong> detail by Dijksterhuis et al. (2002) <strong>in</strong> Talaromyces<br />
macrosporus. This fungus is a candidate to become a model system for<br />
research on heat-resistant ascospores. On oatmeal agar at 30°C<br />
T. macrosporus forms numerous yellow ascoma (Figure 1) with<strong>in</strong> a few<br />
weeks and a dense homogenous suspension of ascospores can be harvested<br />
by a simple procedure. These cells do not germ<strong>in</strong>ate when left<br />
<strong>in</strong> malt extract broth for prolonged times. A 7-10 m<strong>in</strong> treatment at
250 Jan Dijksterhuis and Robert A. Samson<br />
Figure 1. Ascoma of Talaromyces macrosporus. Note the <strong>in</strong>tricate network on the<br />
outside of the structure. The ascospores visible on the outside of the fruit body have<br />
been released from other broken ascomata. Bar = 100 µm.<br />
85°C, however, results <strong>in</strong> germ<strong>in</strong>ation of the majority of the cells. The<br />
ascospores conta<strong>in</strong> high levels of trehalose, low amounts of water and<br />
are bound by a very thick multi-layered cell wall. Upon heat treatment<br />
the trehalose is broken down by a very active trehalase and the product<br />
of hydrolysis, glucose, is accumulated <strong>in</strong>side the spore. Figure 2 shows<br />
6.5<br />
7.0<br />
7.5<br />
8.0<br />
8.5<br />
9.0<br />
9.5<br />
Retention time (m<strong>in</strong>)<br />
Figure 2. HPLC profiles of cell free extracts of broken spores dur<strong>in</strong>g early germ<strong>in</strong>ation<br />
of ascospores. The left peak shows trehalose, the right peak co-elutes with a glucose<br />
standard. The different profiles are at 0, 24, 39, 56, 73, 90, 106 and124 m<strong>in</strong> after<br />
the start of the heat treatment (from front to rear).<br />
10.0<br />
10.5<br />
11.0
Activation of Ascospores by Novel <strong>Food</strong> Preservation Techniques 251<br />
the degradation of trehalose and the simultaneous formation of glucose<br />
early <strong>in</strong> the germ<strong>in</strong>ation process. The glucose is present <strong>in</strong> the<br />
cells for only a short time. After that it occurs <strong>in</strong> measurable quantities<br />
<strong>in</strong> the substrate, <strong>in</strong>dicat<strong>in</strong>g a massive release of glucose from the<br />
germ<strong>in</strong>at<strong>in</strong>g cell.<br />
After 150 m<strong>in</strong> or more the <strong>in</strong>ner cell emerges rapidly from with<strong>in</strong> the<br />
outer cell wall, which is ruptured. The emptied outer cell wall rema<strong>in</strong>s<br />
attached to the protoplast, which is encompassed by the <strong>in</strong>ner layer of<br />
the ascospore cell wall. This process is very sudden, it only takes a second<br />
or less, and is termed prosilition (Lat: prosilire, to jump out).<br />
After this remarkable phenomenon, the respiration of the cells<br />
<strong>in</strong>creases quickly and the cells swell and form a germ tube 6 hours<br />
after the heat treatment. Figure 3 shows snap frozen cells that are <strong>in</strong><br />
various stages of the process, as observed by Cryo-Scann<strong>in</strong>g Electron<br />
Microscopy us<strong>in</strong>g a JEOL JSM 840 scann<strong>in</strong>g electron microscope<br />
(Dijksterhuis et al., 2002).<br />
Recently, prosilition was confirmed <strong>in</strong> other species of<br />
Talaromyces namely T. stipitatus, T. helicus and T. bacillosporus (our<br />
unpublished observations). However, ascospores of Neosartorya<br />
species seem to germ<strong>in</strong>ate by a slow separation of the two shell-like<br />
ornamented halves and subsequent formation of a germ tube.<br />
Apparently, these two genera show different modes of ascospore<br />
germ<strong>in</strong>ation.<br />
3. FUNGI IN FOOD AND HIGH PRESSURE<br />
TREATMENT<br />
Ultra high pressure is a suitable candidate for non-thermal treatment<br />
of food products. These treatments have the benefit that the<br />
organoleptic properties of the food are less affected compared to<br />
pasteurisation or more severe heat treatment. In addition, vitam<strong>in</strong>s<br />
are better preserved after the application of this alternative preservation<br />
technique. While vegetative microbial cells are <strong>in</strong>activated at<br />
relatively low pressures (200-300 MPa), spores are more resistant to<br />
these treatments. Bacterial spores even are activated to germ<strong>in</strong>ate at<br />
a treatment of 200 MPa, but bacterial spores germ<strong>in</strong>ate so quickly<br />
that they are killed dur<strong>in</strong>g prolonged treatment. When higher pressures<br />
are applied the germ<strong>in</strong>ation sequence of the bacterial spores is<br />
blocked render<strong>in</strong>g the spores less vulnerable to ultra high pressures
252 Jan Dijksterhuis and Robert A. Samson<br />
(A)<br />
(B)<br />
(C)<br />
Figure 3. Germ<strong>in</strong>at<strong>in</strong>g ascospores of T. macrosporus. In the top panel (A) an<br />
unprosilited cell (left) and a fully prosilited spore (middle) are shown. At the right a<br />
spore <strong>in</strong> the process of prosilition is captured. The outer cell wall has opened and the<br />
smooth <strong>in</strong>ner cell wall is visible. In the middle panel (B) two fully prosilited spores are<br />
shown. Note the connection between the released cell and the empty outer cell wall.<br />
In the bottom panel (C), spores 6 h after heat treatment are shown, most of the<br />
swollen cells exhibit a germ tube, <strong>in</strong> one case two germ tubes are formed. Bars are<br />
1 µm (A and B) and 10 µm (C).
Activation of Ascospores by Novel <strong>Food</strong> Preservation Techniques 253<br />
(above 600 MPa), but also with a higher sensitivity for heat treatments<br />
(Wuytack et al., 1998).<br />
Do resistant fungal spores such as those produced by heat-resistant<br />
fungi show extended endurance aga<strong>in</strong>st this novel food treatment?<br />
Ascospores of Byssochlames nivea survive pressures at or above 600<br />
MPa for many m<strong>in</strong>utes (Butz et al., 1996), but are killed after repetitive<br />
treatments at these pressures, which are designated as “oscillative<br />
treatments” (Palou et al., 1998). These authors also describe that the<br />
application of elevated temperatures <strong>in</strong> the presence of high pressures<br />
effectively kills the ascospores. However, care is necessary with such<br />
applications to ensure that the organoleptic properties of the food are<br />
changed as m<strong>in</strong>imally as possible. Probably a “happy medium”<br />
approach is important with such treatments.<br />
4. BREAKING ASCOSPORE DORMANCY BY<br />
ULTRA-HIGH PRESSURE<br />
Is heat the only factor that can break the dormancy of these<br />
ascospores? Recently activation of ascospores of T. macrosporus by<br />
high-pressure treatments was reported by Reyns et al. (2003) and<br />
Dijksterhuis and Teunissen (2004). Both studies used the same stra<strong>in</strong> of<br />
fungus and a similar pressurisation equipment. The most important<br />
shared observation was that activation of ascospores occurred after a<br />
pressure treatment and that even a very short treatment at high pressure<br />
caused maximal activation. This is relevant for the food <strong>in</strong>dustry,<br />
because short treatments are important for economic reasons.<br />
Dijksterhuis and Teunissen (2004) observed no activation at 200<br />
MPa and activation of only part of the spores (up to 7% of cells)<br />
between 400 and 800 MPa. This could <strong>in</strong>dicate that treatments at<br />
approx. 300 MPa would be of <strong>in</strong>terest for the food <strong>in</strong>dustry to prevent<br />
contam<strong>in</strong>ation with microorganisms without activat<strong>in</strong>g these<br />
fungi. Reyns et al. (2003), however, observed partial activation of<br />
T. macrosporus at 200 MPa and activation of all spores at 600 MPa<br />
after 15 sec treatment.<br />
A number of factors could have caused of these differences between<br />
the studies. Firstly, the growth conditions of the fungal cultures were<br />
different. As well as age of the culture, growth temperature (Conner<br />
and Beuchat, 1987a) and the growth medium (Beuchat, 1988a) all<br />
<strong>in</strong>fluence the heat resistance of spores. Beuchat (1988a) reported that<br />
ascospores harvested from malt extract agar (which was used by
254 Jan Dijksterhuis and Robert A. Samson<br />
Reyns et al., 2003) exhibited a somewhat lower heat resistance than<br />
spores formed on oatmeal agar (used by Dijksterhuis and Teunissen,<br />
2004). These factors also may have a bear<strong>in</strong>g on the extent of activation<br />
of the spores. The spores used by Reyns et al. (2003) were younger<br />
and grown at a lower temperature. Dijksterhuis and Teunissen (2004)<br />
report that the age of the fungal culture from which ascospores are<br />
harvested correlates with the acquisition of heat resistance, and that<br />
the major <strong>in</strong>crease <strong>in</strong> heat resistance occurs between 20 and 40 days<br />
<strong>in</strong>cubation. The comb<strong>in</strong>ed results of the two papers po<strong>in</strong>t to the<br />
importance of ascospore maturity <strong>in</strong> <strong>in</strong>fluenc<strong>in</strong>g the ability of the cells<br />
to rema<strong>in</strong> dormant.<br />
The second parameter that may affect activation is the treatment of<br />
the spores before and dur<strong>in</strong>g pressurisation. Reyns et al. (2003) pretreated<br />
their ascospore suspension for 20 m<strong>in</strong> at 65°C to kill vegetative<br />
cells. In a buffer at a pH 6.8, this treatment should not activate<br />
ascospores, although at 70°C a significant <strong>in</strong>crease <strong>in</strong> activation is<br />
observed <strong>in</strong> our laboratory (J. Dijksterhuis, unpublished results). At<br />
lower pH (3.0) Reyns et al. (2003) observed that nearly complete activation<br />
occurred after heat treatment at 65°C. This <strong>in</strong>dicates a clear<br />
lower<strong>in</strong>g of the heat activation temperature at low pH. Recently, we<br />
observed activation <strong>in</strong> a low pH medium at room temperature or only<br />
slightly elevated temperatures (J. Dijksterhuis, unpublished results).<br />
Heat resistant ascospores cause problems <strong>in</strong> fruit juices, and this lower<strong>in</strong>g<br />
<strong>in</strong> heat activation could also occur <strong>in</strong> these food products. The<br />
lower pH of the environment <strong>in</strong> fruit juices may reduce the heat resistance<br />
of the ascospores somewhat, but the protective effect of the<br />
<strong>in</strong>creas<strong>in</strong>g the sugar content is much greater (K<strong>in</strong>g and Whitehand,<br />
1990). Organic acids, particularly fumaric acid and to a lesser extent<br />
sorbic, benzoic and acetic acids have clear effect on heat resistance<br />
below pH 4.0 (Beuchat, 1988b). We have observed that the spores<br />
also become more resistant to high pressure under these conditions<br />
(J. Dijksterhuis, unpublished results).<br />
It is also possible that menstruum <strong>in</strong> which the spores are suspended<br />
dur<strong>in</strong>g the high pressure treatments has an effect. Dijksterhuis and<br />
Teunissen (2004) used buffer (10 mM ACES, pH 6.8) whereas Reyns<br />
et al. (2003) suspended ascospores <strong>in</strong> distilled water. Dur<strong>in</strong>g high pressure<br />
treatment the acidity of the medium decreases, but this drop<br />
would be less extensive <strong>in</strong> a buffered system. Spores suspended <strong>in</strong> distilled<br />
water may be confronted with a temporary drop <strong>in</strong> pH, result<strong>in</strong>g<br />
<strong>in</strong> more extensive activation.<br />
The authors of both papers conclude that activation by high pressure<br />
might be related to the barrier function of the ascospore cell wall.
Activation of Ascospores by Novel <strong>Food</strong> Preservation Techniques 255<br />
Dijksterhuis and Teunissen (2004) performed cryo-electron<br />
microscopy on the spores and showed alterations of the cell after very<br />
short treatments at high temperature. Reyns et al. (2003) illustrated<br />
that the spores collapsed when air dried after a high pressure treatments,<br />
whereas untreated spores ma<strong>in</strong>ta<strong>in</strong>ed their shape. These observations<br />
<strong>in</strong>dicate that structural changes occur <strong>in</strong> the cell wall and that<br />
these have a direct <strong>in</strong>fluence of the process of activation.<br />
5. THE CONNECTION BETWEEN THE<br />
DIFFERENT TREATMENTS<br />
Ascospores of Eurotium herbariorum are recognised as heat resistant,<br />
albeit less so than Byssochlamys, Neosartorya and Talaromyces<br />
species (Splittstoesser et al., 1989). Eicher and Ludwig (2002) showed<br />
that a proportion of the spores of Eurotium repens (8%) is activated<br />
from dormancy by a treatment of 200 MPa for 60 m<strong>in</strong>. The<br />
ascospores were also activated by a heat treatment: after 8 m<strong>in</strong> at 60°C<br />
approximately 50% of the spores germ<strong>in</strong>ated (on Sabouraud agar)<br />
after 5 days. At 50°C, 60 m<strong>in</strong> were needed to activate this population<br />
of cells. At room temperature <strong>in</strong> an isotonic salt solution ascospores<br />
did germ<strong>in</strong>ate, though after a delay: after 18 hours approximately 15%<br />
of the cells showed signs of germ<strong>in</strong>ation. Ascospores that were heat<br />
activated (15 m<strong>in</strong>, 60°C) were more sensitive to a subsequent high<br />
pressure treatment at 500 MPa. A 30 m<strong>in</strong> treatment at 500 MPa<br />
reduced the number of colony form<strong>in</strong>g units even compared with the<br />
unactivated spore suspensions. When the pressure treatment was<br />
applied immediately after a heat treatment at 60°C for 15 m<strong>in</strong> the<br />
number of colony form<strong>in</strong>g cells reduced by a factor of 40. When a<br />
pause was <strong>in</strong>troduced between the treatments dur<strong>in</strong>g which the spores<br />
were stored at 20°C <strong>in</strong> an isotonic salt solution, the number of colony<br />
form<strong>in</strong>g units restored 10-fold. This phenomenon was designated<br />
“re-stabilisation” (Eicher and Ludwig, 2002). After treatments with<br />
high pressure (500 MPA, 30 m<strong>in</strong>), heat activation did not result <strong>in</strong> any<br />
enhancement of germ<strong>in</strong>at<strong>in</strong>g cells of E. repens. In fact, heat treatment<br />
resulted <strong>in</strong> some further reduction of colony form<strong>in</strong>g units, viability<br />
counts only reduced further when a pause was present between the<br />
treatments (Eicher and Ludwig, 2002).<br />
In the case of T. macrosporus, Dijksterhuis and Teunissen (2004)<br />
described near total activation of germ<strong>in</strong>ation by heat (7 m<strong>in</strong>, 85°C)<br />
after 5 m<strong>in</strong> pressure treatments up to 800 MPa. Short pressure
256 Jan Dijksterhuis and Robert A. Samson<br />
treatments at least did not lead to heat sensitisation <strong>in</strong> their experiments.<br />
However, Reyns et al. (2003) show clear sensitisation of<br />
ascospores for the activation heat treatment (30 m<strong>in</strong>, 80°C) after all<br />
pressure treatments at 600 MPa and 700 MPa.<br />
6. DORMANCY REVISITED<br />
The observed re-stabilisation phenomenon of E. repens<br />
ascospores poses the question of whether ascospores can return to<br />
their dormant state once activated. We addressed this question <strong>in</strong> a<br />
number of experiments where heat activated ascospores of<br />
T. macrosporus were confronted with a sudden lower<strong>in</strong>g of the temperature<br />
or with dry<strong>in</strong>g conditions. The high amount of trehalose<br />
(10-20% wet weight) and the low water content of the spores (38%)<br />
may <strong>in</strong>troduce a very high viscosity <strong>in</strong>side the spores. This has<br />
recently been confirmed by electron-(para)magnetic-resonance studies<br />
(J. Dijksterhuis, unpublished results). A sudden lower<strong>in</strong>g of the temperature<br />
or a reduction of the water content will most likely <strong>in</strong>troduce a<br />
glass transition situation <strong>in</strong>side the cell. The glassy state is an amorphous<br />
phase characterized by very low movement speeds of the cell<br />
components. Reduction of the water content or lower<strong>in</strong>g of the temperature<br />
are two factors that favour the glass transition of the<br />
ascospores. Plung<strong>in</strong>g of the cells <strong>in</strong>to liquid nitrogen or controlled<br />
dry<strong>in</strong>g below 3% water-content especially will <strong>in</strong>troduce a glassy state<br />
<strong>in</strong> these cells. This transition also may re-establish the dormancy of<br />
these cells. “Biological glasses”, a term <strong>in</strong>troduced by Buit<strong>in</strong>k (2000)<br />
are characterised by a melt<strong>in</strong>g temperature that can be high (Wolkers<br />
et al., 1996) and cells may need to be exposed to high temperatures<br />
aga<strong>in</strong> <strong>in</strong> order to re-activate them.<br />
A number of experiments were done by stor<strong>in</strong>g heat-activated<br />
ascospores (7 m<strong>in</strong>, 85°C) on ice for 15 m<strong>in</strong> or plung<strong>in</strong>g the cells <strong>in</strong>to<br />
liquid nitrogen and keep<strong>in</strong>g them there for 15 m<strong>in</strong>. The samples were<br />
allowed to warm to room temperature and subsequently plated out. In<br />
addition, activated ascospores were <strong>in</strong>cubated at 30°C for 1 h before<br />
the cold treatment. Table 2 summarises these experiments and shows<br />
that dormant ascospores showed only low levels of germ<strong>in</strong>ation<br />
whether they are cooled or not, while heat activated cells under all<br />
cases showed very high percentages of germ<strong>in</strong>at<strong>in</strong>g cells.<br />
Ice-treatment directly after or 1 h follow<strong>in</strong>g heat activation did not<br />
show any effect. These experiments <strong>in</strong>dicate that a sudden lower<strong>in</strong>g of
Activation of Ascospores by Novel <strong>Food</strong> Preservation Techniques 257<br />
Table 2. Influence of a sudden cool<strong>in</strong>g treatment on extent of germ<strong>in</strong>ation of activated<br />
ascospores of T. macrosporus. Numbers represent the ratio between the<br />
treated samples and the untreated controls.<br />
Cooled on ice Cooled <strong>in</strong> liquid nitrogen<br />
Experiment 1 Experiment 2 Experiment 1 Experiment 2<br />
Controls 1 1 1 1<br />
Controls, cooled 1.2 0.3 0.5 0.2<br />
Activated 150 9.7 11 13<br />
Activated, cooled 248 11.2 7.4 14.1<br />
Activated, cooled<br />
after 1 h<br />
198 31 12.9 10.2<br />
Heat activation of ascospores was <strong>in</strong> 3-5 ml suspensions at 85°C for 7 m<strong>in</strong> and shaken<br />
at 140 rpm <strong>in</strong> a waterbath. When spores were <strong>in</strong>cubated for 1 h this was at 30° C (140<br />
rpm). Spores were placed <strong>in</strong> Eppendorf tubes and put on ice or <strong>in</strong>to liquid nitrogen.<br />
Samples were plated out <strong>in</strong> duplicate and colonies counted. The ratio between treated<br />
samples and untreated controls is given <strong>in</strong> this table.<br />
the temperature, irrespective of how fast and large it might be, does<br />
not “reset” the ascospores to the dormant mode.<br />
In a further experiment, ascospores were dried accord<strong>in</strong>g to the<br />
procedures used at the CBS. The latter <strong>in</strong>cludes controlled freez<strong>in</strong>g to<br />
−40°C (1°C/m<strong>in</strong>), storage at −80°C and dry<strong>in</strong>g under vacuum. Dried<br />
dormant ascospores rema<strong>in</strong>ed dormant after dry<strong>in</strong>g and could be<br />
effectively activated by a heat treatment after resuspend<strong>in</strong>g them <strong>in</strong><br />
buffer. Dried freshly activated ascospores produced the similar numbers<br />
of activated spores and the cells showed similar tolerance to the<br />
dry<strong>in</strong>g treatment as dormant cells. When these cells were heat treated<br />
(<strong>in</strong> case dormancy had re-established) no additional <strong>in</strong>crease or<br />
decrease of cell numbers occurred. This <strong>in</strong>dicated that these cells had<br />
reta<strong>in</strong>ed their activated state, but still showed heat resistance. Both<br />
dry<strong>in</strong>g tolerance and heat resistance had decreased markedly after<br />
<strong>in</strong>cubat<strong>in</strong>g the activated cells for 2 h at 30°C. These comb<strong>in</strong>ed observations<br />
show that important phase transitions of the <strong>in</strong>ner cell do not<br />
change the status of activation or dormancy <strong>in</strong> T. macrosporus.<br />
7. THE SPEED OF HEAT ACTIVATION<br />
Accord<strong>in</strong>g to Sussman (1966) dormancy is def<strong>in</strong>ed as a hypometabolic<br />
state; i.e. a rest period or reversible <strong>in</strong>terruption of (phenotypic)<br />
development. He discerns exogenous dormany (quiescence) which<br />
<strong>in</strong>clude delayed development due to physical or chemical cues.
258 Jan Dijksterhuis and Robert A. Samson<br />
Constitutive dormancy is a condition <strong>in</strong> which development is delayed<br />
as an <strong>in</strong>nate property such as a barrier to the penetration of nutrients,<br />
a metabolic block, or a self-<strong>in</strong>hibitory compound. In case of<br />
T. macrosporus, ascospores can be activated and also synchronised to<br />
germ<strong>in</strong>ate by a robust physical signal such as heat and/or high pressure.<br />
Careful exam<strong>in</strong>ation of the data provided by Beuchat (1986)<br />
suggests that the speed of activation is <strong>in</strong>creased at higher temperatures.<br />
Ascospores of Neosartorya fischeri exhibited constant rates of<br />
heat activation between 70° and 85°C (K<strong>in</strong>g and Halbrook, 1987,<br />
Figure 1). In our laboratory we observed that ascospores added to<br />
preheated buffer were fully activated with<strong>in</strong> 2 m<strong>in</strong>.<br />
Kikoku (2003) reported full activation of T. macrosporus ascospores <strong>in</strong><br />
a citrate-phosphate buffer (pH 6.5) with<strong>in</strong> 100 s at 81° and 82.5°C. Above<br />
this temperature this time became even shorter, namely 60 s at 86.5 and<br />
87°C, and 35 s at 91°C. From these data the author extracted rate constants<br />
of heat activation (expressed as k), which range from 1.2 to 4.1/m<strong>in</strong><br />
between 81° and 91°C. At 84°C no difference <strong>in</strong> k was observed between<br />
pH 3.5 or 6.5 (2.9 and 2.8/m<strong>in</strong> respectively) and also <strong>in</strong> phosphate buffer<br />
(pH 6.6) the k value was 2.8/m<strong>in</strong>. However <strong>in</strong> grape juice (5 °Brix) a very<br />
high k value was observed (7.7/m<strong>in</strong>). Thus, the presence of the sugars or<br />
organic acids or some other compound <strong>in</strong> the fruit juice resulted <strong>in</strong> a very<br />
rapid activation at this temperature (100% <strong>in</strong> 20 s).<br />
Activation energy (Ea) can be calculated us<strong>in</strong>g an Arrhenius plot<br />
where ln (0.23303.k) is plotted aga<strong>in</strong>st 1/T. When activation is the result<br />
of the conformation or chemical change of one def<strong>in</strong>ed compound <strong>in</strong><br />
the ascospore, for <strong>in</strong>stance a compound of the (plasma) membrane or<br />
a receptor prote<strong>in</strong>, the Ea reflects the energy needed to convert 1 mole<br />
of such compound. Changes <strong>in</strong> prote<strong>in</strong>aceous compounds do need a<br />
different energy absorption than lipid compounds and the Ea could<br />
give clues about the nature of activation. However, when more systemic<br />
changes <strong>in</strong> the ascospore absorb the energy delivered by the heat, the<br />
Ea calculated does not give much <strong>in</strong>formation. Recent f<strong>in</strong>d<strong>in</strong>gs at our<br />
laboratory show changes <strong>in</strong> the ascospore dur<strong>in</strong>g heat activation<br />
<strong>in</strong>volv<strong>in</strong>g both prote<strong>in</strong>s and cell wall components, and this multitude of<br />
changes may make a <strong>in</strong>terpretation of the Ea value difficult.<br />
8. ACKNOWLEDGEMENT<br />
The authors are <strong>in</strong>debted to Kenneth van Driel for assistance with<br />
Low-Temperature Scann<strong>in</strong>g Electron Microscopy experiments, and
Activation of Ascospores by Novel <strong>Food</strong> Preservation Techniques 259<br />
Yasm<strong>in</strong> Stoop for her assistance with the dormancy experiments.<br />
Joost Eleveld and Mark Kweens are thanked for their data on heat<br />
<strong>in</strong>activation of Talaromyces ascospores.<br />
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MIXTURES OF NATURAL AND SYNTHETIC<br />
ANTIFUNGAL AGENTS<br />
Aurelio López-Malo, Enrique Palou, Reyna León-Cruz and<br />
Stella M. Alzamora *<br />
1. INTRODUCTION<br />
Antimicrobial agents are chemical compounds present <strong>in</strong>, or added<br />
to, foods that retard microbial growth or cause microbial death<br />
(López-Malo et al., 2000). The use of such agents is one of the oldest<br />
and most traditional food preservation techniques (López-Malo et al.,<br />
2005a).<br />
Antimicrobial agents are somewhat arbitrarily classified as traditional<br />
and naturally occurr<strong>in</strong>g (Davidson, 2001). Traditional antimicrobials<br />
are those that have been used for long time, approved by many<br />
countries as antimicrobials <strong>in</strong> foods or are produced by synthetic<br />
means, or are <strong>in</strong>organic. Antimicrobial agents may be either synthetic<br />
compounds <strong>in</strong>tentionally added to foods or naturally occurr<strong>in</strong>g,<br />
biologically derived substances (the so called naturally occurr<strong>in</strong>g<br />
antimicrobials), which may be used commercially as additives for food<br />
preservation as well as exhibit<strong>in</strong>g antimicrobial properties <strong>in</strong> the<br />
biological systems from which they orig<strong>in</strong>ate (Sofos et al., 1998).<br />
However, as Davidson (2001) stated, antimicrobial agents now<br />
produced synthetically are also found <strong>in</strong> nature, <strong>in</strong>clud<strong>in</strong>g acetic,<br />
benzoic or sorbic acids.<br />
*A. López-Malo, E. Palou, R. León-Cruz, Ingeniería Química y Alimentos, Universidad<br />
de las Américas, Puebla, Cholula 72820, Mexico. S. Alzamora, Dept Industrias, Fac. de<br />
Ciencias Exactas y Naturales, Univ. de Buenos Aires, Ciudad Universitaria 1428, Buenos<br />
Aires, Argent<strong>in</strong>a. Correspondence to aurelio.lopezm@udlap.mx<br />
261
262 Aurelio López-Malo et al.<br />
Concerns about the use of antimicrobial agents <strong>in</strong> food products<br />
have been discussed for decades (Parish and Carroll, 1988). The<br />
<strong>in</strong>creas<strong>in</strong>g demand for more “natural” foods with reduced additives<br />
(<strong>in</strong>clud<strong>in</strong>g antimicrobial agents) and the <strong>in</strong>creas<strong>in</strong>g demand for<br />
greater convenience, have promoted the search for alternative antimicrobial<br />
agents or comb<strong>in</strong>ations to be used by the food <strong>in</strong>dustry<br />
(Alzamora et al., 2003; López-Malo et al., 2000, 2005a). In this<br />
search, a wide range of natural systems from animals, plants and<br />
microorganisms have been studied (Beuchat and Golden, 1989;<br />
Board, 1995; Hill, 1995; Nychas, 1995; Sofos et al., 1998; Davidson,<br />
2001; Chik<strong>in</strong>das and Montville, 2002; Gould, 2002; Alzamora<br />
et al., 2003; López-Malo et al., 2000, 2005a), and the studies are cont<strong>in</strong>u<strong>in</strong>g.<br />
However, the strict requirements to obta<strong>in</strong> approval and the<br />
economic cost of gett<strong>in</strong>g the product onto the market restrict the<br />
spectrum of new chemical compounds that can be used <strong>in</strong> the preservation<br />
of foods. These obstacles have prompted the renewed search for<br />
preservatives by exam<strong>in</strong><strong>in</strong>g compounds already used <strong>in</strong> the food<br />
<strong>in</strong>dustry, perhaps for other purposes, but with potential antimicrobial<br />
activity. Such compounds are already approved and not toxic <strong>in</strong> the<br />
levels used; many of them classified <strong>in</strong> the USA as generally recognized<br />
as safe (GRAS). Included <strong>in</strong> these compounds are, for example,<br />
the so-called “green chemicals” present <strong>in</strong> plants that are utilized as<br />
flavour <strong>in</strong>gredients (Davidson, 2001; Alzamora et al., 2003; López-<br />
Malo et al., 2005a).<br />
The antimicrobial activities of several plant derivatives used<br />
today as season<strong>in</strong>g agents <strong>in</strong> foods and beverages have been recognized<br />
for centuries. Although ancient civilizations recognized the<br />
antiseptic or antimicrobial potential of many plant extracts, it was<br />
not until the eighteenth century that scientific <strong>in</strong>formation underp<strong>in</strong>ned<br />
the observed effects. Naturally occurr<strong>in</strong>g antimicrobial compounds<br />
are abundant <strong>in</strong> the environment. Some of these natural<br />
antimicrobial systems are already employed for food preservation,<br />
while many others are still be<strong>in</strong>g studied (Gould, 1995). The exploration<br />
and use of naturally occurr<strong>in</strong>g antimicrobials <strong>in</strong> foods, as<br />
well as their chemistry, food safety and toxicity aspects, antimicrobial<br />
activity and mechanisms of action, are covered <strong>in</strong> excellent<br />
reviews by Branen et al. (1980), Shelef (1983), Zaika (1988),<br />
Beuchat and Golden (1989), Wilk<strong>in</strong>s and Board (1989), Conner<br />
(1993), Board (1995), Nychas (1995), Sofos et al. (1998), Smid and<br />
Gorris (1999), Davidson (2001), Gould (2002), and López-Malo<br />
et al. (2000, 2005a).
Mixtures of Natural and Synthetic Antifungal Agents 263<br />
1.1. Sources of Natural Antimicrobials From Plants<br />
Plants, herbs and spices as well as their derived essential oils and<br />
isolated compounds conta<strong>in</strong> a large number of substances that are<br />
known to <strong>in</strong>hibit various metabolic activities of bacteria, and fungi,<br />
although many have not been fully exploited. Hundreds of plants are<br />
known to be potential sources of antimicrobial compounds (Wilk<strong>in</strong>s<br />
and Board, 1989; Beuchat, 1994; Nychas, 1995). Antimicrobial compounds<br />
<strong>in</strong> plant materials are commonly conta<strong>in</strong>ed <strong>in</strong> the essential oil<br />
fraction of leaves, flowers and flower buds, bulbs, rhizomes, fruit, or<br />
other parts of the plant (Shelef, 1983; Nychas, 1995). Antimicrobial<br />
compounds may be lethal to microorganisms or they may simply<br />
<strong>in</strong>hibit the production of metabolites such as mycotox<strong>in</strong>s (Conner and<br />
Beuchat, 1984a, b; Zaika, 1988; Beuchat, 1994; Davidson, 2001).<br />
Major components with antimicrobial activity found <strong>in</strong> plants,<br />
herbs and spices are phenolic compounds, terpenes, aliphatic alcohols,<br />
aldehydes, ketones, acids and isoflavonoids. As a rule, the antimicrobial<br />
activity of essential oils depends on the chemical structure of<br />
their components and on their concentration (Davidson, 2001; López-<br />
Malo et al., 2005a). Simple and complex derivatives of phenol are the<br />
ma<strong>in</strong> antimicrobial compounds <strong>in</strong> essential oils from spices (Shelef,<br />
1983). The antimicrobial activity of c<strong>in</strong>namon, allspice, and cloves is<br />
attributed to eugenol (2-methoxy-4-allyl phenol) and c<strong>in</strong>namic<br />
aldehyde, which are major constituents of the volatile oils of these<br />
spices (Bullerman et al., 1977; Farrell, 1990). Eugenol, carvacrol, thymol<br />
and vanill<strong>in</strong> have been recognized as active antimicrobial compounds<br />
from plant essential oils (López-Malo et al., 2005a), and<br />
aliphatic alcohols and phenolics have also been reported as fungal<br />
growth <strong>in</strong>hibitors (Katayama and Nagai, 1960; Farag et al., 1989).<br />
A wide antimicrobial spectrum has been found <strong>in</strong> phenolic compounds<br />
such as thymol extracted from thyme and oregano, c<strong>in</strong>namic<br />
aldehyde extracted from c<strong>in</strong>namon, and eugenol extracted from cloves<br />
(Beuchat and Golden, 1989; Wilk<strong>in</strong>s and Board, 1989; Davidson and<br />
Branen, 1993). Vanill<strong>in</strong>, a phenolic compound present <strong>in</strong> vanilla pods<br />
also has antifungal activity (Beuchat, 1976; Zaika, 1988; Beuchat and<br />
Golden, 1989; Farag et al., 1989; Cerrutti and Alzamora, 1996;<br />
Cerrutti et al., 1997; López-Malo et al., 1995, 1997, 1998). C<strong>in</strong>namon<br />
bark is highly <strong>in</strong>hibitory to the moulds Aspergillus flavus, A. parasiticus,<br />
A. versicolor, and A. ochraceus (Hitokoto et al., 1978). Ground c<strong>in</strong>namon<br />
at a concentration of 1-2% <strong>in</strong> broth allowed some growth of<br />
A. parasiticus, but elim<strong>in</strong>ated approximately 99% of the production of
264 Aurelio López-Malo et al.<br />
aflatox<strong>in</strong> (Bullerman, 1974). The active compounds were reported to<br />
be c<strong>in</strong>namic aldehyde and eugenol, which are the major constituents<br />
of essential oils from c<strong>in</strong>namon and clove (Bullerman et al., 1977).<br />
Eugenol has been reported as one of the most effective natural antimicrobials<br />
from plant orig<strong>in</strong> act<strong>in</strong>g as a sporostatic agent (Al-Khayat<br />
and Blank, 1985). Thymol at 100 ppm <strong>in</strong>hibited A. parasiticus growth<br />
for 7 days at 28°C (Buchanan and Shepherd, 1981). A. flavus was<br />
shown to be more sensitive than two other Aspergillus species when<br />
exposed to essential oils of oregano and cloves (Paster et al., 1990).<br />
1.2. Mechanisms of Action<br />
Some essential oils, plant extracts, and oleores<strong>in</strong>s <strong>in</strong>fluence certa<strong>in</strong><br />
biochemical and/or metabolic functions, such as respiration or<br />
production of tox<strong>in</strong>s or acids, <strong>in</strong>dicat<strong>in</strong>g that the active components <strong>in</strong><br />
various oils and oleores<strong>in</strong>s may have different specificities for target<br />
sites on or <strong>in</strong> microbial cells. Much of the research on spices has<br />
<strong>in</strong>cluded speculation on the contribution of the terpene fraction to<br />
their antimicrobial activity. Few of the studies, however, have<br />
attempted to isolate and identify the antimicrobial fraction, and no<br />
references are found which relate to the mechanism by which spices<br />
<strong>in</strong>hibit microorganisms. It seems reasonable that s<strong>in</strong>ce many of the<br />
components of the essential oils, such as eugenol and thymol, are<br />
similar <strong>in</strong> structure to active phenolic antimicrobials, their modes of<br />
action could be assumed to be similar (Davidson, 2001; López-Malo<br />
et al., 2005a).<br />
The possible modes of action of phenolic compounds have been<br />
reported <strong>in</strong> several reviews (Wilk<strong>in</strong>s and Board; 1989; Beuchat, 1994;<br />
Nychas, 1995; Sofos et al., 1998; Davidson, 2001; López-Malo<br />
et al., 2000, 2005a). These mechanisms have not been completely elucidated,<br />
however, the effect of phenolic compounds is concentration<br />
dependent (Pr<strong>in</strong>dle and Wright, 1977). At low concentration, phenols<br />
affected enzyme activity, especially of those enzymes associated with<br />
energy production, whereas at higher concentrations, prote<strong>in</strong> denaturation<br />
occurred. The effect of phenolic antioxidants on microbial<br />
growth and tox<strong>in</strong> production could be the result of the ability of phenolic<br />
compounds to alter microbial cell permeability, permitt<strong>in</strong>g the<br />
loss of macromolecules from the <strong>in</strong>terior. They could also <strong>in</strong>teract<br />
with membrane prote<strong>in</strong>s caus<strong>in</strong>g a deformation <strong>in</strong> structure and functionality<br />
(Fung et al., 1977). Conner and Beuchat (1984a, b) suggested<br />
that antimicrobial activity of essential oils on yeasts could be the<br />
result of disturbance <strong>in</strong> several enzymatic systems <strong>in</strong>volved <strong>in</strong> energy
Mixtures of Natural and Synthetic Antifungal Agents 265<br />
production and synthesis of structural components. Once phenolic<br />
compounds cross the cell membrane, <strong>in</strong>teractions with membrane<br />
enzymes and prote<strong>in</strong>s would cause an opposite flow of protons, affect<strong>in</strong>g<br />
cellular activity.<br />
Increas<strong>in</strong>g the concentration of thyme essential oil, thymol or<br />
carvacrol was not reflected <strong>in</strong> a direct relationship with antimicrobial<br />
effects. However, after exceed<strong>in</strong>g a certa<strong>in</strong> critical concentration, a<br />
rapid and drastic reduction <strong>in</strong> viable cells was observed (Juven et al.,<br />
1994). Phenolic compounds could sensitize cellular membranes and<br />
when sites are saturated, serious damage and a rapid collapse of<br />
cytoplasmatic membrane <strong>in</strong>tegrity could be present, with the consequent<br />
loss of cytoplasmatic constituents. It has been suggested that<br />
the effects of phenolic compound could be at two levels, on cellular<br />
wall and membrane <strong>in</strong>tegrity as well as on microbial physiological<br />
responses (Kabara and Eklund, 1991). Phenolic compounds could<br />
also denature enzymes responsible for spore germ<strong>in</strong>ation or <strong>in</strong>terfere<br />
with am<strong>in</strong>o acids that are necessary <strong>in</strong> germ<strong>in</strong>ation processes<br />
(Nychas, 1995).<br />
1.3. Factors Affect<strong>in</strong>g Activity<br />
The antimicrobial activities of extracts from several types of plants<br />
and plant parts used as flavour<strong>in</strong>g agents <strong>in</strong> foods have been recognized<br />
for many years. However, few studies have reported on the effect<br />
of extracts <strong>in</strong> comb<strong>in</strong>ation with other factors on microbial growth.<br />
The potential of a compound as a total or partial substitute for<br />
common preservatives to <strong>in</strong>hibit growth of spoilage and pathogenic<br />
microorganisms needs to be evaluated alone and <strong>in</strong> comb<strong>in</strong>ation with<br />
traditional preservation factors, such as storage temperature, pH,<br />
water activity (a w ), other antimicrobials, and modified atmospheres.<br />
Results from such studies could be very useful, permitt<strong>in</strong>g researchers<br />
<strong>in</strong>volved <strong>in</strong> the development of multifactorial preservation of foods to<br />
assess quickly the impact of alter<strong>in</strong>g any comb<strong>in</strong>ation of the studied<br />
variables.<br />
1.4. Comb<strong>in</strong>ed Antimicrobial Agents<br />
Traditionally, only one chemical antimicrobial agent was used to<br />
preserve a food (Busta and Foeged<strong>in</strong>g, 1983). However, more recently,<br />
the use of comb<strong>in</strong>ed agents <strong>in</strong> a s<strong>in</strong>gle food system has become more<br />
common. The use of comb<strong>in</strong>ed antimicrobial agents theoretically<br />
provides a greater spectrum of activity, with enhanced action aga<strong>in</strong>st
266 Aurelio López-Malo et al.<br />
pathogenic and/or spoilage microorganisms. It is thought that comb<strong>in</strong>ed<br />
agents will act on different species <strong>in</strong> a mixed microflora or act<br />
on different metabolic elements with<strong>in</strong> similar species or stra<strong>in</strong>s, which<br />
theoretically results <strong>in</strong> improved control compared with the use of one<br />
antimicrobial agent alone. However, actual proof of improved efficacy<br />
requires objective <strong>in</strong>terpretation. Although test<strong>in</strong>g of comb<strong>in</strong>ed<br />
antibiotics for cl<strong>in</strong>ical use is well studied and relatively well standardized<br />
(Barry, 1976; Krogstad and Moeller<strong>in</strong>g, 1986; Eliopoulos and<br />
Moeller<strong>in</strong>g, 1991), application of such methodology to antimicrobials<br />
<strong>in</strong> food systems is poorly developed (Davidson and Parish, 1989;<br />
López-Malo et al., 2005b). The use of antibiotic comb<strong>in</strong>ations <strong>in</strong><br />
medic<strong>in</strong>e cont<strong>in</strong>ues to be a subject of <strong>in</strong>tensive <strong>in</strong>vestigation and a<br />
matter of great cl<strong>in</strong>ical relevance (Eliopoulos and Moeller<strong>in</strong>g, 1991).<br />
Methods of test<strong>in</strong>g comb<strong>in</strong>ed antimicrobials usually <strong>in</strong>volve agar<br />
diffusion, agar or broth dilution, or death time curves (NCCLS, 1999;<br />
2002). Dilution methods yield quantitative data and are often<br />
conducted with various comb<strong>in</strong>ed concentrations of two antimicrobials<br />
arranged <strong>in</strong> a “checkerboard” array. The checkerboard method is the<br />
technique used most frequently to assess antimicrobial comb<strong>in</strong>ations<br />
<strong>in</strong> vitro, presumably because a) its rationale is easy to understand,<br />
b) the mathematics necessary to calculate and <strong>in</strong>terpret the results are<br />
simple, c) it can be readily performed <strong>in</strong> the laboratory us<strong>in</strong>g microdilution<br />
systems, and d) it has been the technique most frequently used<br />
<strong>in</strong> studies that have <strong>in</strong>vestigated synergistic <strong>in</strong>teractions of antibiotics<br />
<strong>in</strong> cl<strong>in</strong>ical treatments (Eliopoulos and Moeller<strong>in</strong>g, 1991). The term<br />
checkerboard refers to the pattern (of tubes, plates or microtitre wells)<br />
formed by multiple dilutions of the two antimicrobials be<strong>in</strong>g tested <strong>in</strong><br />
concentrations equal to, above, and below their MIC. Traditional<br />
cl<strong>in</strong>ical dilution test<strong>in</strong>g uses two-fold dilutions of test compounds, but<br />
test<strong>in</strong>g of food antimicrobials is often conducted with alternate<br />
dilution schemes (Rehm, 1959; Davidson and Parish, 1989; Eliopoulos<br />
and Moeller<strong>in</strong>g, 1991).<br />
Comb<strong>in</strong>ed studies are conducted to determ<strong>in</strong>e if specific types of<br />
<strong>in</strong>teractions occur between the two comb<strong>in</strong>ed antimicrobials.<br />
Traditionally, the terms “additive,” “antagonistic,” and “synergistic”<br />
were used to describe possible antimicrobial <strong>in</strong>teractions. Additivity<br />
occurs when two comb<strong>in</strong>ed antimicrobials give results that are equivalent<br />
to the sum of each antimicrobial act<strong>in</strong>g <strong>in</strong>dependently, i.e. no<br />
enhancement or reduction <strong>in</strong> overall efficacy for the comb<strong>in</strong>ed antimicrobials<br />
occurs compared to the <strong>in</strong>dividual results, and is also sometimes<br />
referred to as “<strong>in</strong>difference” (Krogstad and Moeller<strong>in</strong>g, 1986;<br />
Eliopoulos and Moeller<strong>in</strong>g, 1991). Antagonism refers to a reduced
Mixtures of Natural and Synthetic Antifungal Agents 267<br />
efficacy of the comb<strong>in</strong>ed agents compared to the sum of the <strong>in</strong>dividual<br />
results. Synergism is an <strong>in</strong>crease or enhancement of overall antimicrobial<br />
activity when two agents are comb<strong>in</strong>ed compared to the sum of<br />
<strong>in</strong>dividual results (López-Malo et al., 2005b).<br />
A conclusion that synergism occurs must be approached with<br />
caution s<strong>in</strong>ce it implies that a reduction of overall antimicrobial concentration<br />
might be achieved <strong>in</strong> a food system without a reduction <strong>in</strong><br />
efficacy. Gardner (1977) stated that true synergism is quite rare <strong>in</strong><br />
relation to comb<strong>in</strong>ed antibiotics. Other concerns about the misuse of<br />
the term “synergism” <strong>in</strong> relation to antimicrobials have been cited<br />
(Garrett, 1958; Davidson and Parish, 1989). Most commonly, additive<br />
<strong>in</strong>teractions are misidentified as synergistic. A case <strong>in</strong> which an<br />
<strong>in</strong>crease <strong>in</strong> antimicrobial activity is observed upon the addition of a<br />
second compound to a food system does not necessarily constitute<br />
synergy. A conclusion of synergism requires that the overall efficacy of<br />
the comb<strong>in</strong>ation be significantly greater than the sum of the efficacies<br />
of the <strong>in</strong>dividual compounds.<br />
Additive, synergistic, or antagonistic <strong>in</strong>teractions can be <strong>in</strong>terpreted<br />
with an MIC isobologram. Isobologram construction can be simplified<br />
us<strong>in</strong>g fractional <strong>in</strong>hibitory concentrations (FIC), which are MICs<br />
normalized to unity. The FIC is the concentration of a compound<br />
needed to <strong>in</strong>hibit growth (expressed as a fraction of its MIC) when<br />
comb<strong>in</strong>ed with a known amount of a second antimicrobial compound.<br />
It is calculated as the ratio of the MIC of a compound when<br />
comb<strong>in</strong>ed with a second compound divided by the MIC of the first<br />
compound alone. The FIC of two compounds <strong>in</strong> an <strong>in</strong>hibitory<br />
comb<strong>in</strong>ation may be added to give a total FIC Index . An FIC Index near 1<br />
<strong>in</strong>dicates additivity, whereas an FIC Index less than 1 <strong>in</strong>dicates synergy<br />
and an FIC Index greater than 1 <strong>in</strong>dicates antagonism. The degree to which<br />
a result must be less than or greater than 1, to <strong>in</strong>dicate synergism or<br />
antagonism is a matter of <strong>in</strong>terpretation. Squires and Cleeland (1985)<br />
proposed that for antibiotic test<strong>in</strong>g FIC Index <strong>in</strong>dicates additive results<br />
between 0.5 and 2.0. Synergism and antagonism are <strong>in</strong>dicated by<br />
results FIC Index < 0.5 and FIC Index > 2.0, respectively. Research is<br />
needed to provide a database for proper <strong>in</strong>terpretation of FIC and<br />
FIC Index <strong>in</strong> relation to food antimicrobial systems. Data <strong>in</strong>terpretation<br />
must be conducted conditionally and will depend upon a number of<br />
variables, such as specific test conditions, microbial stra<strong>in</strong>, and target<br />
food system (López-Malo et al., 2005b). It should be noted that<br />
<strong>in</strong>terpretations might also vary depend<strong>in</strong>g upon the specific concentrations<br />
of each antimicrobial used <strong>in</strong> comb<strong>in</strong>ation. Parish and<br />
Carroll (1988) observed additivity between SO 2 and either sorbate or
268 Aurelio López-Malo et al.<br />
butylparaben when the <strong>in</strong>hibitory concentration conta<strong>in</strong>ed less than<br />
0.25 FIC of SO 2 . However, for the same comb<strong>in</strong>ation at higher SO 2 ,<br />
the calculated FIC <strong>in</strong>dicated antagonistic results. Rehm (1959)<br />
observed similar anomalies when sodium sulphite was comb<strong>in</strong>ed with<br />
formate or borate.<br />
An exam<strong>in</strong>ation of these results shows that it is not easy to anticipate<br />
the effects or to expla<strong>in</strong> observed activity when consider<strong>in</strong>g<br />
b<strong>in</strong>ary mixtures of antimicrobials. Moreover, there is an <strong>in</strong>creas<strong>in</strong>g<br />
awareness that many comb<strong>in</strong>ations may result <strong>in</strong> antagonism. Four<br />
mechanisms of antimicrobial <strong>in</strong>teraction that produce synergism are<br />
generally accepted: a) sequential <strong>in</strong>hibition of a common biochemical<br />
pathway, b) <strong>in</strong>hibition of protective enzymes, c) comb<strong>in</strong>ations of<br />
agents active aga<strong>in</strong>st cell walls; and d) the use of cell wall active agents<br />
to enhance the uptake of other antimicrobials (Eliopoulos and<br />
Moeller<strong>in</strong>g 1991). Mechanisms of antimicrobial <strong>in</strong>teraction that<br />
produce antagonism are less well understood and <strong>in</strong>clude: a) comb<strong>in</strong>ations<br />
of bactericidal/fungicidal and bacteriostatic/ fungistatic<br />
agents; b) use of compounds that act on the same target of the<br />
microorganism; and c) chemical (direct or <strong>in</strong>direct) <strong>in</strong>teractions<br />
between the compounds (Larson, 1984). The specific modes of action<br />
of plant constituents on metabolic activities of microorganisms still<br />
need to be clearly def<strong>in</strong>ed, even when they are the only stress factor.<br />
It is not easy to anticipate the effects of b<strong>in</strong>ary mixtures of antimicrobials,<br />
or to expla<strong>in</strong> their activity, and it becomes even more difficult<br />
if more than two antimicrobials are mixed. Despite the lack of scientific<br />
knowledge about the mechanisms of <strong>in</strong>teraction of natural and<br />
synthetic antimicrobials, synergistic comb<strong>in</strong>ations could be useful <strong>in</strong><br />
reduc<strong>in</strong>g the amounts of antimicrobials needed, dim<strong>in</strong>ish<strong>in</strong>g<br />
consumer concerns about the use of chemical preservatives. Our<br />
objective <strong>in</strong> this study was to evaluate the <strong>in</strong>dividual and comb<strong>in</strong>ed<br />
(b<strong>in</strong>ary or ternary) effects of selected natural phenolic compounds<br />
such as eugenol, vanill<strong>in</strong>, thymol, and carvacrol, and a synthetic<br />
antimicrobial agent (potassium sorbate) as antifungal agent mixtures.<br />
2. MATERIALS AND METHODS<br />
2.1. Microorganisms and Preparation of Inocula<br />
Aspergillus flavus (ATCC 16872) was cultivated on potato dextrose<br />
agar slants (PDA; Merck, Mexico City, Mexico) for 10 days at 25°C
Mixtures of Natural and Synthetic Antifungal Agents 269<br />
and the spores were harvested with 10 ml of 0.1% Tween 80 (Merck,<br />
Mexico City, Mexico) solution sterilized by membrane (0.45 µm)<br />
filtration. The spore suspension was adjusted with the same solution<br />
to give a f<strong>in</strong>al spore concentration of 10 6 spore/ml and used the<br />
same day.<br />
2.2. Preparation of Agar Systems<br />
PDA agar systems were prepared with the addition of commercial<br />
sucrose to produce a w 0.99, sterilized for 15 m<strong>in</strong> at 121°C, cooled and<br />
acidified with hydrochloric acid to atta<strong>in</strong> pH 3.5. The amounts of<br />
sucrose and hydrochloric acid needed were previously determ<strong>in</strong>ed.<br />
The sterile acidified agar was aseptically divided and the required<br />
amounts of thymol, carvacrol, vanill<strong>in</strong>, eugenol (0, 25, 50, 75, up to<br />
1300 ppm), and/or potassium sorbate (0, 50, 100, 150 up to 1000 ppm)<br />
were added and mechanically <strong>in</strong>corporated aseptically. Test<br />
compounds were obta<strong>in</strong>ed from Sigma Chemical, Co., St. Louis, MO.<br />
Agar solutions were poured <strong>in</strong>to sterile Petri dishes. The comb<strong>in</strong>ations<br />
tested are given <strong>in</strong> Table 1.<br />
As examples of the checkerboard array employed to evaluate the<br />
effects of b<strong>in</strong>ary mixtures of antimicrobials, Tables 2 and 3 present<br />
the conditions and concentrations evaluated. In the case of ternary<br />
mixtures a general scheme proposed by Berenbaum et al. (1983) was<br />
used where the maximum concentrations (represented as 1) of each<br />
antimicrobial correspond to the MIC and the concentration of every<br />
agent <strong>in</strong> the mixture represents a fraction of the MIC (Table 4).<br />
2.3. Inoculation and Incubation<br />
Triplicate Petri dishes of each system were centrally <strong>in</strong>oculated by<br />
spott<strong>in</strong>g 2 µl of the spore suspension (≈ 2.0 × 10 3 spores/plate) to give<br />
Table 1. Mixtures of phenolic and synthetic antimicrobial agents evaluated to<br />
<strong>in</strong>hibit Aspergillus flavus growth<br />
B<strong>in</strong>ary Mixtures Ternary Mixtures<br />
Vanill<strong>in</strong> -Eugenol Thymol -Carvacrol -Potassium sorbate<br />
Vanill<strong>in</strong> -Carvacrol Thymol -Eugenol -Potassium sorbate<br />
Vanill<strong>in</strong> -Thymol Thymol -Vanill<strong>in</strong> -Potassium sorbate<br />
Vanill<strong>in</strong> -Potassium sorbate Carvacrol -Vanill<strong>in</strong> -Potassium sorbate<br />
Eugenol -Carvacrol Carvacrol -Eugenol -Potassium sorbate<br />
Eugenol -Thymol Eugenol -Vanill<strong>in</strong> -Potassium sorbate<br />
Eugenol -Potassium sorbate<br />
Thymol -Potassium sorbate
270 Aurelio López-Malo et al.<br />
Table 2. Aspergillus flavus growth (G) or no growth (NG) response after one month<br />
of <strong>in</strong>cubation <strong>in</strong> potato dextrose agar formulated with pH 3.5, a w 0.99 and selected<br />
b<strong>in</strong>ary mixtures of vanill<strong>in</strong> and eugenol<br />
Eugenol (ppm)<br />
Vanill<strong>in</strong> (ppm) 100 200 300 400 500 600<br />
100 G G G G NG NG<br />
200 G G G NG NG NG<br />
300 G G G NG NG NG<br />
400 G G G NG NG NG<br />
500 G G NG NG NG NG<br />
600 G G NG NG NG NG<br />
700 G G NG NG NG NG<br />
800 G NG NG NG NG NG<br />
900 NG NG NG NG NG NG<br />
1000 NG NG NG NG NG NG<br />
1100 NG NG NG NG NG NG<br />
1200 NG NG NG NG NG NG<br />
Table 3. Aspergillus flavus growth (G) or no growth (NG) response after one month<br />
of <strong>in</strong>cubation <strong>in</strong> potato dextrose agar formulated with pH 3.5, a w 0.99 and selected<br />
b<strong>in</strong>ary mixtures of carvacrol and thymol.<br />
Thymol (ppm)<br />
Carvacrol (ppm) 50 100 150 200 250 300 350<br />
50 G G G G G G NG<br />
100 G G G G NG NG NG<br />
150 G G G NG NG NG NG<br />
200 G G NG NG NG NG NG<br />
250 G NG NG NG NG NG NG<br />
Table 4. Experimental design utilized to evaluate ternary mixtures<br />
of antimicrobial agents a<br />
Experiment Agent A Agent B Agent C<br />
1 1 0 0<br />
2 0 1 0<br />
3 0 0 1<br />
4 0 1/2 1/2<br />
5 1/2 0 1/2<br />
6 1/2 1/2 0<br />
7 1/3 1/3 1/3<br />
8 1/6 1/6 1/6<br />
9 1/6 1/6 2/3<br />
10 2/3 1/6 1/6<br />
11 1/6 2/3 1/6<br />
12 1/12 1/12 1/3<br />
13 1/3 1/12 1/12<br />
14 1/12 1/3 1/12<br />
a From Berenbaum et al., 1983
Mixtures of Natural and Synthetic Antifungal Agents 271<br />
a circular <strong>in</strong>oculum of 1 mm diameter. Growth controls without<br />
antimicrobials were prepared and <strong>in</strong>oculated as above. Three plates of<br />
every system were ma<strong>in</strong>ta<strong>in</strong>ed without <strong>in</strong>oculation for a w and pH<br />
measurements. The <strong>in</strong>oculated plates and controls were <strong>in</strong>cubated for<br />
1 month at 25°C <strong>in</strong> hermetically closed plastic conta<strong>in</strong>ers to avoid<br />
dehydration. Enough headspace was left <strong>in</strong> the conta<strong>in</strong>ers to<br />
avoid anoxic conditions. Periodically, the <strong>in</strong>oculated plates were<br />
removed briefly to observe them and immediately re-<strong>in</strong>cubated.<br />
Water activity was measured with an AquaLab CX-2 <strong>in</strong>strument<br />
(Decagon Devices, Inc., Pullman, WA) calibrated and operated follow<strong>in</strong>g<br />
the procedure described by López-Malo et al. (1994). pH was determ<strong>in</strong>ed<br />
with a Beckman pH meter model 50 (Beckman Instruments,<br />
Inc., Fullerton, CA). Measurements were made <strong>in</strong> triplicate.<br />
2.4. Calculation of the Mould Growth Responses<br />
The <strong>in</strong>oculated systems were exam<strong>in</strong>ed daily us<strong>in</strong>g a stereoscopic<br />
microscope (American Optical, model Forty), if no growth was<br />
observed the plates were re-<strong>in</strong>cubated and if mould growth<br />
was detected plates were discarded. Inhibition was def<strong>in</strong>ed as no<br />
observable mould growth after one month of <strong>in</strong>cubation.<br />
M<strong>in</strong>imal <strong>in</strong>hibitory concentrations (MIC) for <strong>in</strong>dividual antimicrobials<br />
were def<strong>in</strong>ed as the m<strong>in</strong>imal concentration of the compounds<br />
used required to <strong>in</strong>hibit mould growth. MIC data was transformed to<br />
fractional <strong>in</strong>hibitory concentration (FIC), as def<strong>in</strong>ed by Davidson and<br />
Parish (1989) for b<strong>in</strong>ary mixtures:<br />
FIC A = (MIC compound A with compound B ) / (MIC compound A ) (1)<br />
FIC B = (MIC compound B with compound A ) / (MIC compound B ) (2)<br />
that can be extended to ternary mixtures (López-Malo et al.,<br />
2005b):<br />
FIC A = (MIC compound A with compounds B and C ) / (MIC compound A ) (3)<br />
FIC B = (MIC compound B with compounds A and C ) / (MIC compound B ) (4)<br />
FIC C = (MIC compound C with compounds A and B ) / (MIC compound C ) (5)<br />
FIC Index was calculated with the FICs for <strong>in</strong>dividual antimicrobials<br />
as follows:<br />
FIC Index = FIC A + FIC B + FIC C<br />
(6)
272 Aurelio López-Malo et al.<br />
3. RESULTS AND DISCUSSION<br />
The pH and a w of the PDA systems without <strong>in</strong>oculation determ<strong>in</strong>ed<br />
at the beg<strong>in</strong>n<strong>in</strong>g and at the end of <strong>in</strong>cubation demonstrated<br />
that the desired values rema<strong>in</strong>ed constant under the storage conditions.<br />
In control systems without antimicrobials, A. flavus grew at a w<br />
0.99 and pH 3.5.<br />
Individual effects of the synthetic antimicrobial (potassium sorbate)<br />
and naturally occurr<strong>in</strong>g antimicrobials (thymol, carvacrol, vanill<strong>in</strong>,<br />
eugenol) on A. flavus growth response <strong>in</strong> PDA at a w 0.99 and pH 3.5,<br />
represented as the m<strong>in</strong>imal concentrations (MIC) that <strong>in</strong>hibit the<br />
mould growth for <strong>in</strong>dividual antimicrobials, are presented <strong>in</strong> Table 5.<br />
A. flavus exhibited higher sensitivity to thymol, eugenol, carvacrol and<br />
potassium sorbate than to vanill<strong>in</strong>. MICs varied from 300 ppm for<br />
carvacrol to 1300 ppm for vanill<strong>in</strong>.<br />
3.1. B<strong>in</strong>ary Mixtures<br />
The comb<strong>in</strong>ed effects of vanill<strong>in</strong> and eugenol and carvacrol and<br />
thymol resulted <strong>in</strong> the <strong>in</strong>hibitory conditions presented <strong>in</strong> Tables 2 and<br />
3, respectively. In the same manner, we obta<strong>in</strong>ed growth/no growth<br />
responses for every b<strong>in</strong>ary mixture evaluated (data not shown). In<br />
many cases, comb<strong>in</strong><strong>in</strong>g antimicrobial agents resulted <strong>in</strong> no growth<br />
observations with lower <strong>in</strong>hibitory antimicrobial concentrations than<br />
when <strong>in</strong>dividually evaluated (Tables 2 and 3). These no growth results<br />
were transformed <strong>in</strong> fractional <strong>in</strong>hibitory concentrations to construct<br />
isobolograms and calculate each FIC Index .<br />
An isobologram may be thought of as an array of differ<strong>in</strong>g<br />
concentrations of two compounds, where one compound ranges from<br />
lowest to highest concentration on the x-axis and the other on the<br />
y-axis. All possible permutations of comb<strong>in</strong>ed concentrations are<br />
reflected with<strong>in</strong> the array. If those concentrations that <strong>in</strong>hibit the<br />
Table 5. M<strong>in</strong>imal <strong>in</strong>hibitory concentrations a (MIC) of selected antimicrobials for<br />
Aspergillus flavus <strong>in</strong> potato dextrose agar formulated with a w 0.99 and pH 3.5<br />
Antimicrobial MIC (ppm)<br />
Vanill<strong>in</strong> 1300<br />
Eugenol 600<br />
Carvacrol 300<br />
Thymol 400<br />
Potassium sorbate 400<br />
a M<strong>in</strong>imal concentration required for <strong>in</strong>hibit<strong>in</strong>g mould growth for two months at 25°C
Mixtures of Natural and Synthetic Antifungal Agents 273<br />
growth of the test organism fall on an approximately straight l<strong>in</strong>e that<br />
connects the <strong>in</strong>dividual MIC, or the FIC, on the x and y axes, the<br />
comb<strong>in</strong>ed effect is additive. Deviation of l<strong>in</strong>earity to the left or right<br />
of the additive l<strong>in</strong>e is <strong>in</strong>terpreted as synergism or antagonism, respectively.<br />
As examples, FIC isobolograms are presented for comb<strong>in</strong>ations<br />
of vanill<strong>in</strong> and potassium sorbate (Figure 1), potassium sorbate<br />
and eugenol (Figure 2), and eugenol and carvacrol (Figure 3) that<br />
<strong>in</strong>hibited A. flavus. Depend<strong>in</strong>g on the antimicrobials used <strong>in</strong> the<br />
b<strong>in</strong>ary mixture, different isobolograms were obta<strong>in</strong>ed (Figures 1 to<br />
3), <strong>in</strong>dicat<strong>in</strong>g differences or similarities <strong>in</strong> the ability of the<br />
compounds to <strong>in</strong>hibit mould growth. Po<strong>in</strong>ts represent<strong>in</strong>g no growth<br />
comb<strong>in</strong>ations do not always follow a well-def<strong>in</strong>ed pattern and <strong>in</strong><br />
some cases the shape of the curve is concentration dependent. In<br />
other words, mould <strong>in</strong>hibition can be obta<strong>in</strong>ed with different comb<strong>in</strong>ations<br />
of two antimicrobials but the overall result (synergic, additive<br />
or antagonist) depends on the concentration of each antimicrobial <strong>in</strong><br />
the mixture. This is also observed <strong>in</strong> Tables 6-10. Calculated FIC Index<br />
for each b<strong>in</strong>ary mixture are also presented <strong>in</strong> Tables 6-10. In a similar<br />
way as isobolograms, a FIC Index near 1 implicates additivity; FIC Index<br />
< 1 implies synergy; and FIC Index > 1 implies antagonism (López-<br />
Malo et al., 2005b).<br />
FIC Vanill<strong>in</strong><br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
0 0.2 0.4 0.6 0.8 1<br />
FIC Potassium sorbate<br />
Figure 1. Fractional <strong>in</strong>hibitory concentration (FIC) isobologram for potassium<br />
sorbate and vanill<strong>in</strong> comb<strong>in</strong>ations to <strong>in</strong>hibit Aspergillus flavus <strong>in</strong> potato dextrose agar<br />
(a w 0.99 and pH 3.5) after 30 days <strong>in</strong>cubation at 25°C.
274 Aurelio López-Malo et al.<br />
FIC Potassium sorbate<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
0 0.2 0.4 0.6 0.8 1<br />
FIC Eugenol<br />
Figure 2. Fractional <strong>in</strong>hibitory concentration (FIC) isobologram for eugenol and<br />
potassium sorbate comb<strong>in</strong>ations to <strong>in</strong>hibit Aspergillus flavus <strong>in</strong> potato dextrose agar<br />
(a w 0.99 and pH 3.5) after 30 days <strong>in</strong>cubation at 25°C.<br />
Several comb<strong>in</strong>ations of vanill<strong>in</strong> and potassium sorbate were synergistic<br />
(Figure 1, Table 6) as were some other b<strong>in</strong>ary mixtures (Tables 7-10).<br />
Others, such as comb<strong>in</strong>ations of thymol and carvacrol, demonstrated<br />
only additive effects (Table 10). In summary, the follow<strong>in</strong>g antimicrobial<br />
FIC Eugenol<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
0 0.2 0.4 0.6 0.8 1<br />
FIC Carvacrol<br />
Figure 3. Fractional <strong>in</strong>hibitory concentration (FIC) isobologram for carvacrol and<br />
eugenol comb<strong>in</strong>ations to <strong>in</strong>hibit Aspergillus flavus <strong>in</strong> potato dextrose agar (a w 0.99<br />
and pH 3.5) after 30 days <strong>in</strong>cubation at 25°C.
Mixtures of Natural and Synthetic Antifungal Agents 275<br />
Table 6. Fractional <strong>in</strong>hibitory concentration <strong>in</strong>dex (FIC Index ) for Aspergillus flavus<br />
us<strong>in</strong>g b<strong>in</strong>ary mixtures of antimicrobials <strong>in</strong> potato dextrose agar formulated at a w<br />
0.99, pH 3.5.<br />
Potassium Vanill<strong>in</strong> Eugenol Vanill<strong>in</strong><br />
sorbate (ppm) (ppm) FIC Index (ppm) (ppm) FIC Index<br />
50 900 0.817 100 900 0.859<br />
100 700 0.788 200 800 0.949<br />
150 500 0.760 300 500 0.885<br />
200 400 0.808 400 200 0.821<br />
250 200 0.779 500 100 0.910<br />
300 100 0.827<br />
350 100 0.952<br />
Table 7. Fractional <strong>in</strong>hibitory concentration <strong>in</strong>dex (FIC Index ) for Aspergillus flavus<br />
us<strong>in</strong>g b<strong>in</strong>ary mixtures of antimicrobials <strong>in</strong> potato dextrose agar formulated at a w<br />
0.99, pH 3.5.<br />
Carvacrol Vanill<strong>in</strong> Thymol Vanill<strong>in</strong><br />
(ppm) (ppm) FIC Index (ppm) (ppm) FIC Index<br />
50 800 0.782 50 900 0.817<br />
100 700 0.872 100 800 0.865<br />
150 500 0.885 150 600 0.837<br />
200 500 1.051 200 500 0.885<br />
250 200 0.987 250 200 0.779<br />
300 100 0.827<br />
comb<strong>in</strong>ations were synergistic, <strong>in</strong> at least one comb<strong>in</strong>ation, <strong>in</strong> <strong>in</strong>hibit<strong>in</strong>g<br />
A. flavus for at least 30 days of <strong>in</strong>cubation at a w 0.99 and pH 3.5: vanill<strong>in</strong>potassium<br />
sorbate (FIC Index 0.76), vanill<strong>in</strong>-eugenol (FIC Index 0.82),<br />
vanill<strong>in</strong>-thymol (FIC Index 0.82), vanill<strong>in</strong>-carvacrol (FIC Index 0.78);<br />
eugenol-carvacrol (FIC Index 0.83), thymol-potassium sorbate<br />
(FIC Index 0.75), eugenol-potassium sorbate (FIC Index 0.67), and eugenolthymol<br />
(FIC Index 0.83). Antimicrobial comb<strong>in</strong>ations <strong>in</strong>clud<strong>in</strong>g potassium<br />
Table 8. Fractional <strong>in</strong>hibitory concentration <strong>in</strong>dex (FIC Index ) for Aspergillus flavus<br />
us<strong>in</strong>g b<strong>in</strong>ary mixtures of antimicrobials <strong>in</strong> potato dextrose agar formulated at a w<br />
0.99, pH 3.5.<br />
Potassium Potassium<br />
Eugenol sorbate Carvacrol sorbate<br />
(ppm) (ppm) FIC Index (ppm) (ppm) FIC Index<br />
100 200 0.667 50 250 0.792<br />
200 200 0.833 100 200 0.833<br />
300 150 0.875 150 200 1.000<br />
400 100 0.917 200 150 1.042<br />
500 50 0.958 250 100 1.083
276 Aurelio López-Malo et al.<br />
Table 9. Fractional <strong>in</strong>hibitory concentration <strong>in</strong>dex (FIC Index ) for Aspergillus flavus<br />
us<strong>in</strong>g b<strong>in</strong>ary mixtures of antimicrobials <strong>in</strong> potato dextrose agar formulated at a w<br />
0.99, pH 3.5.<br />
Potassium<br />
Thymol sorbate Carvacrol Eugenol<br />
(ppm) (ppm) FIC Index (ppm) (ppm) FIC Index<br />
50 250 0.750 50 400 0.833<br />
100 200 0.750 100 300 0.833<br />
150 200 0.875 150 200 0.833<br />
200 200 1.000 200 200 1.000<br />
250 150 1.000 250 100 1.000<br />
300 100 1.000<br />
350 50 1.000<br />
sorbate and carvacrol or thymol were synergistic when small concentrations<br />
of phenolics were comb<strong>in</strong>ed with > 150 ppm potassium sorbate.<br />
Fungal <strong>in</strong>hibition can be achieved by comb<strong>in</strong><strong>in</strong>g spices (or their<br />
phenolic compounds) and traditional antimicrobials, reduc<strong>in</strong>g the<br />
concentrations needed to achieve the same effect than when us<strong>in</strong>g<br />
only one antimicrobial agent. In previous studies, Azzous and<br />
Bullerman (1982) reported that clove was an efficient antimycotic<br />
agent aga<strong>in</strong>st A. flavus, A. parasiticus and A. ochraceus and four<br />
stra<strong>in</strong>s of Penicillium, delay<strong>in</strong>g mould growth by more than 21 days.<br />
These authors also observed additive and synergic effects comb<strong>in</strong><strong>in</strong>g<br />
0.1% clove with 0.1-0.3% potassium sorbate, delay<strong>in</strong>g mould germ<strong>in</strong>ation<br />
time. Sebti and Tantaoui-Elaraki (1994) reported that the<br />
comb<strong>in</strong>ation of sorbic acid (0.75 g/kg) with an aqueous c<strong>in</strong>namon<br />
extract (20 g/kg) <strong>in</strong>hibited growth of 151 mould and yeast stra<strong>in</strong>s<br />
isolated from a Moroccan bakery product. In contrast, when us<strong>in</strong>g<br />
Table 10. Fractional <strong>in</strong>hibitory concentration <strong>in</strong>dex (FIC Index ) for Aspergillus flavus<br />
us<strong>in</strong>g b<strong>in</strong>ary mixtures of antimicrobials <strong>in</strong> potato dextrose agar formulated at a w<br />
0.99, pH 3.5.<br />
Thymol Eugenol Thymol Carvacrol<br />
(ppm) (ppm) FIC Index (ppm) (ppm) FIC Index<br />
50 500 0.958 100 250 1.083<br />
100 400 0.917 150 200 1.042<br />
150 300 0.875 200 150 1.000<br />
200 200 0.833 250 100 0.958<br />
250 100 0.792 300 100 1.083<br />
300 100 0.917 350 50 1.042<br />
350 100 1.042
Mixtures of Natural and Synthetic Antifungal Agents 277<br />
only one antimicrobial agent to <strong>in</strong>hibit the studied microorganisms,<br />
2000 ppm of sorbic acid was needed. Matamoros-León et al. (1999)<br />
evaluated <strong>in</strong>dividual and comb<strong>in</strong>ed effects of potassium sorbate and<br />
vanill<strong>in</strong> concentrations on the growth of Penicillium digitatum, P.<br />
glabrum and P. italicum <strong>in</strong> PDA adjusted to a w 0.98 and pH 3.5, and<br />
observed that 150 ppm potassium sorbate <strong>in</strong>hibited P. digitatum while<br />
700 ppm were needed to <strong>in</strong>hibit P. glabrum. Us<strong>in</strong>g vanill<strong>in</strong>, <strong>in</strong>hibitory<br />
concentrations varied from 1100 ppm for P. digitatum and P. italicum to<br />
1300 ppm for P. glabrum. When used <strong>in</strong> comb<strong>in</strong>ation, m<strong>in</strong>imal<br />
<strong>in</strong>hibitory concentration (MIC) isobolograms illustrated that curves<br />
deviated to the left of the additive l<strong>in</strong>e. Also, calculated FIC Index values<br />
varied from 0.60 to 0.84. FIC Index as well as isobolograms demonstrated<br />
synergistic effects on mould <strong>in</strong>hibition when vanill<strong>in</strong> and<br />
potassium sorbate were applied <strong>in</strong> comb<strong>in</strong>ation (Matamoros-León<br />
et al., 1999).<br />
3.2. Ternary Mixtures<br />
Several of the tested comb<strong>in</strong>ations of two antimicrobials exhibited<br />
synergy <strong>in</strong> an experimental system. The question arises as to whether<br />
comb<strong>in</strong>ations of more than two agents might show even greater<br />
synergy. However it is not easy to answer this question (Berenbaum<br />
et al., 1983). An alternative approach to improv<strong>in</strong>g fungal <strong>in</strong>hibition<br />
could be to comb<strong>in</strong>e three antimicrobials agents, especially for resistant<br />
stra<strong>in</strong>s which cannot be <strong>in</strong>hibited with <strong>in</strong>dividual or b<strong>in</strong>ary mixtures<br />
of antimicrobials. As already mentioned, little is known about<br />
the <strong>in</strong>teraction between antifungal agents aga<strong>in</strong>st filamentous fungi.<br />
In order to determ<strong>in</strong>e the potential use of ternary comb<strong>in</strong>ations of<br />
antifungal agents to <strong>in</strong>hibit growth of A. flavus we decided to study<br />
the <strong>in</strong>teractions among selected antimicrobials (Table 4).<br />
Tables 11-16 present the growth/no growth results for the evaluated<br />
ternary mixtures, as well as FIC of each antimicrobial <strong>in</strong> the mixture<br />
and FIC Index . The experimental design used, proposed by Berenbaum<br />
et al. (1983), has been used for antibiotics and is focused on determ<strong>in</strong><strong>in</strong>g<br />
synergistic mixtures and establish<strong>in</strong>g if they are consistently synergistic,<br />
i.e. results are not dependent on concentration. Several<br />
experiments (mixtures) proposed <strong>in</strong> the design (Table 4) and evaluated<br />
(Tables 11-16) have by def<strong>in</strong>ition an FIC Index equal to 1, and are<br />
<strong>in</strong>cluded to corroborate <strong>in</strong>dividual and b<strong>in</strong>ary <strong>in</strong>hibitory effects, as well<br />
as ternary comb<strong>in</strong>ations <strong>in</strong> which the proportions of antimicrobials<br />
(fractions of MIC) add up to 1. Two thirds of the MIC of one<br />
agent and 1/6 of the MIC of the other two, or 1/3 of the MIC of each
278 Aurelio López-Malo et al.<br />
Table 11. Fractional <strong>in</strong>hibitory concentration (FIC) and FIC Index for Aspergillus<br />
flavus us<strong>in</strong>g ternary mixtures of potassium sorbate (KS), carvacrol and thymol <strong>in</strong><br />
potato dextrose agar formulated at a w 0.99, pH 3.5.<br />
KS Carvacrol Thymol Growth FIC FIC FIC<br />
(ppm) (ppm) (ppm) Response KS Carvacrol Thymol FIC Index<br />
400 0 0 NG 1.00 0.00 0.00 1.00<br />
0 300 0 NG 0.00 1.00 0.00 1.00<br />
0 0 400 NG 0.00 0.00 1.00 1.00<br />
0 150 200 NG 0.00 0.50 0.50 1.00<br />
200 0 200 NG 0.50 0.00 0.50 1.00<br />
200 150 0 NG 0.50 0.50 0.00 1.00<br />
132 99 132 NG 0.33 0.33 0.33 1.00<br />
68 51 68 NG 0.17 0.17 0.17 0.50<br />
68 51 268 NG 0.17 0.17 0.67 1.00<br />
268 51 68 NG 0.67 0.17 0.17 1.00<br />
68 201 68 NG 0.17 0.67 0.17 1.00<br />
32 24 132 NG 0.08 0.08 0.33 0.50<br />
132 24 32 G<br />
32 99 32 NG 0.08 0.33 0.08 0.50<br />
agent are examples of these ternary mixtures. The rest of the experiments<br />
are used to test synergy by comb<strong>in</strong><strong>in</strong>g 1/6 MIC of each agent or<br />
1/3 MIC of one antimicrobial with 1/12 MIC of the other two.<br />
Berenbaum et al. (1983) <strong>in</strong>dicated that if no growth is obta<strong>in</strong>ed <strong>in</strong> every<br />
comb<strong>in</strong>ation tested, the ternary mixture is synergic <strong>in</strong> a consistent way.<br />
Table 12. Fractional <strong>in</strong>hibitory concentration (FIC) and FIC Index for Aspergillus<br />
flavus us<strong>in</strong>g ternary mixtures of potassium sorbate (KS), eugenol and thymol <strong>in</strong><br />
potato dextrose agar formulated at a w 0.99, pH 3.5.<br />
KS Eugenol Thymol Growth FIC FIC FIC<br />
(ppm) (ppm) (ppm) Response KS Eugenol Thymol FIC Index<br />
400 0 0 NG 1.00 0.00 0.00 1.00<br />
0 600 0 NG 0.00 1.00 0.00 1.00<br />
0 0 400 NG 0.00 0.00 1.00 1.00<br />
0 300 200 NG 0.00 0.50 0.50 1.00<br />
200 0 200 NG 0.50 0.00 0.50 1.00<br />
200 300 0 NG 0.50 0.50 0.00 1.00<br />
132 198 132 NG 0.33 0.33 0.33 1.00<br />
68 102 68 NG 0.17 0.17 0.17 0.50<br />
68 102 268 NG 0.17 0.17 0.67 1.00<br />
268 102 68 NG 0.67 0.17 0.17 1.00<br />
68 402 68 NG 0.17 0.67 0.17 1.00<br />
32 48 132 NG 0.08 0.08 0.33 0.50<br />
132 48 32 G<br />
32 198 32 NG 0.08 0.33 0.08 0.50
Mixtures of Natural and Synthetic Antifungal Agents 279<br />
Table 13. Fractional <strong>in</strong>hibitory concentration (FIC) and FIC Index for Aspergillus<br />
flavus us<strong>in</strong>g ternary mixtures of potassium sorbate (KS), vanill<strong>in</strong> and thymol <strong>in</strong><br />
potato dextrose agar formulated at aw 0.99, pH 3.5.<br />
KS Vanill<strong>in</strong> Thymol Growth FIC FIC FIC<br />
(ppm) (ppm) (ppm) Response KS Vanill<strong>in</strong> Thymol FIC Index<br />
400 0 0 NG 1.00 0.00 0.00 1.00<br />
0 1300 0 NG 0.00 1.00 0.00 1.00<br />
0 0 400 NG 0.00 0.00 1.00 1.00<br />
0 650 200 NG 0.00 0.50 0.50 1.00<br />
200 0 200 NG 0.50 0.00 0.50 1.00<br />
200 650 0 NG 0.50 0.50 0.00 1.00<br />
132 429 132 NG 0.33 0.33 0.33 1.00<br />
68 221 68 G<br />
68 221 268 G<br />
268 221 68 NG 0.67 0.17 0.17 1.00<br />
68 871 68 NG 0.17 0.67 0.17 1.00<br />
32 104 132 G<br />
132 104 32 G<br />
32 429 32 G<br />
Therefore, above the lowest concentration of every antimicrobial tested<br />
<strong>in</strong> the mixture, the comb<strong>in</strong>ation will be synergistic.<br />
In ternary mixtures <strong>in</strong>clud<strong>in</strong>g potassium sorbate-thymol-carvacrol<br />
(Table 11) and potassium sorbate-thymol-eugenol (Table 12), mould<br />
growth was observed only when 1/3 MIC of potassium sorbate was<br />
Table 14. Fractional <strong>in</strong>hibitory concentration (FIC) and FIC Index for Aspergillus<br />
flavus us<strong>in</strong>g ternary mixtures of potassium sorbate (KS), eugenol and carvacrol <strong>in</strong><br />
potato dextrose agar formulated at a w 0.99, pH 3.5.<br />
KS Eugenol Carvacrol Growth FIC FIC FIC<br />
(ppm) (ppm) (ppm) Response KS Eugenol Carvacrol FIC Index<br />
400 0 0 NG 1.00 0.00 0.00 1.00<br />
0 600 0 NG 0.00 1.00 0.00 1.00<br />
0 0 300 NG 0.00 0.00 1.00 1.00<br />
0 300 150 NG 0.00 0.50 0.50 1.00<br />
200 0 150 NG 0.50 0.00 0.50 1.00<br />
200 300 0 NG 0.50 0.50 0.00 1.00<br />
132 198 99 NG 0.33 0.33 0.33 1.00<br />
68 102 51 NG 0.17 0.17 0.17 0.50<br />
68 102 201 NG 0.17 0.17 0.67 1.00<br />
268 102 51 NG 0.67 0.17 0.17 1.00<br />
68 402 51 NG 0.17 0.67 0.17 1.00<br />
32 48 99 G<br />
132 48 24 G<br />
32 198 24 NG 0.08 0.33 0.08 0.50
280 Aurelio López-Malo et al.<br />
Table 15. Fractional <strong>in</strong>hibitory concentration (FIC) and FIC Index for Aspergillus<br />
flavus us<strong>in</strong>g ternary mixtures of potassium sorbate (KS), vanill<strong>in</strong> and carvacrol <strong>in</strong><br />
potato dextrose agar formulated at a w 0.99, pH 3.5.<br />
KS Vanill<strong>in</strong> Carvacrol Growth FIC FIC FIC<br />
(ppm) (ppm) (ppm) Response KS Vanill<strong>in</strong> Carvacrol FIC Index<br />
400 0 0 NG 1.00 0.00 0.00 1.00<br />
0 1300 0 NG 0.00 1.00 0.00 1.00<br />
0 0 300 NG 0.00 0.00 1.00 1.00<br />
0 650 150 NG 0.00 0.50 0.50 1.00<br />
200 0 150 NG 0.50 0.00 0.50 1.00<br />
200 650 0 NG 0.50 0.50 0.00 1.00<br />
132 429 99 G<br />
68 221 51 G<br />
68 221 201 NG 0.17 0.17 0.67 1.00<br />
268 221 51 G<br />
68 871 51 NG 0.17 0.67 0.17 1.00<br />
32 104 99 G<br />
132 104 24 G<br />
32 429 24 G<br />
comb<strong>in</strong>ed with 1/12 MIC of thymol and 1/12 MIC of carvacrol or<br />
eugenol. In both ternary mixtures when growth was observed the<br />
phenolic compounds represent the lowest MIC fraction tested (1/12).<br />
Comb<strong>in</strong>ations that result <strong>in</strong> synergism (FIC = 0.5) <strong>in</strong>clude at least one<br />
phenolic <strong>in</strong> a fraction higher than 1/12 MIC, Therefore, we can<br />
Table 16. Fractional <strong>in</strong>hibitory concentration (FIC) and FIC Index for Aspergillus<br />
flavus us<strong>in</strong>g ternary mixtures of potassium sorbate (KS), vanill<strong>in</strong> and eugenol <strong>in</strong><br />
potato dextrose agar formulated at a w 0.99, pH 3.5.<br />
KS Vanill<strong>in</strong> Eugenol Growth FIC FIC FIC<br />
(ppm) (ppm) (ppm) Response KS Vanill<strong>in</strong> Eugenol FIC Index<br />
400 0 0 NG 1.00 0.00 0.00 1.00<br />
0 1300 0 NG 0.00 1.00 0.00 1.00<br />
0 0 600 NG 0.00 0.00 1.00 1.00<br />
0 650 300 NG 0.00 0.50 0.50 1.00<br />
200 0 300 NG 0.50 0.00 0.50 1.00<br />
200 650 0 NG 0.50 0.50 0.00 1.00<br />
132 429 198 NG 0.33 0.33 0.33 1.00<br />
68 221 102 G<br />
68 221 402 G<br />
268 221 102 G<br />
68 871 102 NG 0.17 0.67 0.17 1.00<br />
32 104 198 G<br />
132 104 48 G<br />
32 429 48 G
Mixtures of Natural and Synthetic Antifungal Agents 281<br />
conclude that synergistic antifungal comb<strong>in</strong>ations of these agents must<br />
<strong>in</strong>clude: 1/12 MIC < phenolic concentrations < 1/6 MIC. Compar<strong>in</strong>g<br />
b<strong>in</strong>ary and ternary mixture results, it can be observed that a considerable<br />
reduction of potassium sorbate concentration from 150-250 ppm<br />
to 32-68 ppm is possible. Ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g a potassium sorbate concentration<br />
near 100 ppm, allows a considerable reduction <strong>in</strong> phenolic antimicrobial<br />
concentrations <strong>in</strong> the <strong>in</strong>hibitory ternary mixtures. Figure 4<br />
presents a representation of FIC of potassium sorbate-thymol-eugenol<br />
<strong>in</strong>hibitory comb<strong>in</strong>ations that <strong>in</strong>hibit A. flavus, this 3-D isobologram as<br />
well as FIC Index illustrate the synergistic comb<strong>in</strong>ations.<br />
Ternary mixtures where vanill<strong>in</strong> is <strong>in</strong>cluded (Tables 13, 15 and 16)<br />
have <strong>in</strong> common that growth was observed <strong>in</strong> those comb<strong>in</strong>ations<br />
where synergism was expected. However, some ternary comb<strong>in</strong>ations<br />
presented an additive result (FIC Index = 1). Observ<strong>in</strong>g b<strong>in</strong>ary results of<br />
those mixtures that <strong>in</strong>clude vanill<strong>in</strong>, synergism was anticipated <strong>in</strong><br />
ternary mixtures but the results cannot be predicted, as can be seen <strong>in</strong><br />
Tables 13, 15 and 16. Monzón et al. (2001) reported that <strong>in</strong>dividual or<br />
b<strong>in</strong>ary antimicrobial agents that exhibit synergistic results do not<br />
necessary generate similar outcomes <strong>in</strong> ternary antimicrobial mixtures.<br />
1.00<br />
0.87<br />
FIC Eugenol<br />
0.73<br />
0.60<br />
0.47<br />
0.33<br />
0.20<br />
0.07<br />
1<br />
0.87<br />
0.73<br />
0.60<br />
0.47<br />
0.33<br />
0.20<br />
0.07<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
FIC Thymol<br />
FIC KS<br />
Figure 4. Fractional <strong>in</strong>hibitory concentration (FIC) isobologram for potassium<br />
sorbate (KS), eugenol and thymol comb<strong>in</strong>ations to <strong>in</strong>hibit Aspergillus flavus <strong>in</strong> potato<br />
dextrose agar (a w 0.99 and pH 3.5) after 30 days <strong>in</strong>cubation at 25°C.
282 Aurelio López-Malo et al.<br />
4. CONCLUSIONS<br />
Comb<strong>in</strong>ations of antimicrobials can be selected when identified<br />
microorganisms are resistant to <strong>in</strong>hibition and/or <strong>in</strong>activation by legal<br />
levels of s<strong>in</strong>gle, conventional antimicrobials. The comb<strong>in</strong>ation may exert<br />
the desired antimicrobial activity. Also, and more frequently, antimicrobial<br />
comb<strong>in</strong>ations can be selected to provide broad-spectrum preservation<br />
(Alzamora et al., 2003). Relatively little work has been reported to<br />
date on the comb<strong>in</strong>ed action of mixtures of conventional and natural<br />
antimicrobials aga<strong>in</strong>st microorganisms, even <strong>in</strong> model systems.<br />
Moreover, little attention has been given to the study of the mechanisms<br />
underly<strong>in</strong>g their toxicity and modes of resistance, particularly for<br />
microorganisms of concern <strong>in</strong> fruit products, among them moulds and<br />
yeasts. This lack of understand<strong>in</strong>g of the relative contribution of factors<br />
to obta<strong>in</strong> safe, high quality foods is surpris<strong>in</strong>g, because the necessity for<br />
an adequate database on which to develop safe multifactorial preservation<br />
systems was po<strong>in</strong>ted out some time ago (Roberts, 1989).<br />
Further work to comb<strong>in</strong>e natural antimicrobials with conventional<br />
ones <strong>in</strong> foods is still required. Several comb<strong>in</strong>ations could be exploited<br />
for mild food preservation techniques <strong>in</strong> the near future. However,<br />
their mode of action <strong>in</strong> model systems and <strong>in</strong> food matrices is still not<br />
well understood and represents a barrier to their application. Results<br />
of the study reported here, and various others <strong>in</strong> the literature, raise<br />
certa<strong>in</strong> questions about the use of antimicrobial mixtures (Alzamora<br />
and López-Malo, 2002; Alzamora et al., 2003). Conversely, a better<br />
understand<strong>in</strong>g of microbial ecology and the physiological response<br />
of microorganisms to <strong>in</strong>dividual preservation factors as well as to<br />
comb<strong>in</strong>ations of natural and conventional antimicrobials <strong>in</strong> different<br />
food environments will offer new opportunities and provide greater<br />
precision for a rational selection of antimicrobial comb<strong>in</strong>ations. The<br />
answer to these po<strong>in</strong>ts can be established by appropriate scientific and<br />
experimental <strong>in</strong>quiry and it is part of the challenge for the future of<br />
antifungal comb<strong>in</strong>ations.<br />
5. ACKNOWLEDGMENTS<br />
We acknowledge f<strong>in</strong>ancial support from CONACyT -Mexico<br />
(Projects 32020-B, 33405-B and 44088), Universidad de las Américas-<br />
Puebla, and CYTED Program (Project XI.15).
Mixtures of Natural and Synthetic Antifungal Agents 283<br />
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activity assay and evaluation of results, <strong>in</strong>: Antimicrobials <strong>in</strong> <strong>Food</strong>, 3rd edition, P. M.<br />
Davidson, J. N. Sofos, and A. L. Branen, eds, CRC Press, New York, pp 659-680.<br />
Matamoros-León, B., Argaiz, A., and López-Malo, A., 1999, Individual and<br />
comb<strong>in</strong>ed effects of vanill<strong>in</strong> and potassium sorbate on Penicillium digitatum,<br />
P. glabrum and P. italicum growth, J. <strong>Food</strong> Prot. 62:540-542.<br />
Monzón, M., Oteiza, C., Leiva, J., and Amorena, B., 2001, Synergy of different<br />
antibiotic comb<strong>in</strong>ations <strong>in</strong> biofilms of Staphylococcus epidermis, J. Antimicrob.<br />
Chemother. 48:793-801.<br />
NCCLS, 1999, Methods for Determ<strong>in</strong><strong>in</strong>g Bactericidal Activity of Antimicrobial<br />
Agents; Approved Guidel<strong>in</strong>e, National Committee for Cl<strong>in</strong>ical Laboratory<br />
Standards, Wayne, PA.<br />
NCCLS, 2002, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria<br />
That Grow Aerobically; Approved Standard, 6th edition, National Committee for<br />
Cl<strong>in</strong>ical Laboratory Standards, Wayne, PA.<br />
Nychas, G. J. E., 1995, Natural antimicrobials from plants, <strong>in</strong>: New Methods of <strong>Food</strong><br />
Preservation, G. W. Gould, ed., Blackie Academic & Professional, Glasgow, UK,<br />
pp. 58-89.<br />
Parish, M. E., and Carroll, D. E., 1988, Effects of comb<strong>in</strong>ed antimicrobial agents on<br />
fermentation <strong>in</strong>itiation by Saccharomyces cerevisae <strong>in</strong> a model broth system,<br />
J. <strong>Food</strong> Sci. 53:240.<br />
Paster, N., Juven, B. J., Shaaya, E., Menasherov, M., 1990, Inhibitory effect of<br />
oregano and thyme essential oils on moulds and foodborne bacteria, Lett. Appl.<br />
Microbiol. 11:33-37.<br />
Pr<strong>in</strong>dle, R. F., and Wright, E. S., 1977, Phenolic compounds, <strong>in</strong>: Dis<strong>in</strong>fection,<br />
Sterilization and Preservation, 2nd edition, S. S. Block, ed., Lea & Febiger,<br />
Philadelphia, PA.<br />
Rehm, H., 1959, Untersuchung zur Sirkung von Konservierungsmittelkomb<strong>in</strong>ationen,<br />
Z. Lebesm. Untersuch. Forsch. 110:356.
286 Aurelio López-Malo et al.<br />
Roberts, T. A., 1989, Comb<strong>in</strong>ations of antimicrobials and process<strong>in</strong>g methods, <strong>Food</strong><br />
Technol. 42:156-163.<br />
Sebti F., and Tantaoui-Elaraki, A., 1994, In vitro <strong>in</strong>hibition of fungi isolated from<br />
“Pastilla” papers by organic acids and c<strong>in</strong>namon, Lebensm.-Wiss. u-Technol.<br />
27:370-74.<br />
Shelef, L.A., 1983, Antimicrobial effects of spices, J. <strong>Food</strong> Safety 6:29-44.<br />
Smid, E. J., and Gorris, L. G. M., 1999, Natural antimicrobials for food preservation,<br />
<strong>in</strong>: Handbook of <strong>Food</strong> Preservation, M. S. Rahman, ed., Marcel Dekker, New York,<br />
pp. 285-308.<br />
Sofos, J. N., Beuchat, L. R., Davidson, P. M., and Johnson, E. A., 1998, Naturally<br />
Occurr<strong>in</strong>g Antimicrobials <strong>in</strong> <strong>Food</strong>, Council for Agricultural Science and<br />
Technology, Task Force Report No 132.<br />
Squires, E., and Cleeland, R., 1985, Methods of Test<strong>in</strong>g Comb<strong>in</strong>ations of<br />
Antimicrobial Agents, Hoffman-LaRoche, Nutley, NJ.<br />
Wilk<strong>in</strong>s, K. M., and Board, R. G., 1989, Natural antimicrobial systems, <strong>in</strong>:<br />
Mechanisms of Action of <strong>Food</strong> Preservation Procedures, G. W. Gould, ed., Elsevier,<br />
New York, pp. 285-362.<br />
Zaika, L. L., 1988, Spices and herbs: their antimicrobial activity and its determ<strong>in</strong>ation,<br />
J. <strong>Food</strong> Safety 6:29-44.
PROBABILISTIC MODELLING OF<br />
ASPERGILLUS GROWTH<br />
Enrique Palou and Aurelio López-Malo *<br />
1. INTRODUCTION<br />
Filamentous fungi are of concern to the food <strong>in</strong>dustry as potential<br />
spoilage micro-organisms (Pitt, 1989; Samson, 1989) and mycotox<strong>in</strong><br />
producers (Smith and Moss, 1985). It is important to understand the<br />
growth k<strong>in</strong>etics of these fungi <strong>in</strong> the food context, <strong>in</strong> order to control<br />
product quality from formulation to storage. This is especially applicable<br />
to long shelf life products, but also <strong>in</strong> those food products where<br />
formulation <strong>in</strong>gredients could be source of fungal contam<strong>in</strong>ation<br />
which may cause spoilage dur<strong>in</strong>g process<strong>in</strong>g or storage. In low water<br />
activity foods, mould spoilage is controlled by controll<strong>in</strong>g a w , either by<br />
dry<strong>in</strong>g or the addition of solutes (NaCl, sucrose, glucose or fructose).<br />
However mould growth can also occur at a w values, especially when<br />
the preservation factors <strong>in</strong>hibit bacteria and allow fungal growth<br />
(Gould, 1989; Pitt and Miscamble, 1995). Mould growth <strong>in</strong> these<br />
products depends on the pH, a w and antimicrobial agents, which are<br />
directly a function of the product formulation (Rosso and Rob<strong>in</strong>son,<br />
2001), the solutes used (ICMSF, 1980; Pitt and Hock<strong>in</strong>g, 1977), and<br />
the storage temperature. Several models describ<strong>in</strong>g the effect of a w or<br />
solute concentration on the growth of moulds have been published<br />
(Gibson et al., 1994; Cuppers et al., 1997; Valik et al., 1999; Rosso and<br />
Rob<strong>in</strong>son, 2001).<br />
*Ingeniería Química y Alimentos, Universidad de las Américas, Puebla. Cholula<br />
72820, Mexico. Correspondence to epalou@mail.udlap.mx<br />
287
288 Enrique Palou and Aurelio López-Malo<br />
Predictive modell<strong>in</strong>g as def<strong>in</strong>ed by the US Advisory Committee on<br />
Microbiological Criteria for <strong>Food</strong>s is the use of mathematical expressions<br />
to describe the likely behaviour of biological agents.<br />
Mathematical modell<strong>in</strong>g of microbial growth or decl<strong>in</strong>e (also called<br />
“predictive microbiology”) is receiv<strong>in</strong>g a great deal of attention<br />
because of its enormous potential with<strong>in</strong> the food <strong>in</strong>dustry. Predictive<br />
modell<strong>in</strong>g provides a fast and relatively <strong>in</strong>expensive way to get reliable<br />
first estimates on microbial growth and survival (McMeek<strong>in</strong> et al.<br />
1993). Predictive microbiology is ga<strong>in</strong><strong>in</strong>g importance as a powerful<br />
tool <strong>in</strong> food microbiology. Predictive modell<strong>in</strong>g can be used for<br />
describ<strong>in</strong>g behaviour of microorganisms under different conditions, as<br />
well as assist<strong>in</strong>g process design and optimization for production and<br />
distribution cha<strong>in</strong>s, based on microbial safety and shelf-life (Alavi<br />
et al., 1999; Alzamora and López-Malo, 2002). The ma<strong>in</strong> driv<strong>in</strong>g<br />
forces for the impressive progress <strong>in</strong> microbial modell<strong>in</strong>g have<br />
been: the advent of reasonably priced microcomputers that has facilitated<br />
multifactorial data analysis, and the great improvement <strong>in</strong> techniques<br />
to establish mathematical models <strong>in</strong> the area of predictive<br />
microbiology.<br />
Predictive microbiology <strong>in</strong>volves the use of mathematical expressions<br />
to describe microbial behaviour. These <strong>in</strong>clude functions that<br />
relate microbial density to time, and growth rate to environmental<br />
conditions such as temperature, pH, a w , and presence of antimicrobial<br />
agents. Predictive models <strong>in</strong> food microbiology can be divided,<br />
accord<strong>in</strong>g to their aim, <strong>in</strong>to two ma<strong>in</strong> categories: k<strong>in</strong>etic models and<br />
probability models. K<strong>in</strong>etic models that predict growth of foodborne<br />
microorganisms are effective under a wide range of conditions; however,<br />
they are less useful close to the boundary between growth and no<br />
growth. Probabilistic models are useful where the objective is to determ<strong>in</strong>e<br />
whether or not microbial growth can occur under specific conditions.<br />
Much of the effort spent on generat<strong>in</strong>g predictive microbiology<br />
databases has focused on k<strong>in</strong>etic data, <strong>in</strong> which growth rates of<br />
microorganisms are determ<strong>in</strong>ed <strong>in</strong> the normal temperature range and<br />
<strong>in</strong> comb<strong>in</strong>ation with a w , pH and nutrient levels that do not prevent<br />
growth of the modelled organism (Salter et al., 2000). This strategy is<br />
adequate when the desired <strong>in</strong>formation is the extent of growth of food<br />
spoilage organisms, or of pathogens for which some tolerance of<br />
growth is acceptable. However, <strong>in</strong> many situations it is important to<br />
ensure that microorganisms do not contam<strong>in</strong>ate foods (Ross and<br />
McMeek<strong>in</strong>, 1994; Tienungoon et al., 2000).<br />
The goal of analysis us<strong>in</strong>g any statistical model-build<strong>in</strong>g technique<br />
is to f<strong>in</strong>d the best suited and most parsimonious, yet biologically
Probabilistic Modell<strong>in</strong>g of Aspergillus Growth 289<br />
reasonable, model to describe the relationship between a dependent<br />
variable and a set of <strong>in</strong>dependent variables. However, while k<strong>in</strong>etic<br />
models make possible the calculation of the food shelf-life or the prediction<br />
of the time span <strong>in</strong> which significant microbial growth might<br />
occur, the probabilistic models focus their attention towards decid<strong>in</strong>g<br />
whether a microorganism might or might not grow. Consequently,<br />
probability modell<strong>in</strong>g is particularly useful when pathogenic or mycotox<strong>in</strong>-produc<strong>in</strong>g<br />
species are <strong>in</strong>volved. In this case, the growth rate of a<br />
microorganism is of lesser importance than the fact that it is present,<br />
and potentially able to multiply up to <strong>in</strong>fectious dose or toxic levels.<br />
Ratkowsky and Ross (1995) proposed the application <strong>in</strong> food microbiology<br />
of the logistic regression model, which enables modell<strong>in</strong>g of<br />
the boundary between growth and no growth for selected microbial<br />
species when one or more growth controll<strong>in</strong>g factors are used. This<br />
approach was subsequently used by Presser et al. (1998), Bolton and<br />
Frank (1999), López-Malo et al. (2000), López-Malo and Palou<br />
(2000a, b), McMeek<strong>in</strong> et al. (2000), Lanciotti et al. (2001), and Palou<br />
and López-Malo (2003). In recent years, there has been a cont<strong>in</strong>u<strong>in</strong>g<br />
<strong>in</strong>terest <strong>in</strong> the development of predictive microbiology models<br />
describ<strong>in</strong>g microbial responses <strong>in</strong> food. The benefits of their application<br />
<strong>in</strong> the food <strong>in</strong>dustry could be substantial and various, such as prediction<br />
of shelf life, or as an aid to the elaboration of m<strong>in</strong>imally<br />
processed foods (Alzamora and López-Malo, 2002). Several<br />
researchers have <strong>in</strong>dicated that a need exists for predictive models with<br />
advantageous mathematical characteristics such as parsimony, robustness<br />
and stability (McMeek<strong>in</strong> et al., 1992; McMeek<strong>in</strong> et al., 1993;<br />
Ratkowsky, 1993; Whit<strong>in</strong>g and Call, 1993; Baranyi and Roberts, 1994;<br />
Massana and Baranyi, 2000a). These properties would decrease the<br />
error of predictions and would <strong>in</strong>crease the confidence <strong>in</strong> us<strong>in</strong>g predictive<br />
models. Multivariate polynomials are commonly used <strong>in</strong><br />
predictive microbiology to summarise experimental results on the<br />
effect of environmental conditions on fungal growth (López-Malo<br />
and Palou, 2000b). They allow the use of l<strong>in</strong>ear regression for curve<br />
fitt<strong>in</strong>g procedures, which results <strong>in</strong> ease of computation and well<br />
established statistical analyses.<br />
The growth of microorganisms <strong>in</strong> food can be fully described only<br />
by a comb<strong>in</strong>ation of the two k<strong>in</strong>ds of models, k<strong>in</strong>etic and probabilistic.<br />
An <strong>in</strong>tegrated description of the microbial response could be given<br />
by first establish<strong>in</strong>g the likelihood of growth through a probability<br />
model (growth/no growth boundary model), and then predict<strong>in</strong>g the<br />
growth parameters; specific rate and lag time, if growth is expected<br />
(López-Malo and Palou, 2000a, b; Massana and Baranyi, 2000b;
290 Enrique Palou and Aurelio López-Malo<br />
Palou and López-Malo, 2003). Boundary models will then help to<br />
def<strong>in</strong>e the range of applicability of k<strong>in</strong>etic models and may also be<br />
important for establish<strong>in</strong>g food safety regulations as highlighted by<br />
Schaffner and Labuza (1997). Boundary models can predict the most<br />
suitable comb<strong>in</strong>ations of factors to prevent microbial growth, thus<br />
giv<strong>in</strong>g a significant degree of quality and safety from spoilage or food<br />
borne disease. This was also the aim of the hurdle approach proposed<br />
by Leistner (1985). Despite their potential importance, until now there<br />
have been only a few attempts to model the growth/no growth boundary<br />
for vegetative microorganisms.<br />
In this study, selected experimental designs, i.e. central composite or<br />
factorials, and the comb<strong>in</strong>ed effects of <strong>in</strong>cubation temperature, a w ,pH<br />
and concentration of antimicrobial agent (vanill<strong>in</strong> or sodium<br />
benzoate) were <strong>in</strong>corporated <strong>in</strong>to laboratory media to evaluate<br />
the growth/no growth response of three important mycotoxigenic<br />
Aspergillus species, namely Aspergillus flavus, A. ochraceus and<br />
A. parasiticus.<br />
2. MATERIALS AND METHODS<br />
2.1. Microorganisms and Preparation of Inocula<br />
Aspergillus flavus ATCC 16872, A. ochraceus ATCC 22947 and<br />
A. parasiticus ATCC 26691 were cultivated on potato dextrose agar<br />
(PDA; Merck, Mexico) slants for 10 days at 25°C and the spores harvested<br />
with 10 ml of 0.1% Tween 80 (Merck, Mexico) solution sterilized<br />
by membrane (0.45 µm) filtration. Spore suspensions were<br />
adjusted with the same solution to give a f<strong>in</strong>al spore concentration of<br />
10 6 spores/ml and were used the same day. Depend<strong>in</strong>g on the experimental<br />
design, a cocktail of these three species (A. flavus, A. parasiticus<br />
and A. ochraceus) or A. flavus alone were used.<br />
2.2. Experimental Designs<br />
A three level central composite design (Montgomery, 1984) was<br />
employed <strong>in</strong> a first study to assess the effects of pH, <strong>in</strong>cubation temperature<br />
and vanill<strong>in</strong> concentration on mould growth response at 0.98<br />
a w . Independent variable levels are presented <strong>in</strong> Table 1. The results of<br />
this first set of experiments were used to select levels, <strong>in</strong>clud<strong>in</strong>g a w ,of<br />
each variable <strong>in</strong> a range where fungi might or might not grow. A facto-
Probabilistic Modell<strong>in</strong>g of Aspergillus Growth 291<br />
Table 1. Central composite design utilized to evaluate growth response of a cocktail<br />
of conidia of Aspergillus flavus, A. parasiticus and A. ochraceus <strong>in</strong> laboratory media<br />
formulated with a w 0.98, selected pH, vanill<strong>in</strong> concentration and <strong>in</strong>cubated at<br />
different temperatures.<br />
Incubation Vanill<strong>in</strong><br />
Temperature Concentration<br />
pH (°C) (ppm)<br />
3.0 10.0 0<br />
4.0 10.0 0<br />
3.0 25.0 0<br />
4.0 25.0 0<br />
2.7 17.5 0<br />
4.3 17.5 0<br />
3.5 4.9 0<br />
3.5 30.1 0<br />
3.5 17.5 0<br />
3.0 10.0 500<br />
4.0 10.0 500<br />
3.0 25.0 500<br />
4.0 25.0 500<br />
3.0 10.0 1000<br />
4.0 10.0 1000<br />
3.0 25.0 1000<br />
4.0 25.0 1000<br />
2.7 17.5 750<br />
4.3 17.5 750<br />
3.5 4.9 750<br />
3.5 30.1 750<br />
3.5 17.5 330<br />
3.5 17.5 1170<br />
3.5 17.5 750<br />
rial design was employed to assess the effects of a w , pH, antimicrobial<br />
concentration, antimicrobial type (sodium benzoate or vanill<strong>in</strong>) and<br />
<strong>in</strong>cubation temperature, on Aspergillus flavus growth response, the levels<br />
of every variable are presented <strong>in</strong> Table 2. Triplicate systems were<br />
prepared with the result<strong>in</strong>g variable comb<strong>in</strong>ations (Tables 1 and 2).<br />
2.3. Laboratory Media<br />
Follow<strong>in</strong>g the experimental designs, PDA systems were prepared<br />
with commercial sucrose to reach a w 0.98, 0.96 or 0.94, sterilized for 15<br />
m<strong>in</strong> at 121°C, cooled and acidified with hydrochloric acid to the desired<br />
pH. The amounts of sucrose and hydrochloric acid needed <strong>in</strong> every<br />
case had been previously determ<strong>in</strong>ed. The sterilized and acidified agar
292 Enrique Palou and Aurelio López-Malo<br />
Table 2. Factorial design utilized to evaluate the growth response of Aspergillus<br />
flavus <strong>in</strong> laboratory media formulated with selected water activity, pH, sodium benzoate<br />
or vanill<strong>in</strong> concentration and <strong>in</strong>cubated at different temperatures.<br />
0.98 a w 0.96 a w 0.94 a w<br />
Incu- Incu- Incubation<br />
bation bation<br />
Temp Conc a Temp Conc Temp Conc<br />
pH (°C) (ppm) pH (°C) (ppm) pH (°C) (ppm)<br />
3 15 0 3 15 0 3 15 0<br />
4 15 0 4 15 0 4 15 0<br />
5 15 0 5 15 0 5 15 0<br />
3 25 0 3 25 0 3 25 0<br />
4 25 0 4 25 0 4 25 0<br />
5 25 0 5 25 0 5 25 0<br />
3 15 100 3 15 100 3 15 100<br />
4 15 100 4 15 100 4 15 100<br />
5 15 100 5 15 100 5 15 100<br />
3 25 100 3 25 100 3 25 100<br />
4 25 100 4 25 100 4 25 100<br />
5 25 100 5 25 100 5 25 100<br />
3 15 200 3 15 200 3 15 200<br />
4 15 200 4 15 200 4 15 200<br />
5 15 200 5 15 200 5 15 200<br />
3 25 200 3 25 200 3 25 200<br />
4 25 200 4 25 200 4 25 200<br />
5 25 200 5 25 200 5 25 200<br />
. . . . . . . . .<br />
. . . . . . . . .<br />
. . . . . . . . .<br />
3 15 1000 3 15 1000 3 15 1000<br />
4 15 1000 4 15 1000 4 15 1000<br />
5 15 1000 5 15 1000 5 15 1000<br />
3 25 1000 3 25 1000 3 25 1000<br />
4 25 1000 4 25 1000 4 25 1000<br />
5 25 1000 5 25 1000 5 25 1000<br />
a Concentration of sodium benzoate or vanill<strong>in</strong><br />
solutions were aseptically divided and depend<strong>in</strong>g on the experimental<br />
design, the necessary amount of vanill<strong>in</strong> or sodium benzoate (Sigma<br />
Chemical, Co., St. Louis, MO, was added and mechanically <strong>in</strong>corporated<br />
under sterile conditions, then poured <strong>in</strong>to sterile Petri dishes.<br />
2.4. Inoculation and Incubation<br />
Triplicate Petri dishes of every system were centrally <strong>in</strong>oculated by<br />
pour<strong>in</strong>g 2 µl of the spore suspension (≈ 2.0 × 10 3 spores/plate) to give
Probabilistic Modell<strong>in</strong>g of Aspergillus Growth 293<br />
a circular <strong>in</strong>oculum (1 mm diameter). For every tested pH and a w ,<br />
growth controls without antimicrobial were prepared and <strong>in</strong>oculated<br />
as above, <strong>in</strong>clud<strong>in</strong>g one control without pH adjustment (pH = 5.5) or<br />
a w change (a w = 0.998). Three plates of each system were ma<strong>in</strong>ta<strong>in</strong>ed<br />
without <strong>in</strong>oculation for a w and pH measurement. The <strong>in</strong>oculated<br />
plates and controls were <strong>in</strong>cubated for 1 month at selected temperatures<br />
(Tables 1 and 2) <strong>in</strong> hermetically closed plastic conta<strong>in</strong>ers to avoid<br />
dehydration. A sufficient headspace was left <strong>in</strong> the conta<strong>in</strong>ers to avoid<br />
anoxic conditions. Periodically, <strong>in</strong>oculated plates were removed briefly<br />
to observe them and determ<strong>in</strong>e if growth had occurred and immediately<br />
re-<strong>in</strong>cubated. Water activity was measured with a Decagon CX-<br />
1 <strong>in</strong>strument (Decagon Devices, Inc., Pullman, WA) calibrated and<br />
operated follow<strong>in</strong>g the procedure described by López-Malo et al.<br />
(1993). pH was determ<strong>in</strong>ed with a Beckman pH meter model 50<br />
(Beckman Instruments, Inc., Fullerton, CA). Measurements were<br />
made by triplicate. The pH and a w of the PDA systems without <strong>in</strong>oculation<br />
determ<strong>in</strong>ed at the beg<strong>in</strong>n<strong>in</strong>g and at the end of <strong>in</strong>cubation<br />
demonstrated that the desired values rema<strong>in</strong>ed constant under<br />
<strong>in</strong>cubation conditions.<br />
2.5. Mould Growth Response<br />
The <strong>in</strong>oculated systems were exam<strong>in</strong>ed daily us<strong>in</strong>g a stereoscopic<br />
microscope (American Optical, model Forty). A diameter of approximately<br />
2 mm was def<strong>in</strong>ed as a positive sign of growth (López-Malo<br />
et al., 1998) and registered as “1.” If no growth was observed dur<strong>in</strong>g<br />
the <strong>in</strong>cubation period (one month), the response was registered as “0”.<br />
2.6. Model Construction<br />
A logistic regression model relates the probability of occurrence of<br />
an event, Y, conditional on a vector, x, of explanatory variables<br />
(Hosmer and Lemeshow, 1989). The quantity p (x) = E (Y⎪x) represents<br />
the conditional mean of Y given x when the logistic distribution<br />
is used. The specific model of the logistic regression is as follows:<br />
p (x) = [exp (Σ b i x i )] / [1+ exp (Σ b i x i )] (1)<br />
The logit transformation of p (x) is def<strong>in</strong>ed as:<br />
logit (p) = g (x) = ln {[p (x) ] / [1-p (x)]} = Σ b x (2)<br />
i i<br />
For our particular case, a , pH, <strong>in</strong>cubation temperature (T),<br />
w<br />
antimicrobial type (A), antimicrobial concentration (C) and their
294 Enrique Palou and Aurelio López-Malo<br />
<strong>in</strong>teractions are the <strong>in</strong>dependent variables and the outcome or<br />
dependent variable is mould response. In order to fit the logistic model<br />
the follow<strong>in</strong>g equations were selected:<br />
Central composite design – Aspergillus flavus, A. ochraceus and<br />
A. parasiticus growth response<br />
g(x)=β 0 +β 1 pH+β 2 pH 2 +β 3 T+β 4 T 2 +β 5 C+β 6 C 2 +β 7 pHT+<br />
β 8 pHC+β 9 TC+β 10 pHTC (3)<br />
Factorial design -Aspergillus flavus growth response<br />
g(x)=β 0 +β 1 T+β 2 a w +β 3 pH+β 4 C+β 5 A+β 6 Ta w +β 7 TpH+<br />
β 8 TC+β 9 TA + β 10 a w pH+β 11 a w C+β 12 a w A+β 13 pHC+<br />
β 14 pHA+β 15 CA+β 16 Ta w pH+β 17 TpHC+<br />
β 18 TCA+β 19 Ta w C+β 20 TpHA+β 21 a w pHC+<br />
β 22 a w CA+β 23 pHCA+β 24 Ta w pHC+β 25 Ta w pHA+<br />
β 26 a w pHCA+β 27 Ta w pHCA (4)<br />
where the coefficients (β i ) are the parameters to be estimated by fitt<strong>in</strong>g<br />
the models to our experimental data.<br />
If an <strong>in</strong>dependent variable is discrete, then it is <strong>in</strong>appropriate to<br />
<strong>in</strong>clude it <strong>in</strong> the model as if it was <strong>in</strong>terval scaled. In this situation, the<br />
method of choice is to use a collection of design or dummy variables<br />
(Hosmer and Lemeshow, 1989). For antimicrobial type (A) which is a<br />
discrete variable the codification we used was as follows: “1” for<br />
vanill<strong>in</strong> and “0” for sodium benzoate. Logistic regression was performed<br />
with the logistic subrout<strong>in</strong>e <strong>in</strong> SPSS 10.0 (SPSS Inc., Chicago,<br />
IL). A forward stepwise selection procedure was performed to fit the<br />
logistic regression equation. The significance of the coefficients was<br />
evaluated and were elim<strong>in</strong>ated from the model if the probability of<br />
be<strong>in</strong>g zero was greater than 0.1.<br />
After fitt<strong>in</strong>g the logistic regression equation, predictions of the<br />
growth/no growth <strong>in</strong>terface were made at probability levels of 0.50,<br />
0.10 and 0.05, by substitut<strong>in</strong>g the value of logit (p) <strong>in</strong> the model and<br />
f<strong>in</strong>d<strong>in</strong>g the value of one <strong>in</strong>dependent variable ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g fixed the<br />
other <strong>in</strong>dependent variables. Also probability of growth was calculated<br />
us<strong>in</strong>g the logistic equation for the evaluated conditions.<br />
3. RESULTS AND DISCUSSION<br />
For every tested pH and a w , controls prepared without antimicrobials<br />
produced growth when the <strong>in</strong>cubation temperature was 15°C or
Probabilistic Modell<strong>in</strong>g of Aspergillus Growth 295<br />
higher, but at temperatures of 10°C or lower no growth was observed<br />
after one month of <strong>in</strong>cubation (Table 3). Growth/no growth results for<br />
the conditions of central composite design (Table 1) and factorial<br />
design (Table 2) are summarized <strong>in</strong> Tables 3 and 4, respectively.<br />
Results demonstrate that mould <strong>in</strong>hibition (no observable growth) can<br />
be obta<strong>in</strong>ed with several pH, <strong>in</strong>cubation temperature and vanill<strong>in</strong> concentration<br />
comb<strong>in</strong>ations (Table 3) or with comb<strong>in</strong>ations of a w , pH,<br />
<strong>in</strong>cubation temperature and antimicrobial (sodium benzoate or<br />
vanill<strong>in</strong>) concentration (Table 4). In most cases once a replicate from<br />
a comb<strong>in</strong>ation produced growth eventually all the others also grew.<br />
Therefore, the probabilities observed were, with some exceptions,<br />
either close to 1 or 0. This observation agrees with Ratkowsky et al.<br />
(1991), they reported higher variance of the microbial response under<br />
more stressful conditions. Some synergistic comb<strong>in</strong>ations can be<br />
Table 3. Response (growth=1, no growth=0) of an Aspergillus flavus A. parasiticus<br />
and A. ochraceus cocktail <strong>in</strong>oculated <strong>in</strong> potato dextose agar formulated at a w 0.98,<br />
selected pH values and different concentrations of vanill<strong>in</strong> <strong>in</strong>cubated at different<br />
temperatures.<br />
Incubation Vanill<strong>in</strong> Mould Cocktail<br />
pH Temperature (°C) Concentration (ppm) Growth Response<br />
2.7 17.5 0 1<br />
2.7 17.5 750 0<br />
3.0 10.0 0 0<br />
3.0 10.0 500 0<br />
3.0 10.0 1000 0<br />
3.0 25.0 0 1<br />
3.0 25.0 500 1<br />
3.0 25.0 1000 0<br />
3.5 4.9 0 0<br />
3.5 4.9 750 0<br />
3.5 17.5 0 1<br />
3.5 17.5 330 1<br />
3.5 17.5 750 1<br />
3.5 17.5 1170 0<br />
3.5 30.1 0 1<br />
3.5 30.1 750 1<br />
4.0 10.0 0 0<br />
4.0 10.0 500 0<br />
4.0 10.0 1000 0<br />
4.0 25.0 0 1<br />
4.0 25.0 500 1<br />
4.0 25.0 1000 1<br />
4.3 17.5 0 1<br />
4.3 17.5 750 1
296 Enrique Palou and Aurelio López-Malo<br />
Table 4. Response (growth=1, no growth=0) of Aspergillus flavus <strong>in</strong>oculated <strong>in</strong><br />
potato dextrose agar formulated with selected a w , pH values and different concentrations<br />
of sodium benzoate or vanill<strong>in</strong> <strong>in</strong>cubated at 25 or 15°C.<br />
0.94 a w 0.96 a w 0.98 a w<br />
Temp Conc. a Sodium Sodium Sodium<br />
(°C) pH (ppm) Benzoate Vanill<strong>in</strong> Benzoate Vanill<strong>in</strong> Benzoate Vanill<strong>in</strong><br />
25 3 0 1 1 1 1 1 1<br />
200 1 1 1 1 1 1<br />
400 0 1 0 1 1 1<br />
600 0 1 0 1 0 1<br />
800 0 1 0 1 0 1<br />
1000 0 0 0 0 0 0<br />
4 0 1 1 1 1 1 1<br />
200 1 1 1 1 1 1<br />
400 1 1 0 1 1 1<br />
600 0 1 0 1 1 1<br />
800 0 1 0 1 0 1<br />
1000 0 1 0 1 0 1<br />
5 0 1 1 1 1 1 1<br />
200 1 1 1 1 1 1<br />
400 1 1 1 1 1 1<br />
600 1 1 1 1 1 1<br />
800 1 1 1 1 1 1<br />
1000 1 0 1 1 1 1<br />
15 3 0 1 1 1 1 1 1<br />
200 0 1 0 1 1 1<br />
400 0 0 0 0 0 0<br />
600 0 0 0 0 0 0<br />
800 0 0 0 0 0 0<br />
1000 0 0 0 0 0 0<br />
4 0 1 1 1 1 1 1<br />
200 0 1 1 1 1 1<br />
400 0 0 0 0 0 1<br />
600 0 0 0 0 0 0<br />
800 0 0 0 0 0 0<br />
1000 0 0 0 0 0 0<br />
5 0 1 1 1 1 1 1<br />
200 0 1 1 1 1 1<br />
400 0 0 0 0 0 1<br />
600 0 0 0 0 0 1<br />
800 0 0 0 0 0 0<br />
1000 0 0 0 0 0 0<br />
a Concentration of antimicrobial compound (sodium benzoate or vanill<strong>in</strong>)<br />
detected <strong>in</strong> Tables 3 and 4 where fungal growth was <strong>in</strong>hibited at relatively<br />
high pH values or <strong>in</strong>cubation temperatures when comb<strong>in</strong>ed with<br />
reduced a w and selected antimicrobial concentrations. This is the pr<strong>in</strong>ciple<br />
of the multifactorial preservation (or hurdle technology)
Probabilistic Modell<strong>in</strong>g of Aspergillus Growth 297<br />
approach: the search for those “m<strong>in</strong>imal” comb<strong>in</strong>ations of factors<br />
that <strong>in</strong>hibit microbial growth.<br />
Fitt<strong>in</strong>g Eqs. (3) and (4) to the growth/no growth data by logistic<br />
regression and elim<strong>in</strong>at<strong>in</strong>g non-significant (p>0.10) terms resulted <strong>in</strong><br />
the reduced models presented <strong>in</strong> Table 5. Variables <strong>in</strong>cluded <strong>in</strong> the<br />
models were statistically significant (p>0.005). The goodness of fit of<br />
every model was tested by log likelihood ratio and Chi-square tests,<br />
both be<strong>in</strong>g significant, which <strong>in</strong>dicates that models are useful to predict<br />
the outcome variable (growth or no growth). The models’ goodness<br />
of fit was also evaluated compar<strong>in</strong>g predicted values and<br />
experimental observations. A predicted probability of growth (cut<br />
value) ≥ 0.50 was considered as a growth prediction. Us<strong>in</strong>g this criterion,<br />
the overall correct observation was 99.2% for the central composite<br />
design; with only 3 misclassified predictions from a total of 171<br />
observations. In only one of these three disagreements was growth<br />
predicted when no growth was observed, and <strong>in</strong> the other two cases<br />
the model predicted no growth when growth was observed. For the<br />
factorial experimental design, the overall correct observation was<br />
Table 5. Reduced logistic model coefficients utilized to predict mould growth/nogrowth<br />
for the evaluated experimental designs.<br />
Factorial design Central composite design<br />
Coefficient Term Estimate Coefficient Term Estimate<br />
β0 β1 β2 Constant<br />
T<br />
aw −9876.684<br />
744.823<br />
9985.567<br />
β0 β5 β7 Constant<br />
C<br />
pH<br />
−201.673<br />
1070.559<br />
* β3 pH 2182.488 β9 T<br />
T<br />
4.624<br />
* β5 β6 A<br />
T<br />
−7002.668<br />
C −109.451<br />
* β7 aw T<br />
−753.751<br />
* β8 pH<br />
T<br />
−156.855<br />
* β9 C<br />
T<br />
−0.336<br />
* β10 β12 β13 A<br />
* a pH w<br />
* a A w<br />
pH<br />
−19.818<br />
−2201.049<br />
7619.495<br />
* β15 C<br />
C<br />
−1.523<br />
* β16 A<br />
T<br />
−1.274<br />
* β17 * a pH w<br />
T<br />
158.516<br />
* pH * β18 C<br />
T<br />
0.126<br />
* C * β19 A<br />
T<br />
0.051<br />
* β20 * a C w<br />
T<br />
0.340<br />
* pH * β21 β24 A<br />
* * a pH C w<br />
T<br />
63.797<br />
1.541<br />
* β25 * * a pH C w<br />
T<br />
−0.128<br />
* * * a pH A w<br />
−64.832
298 Enrique Palou and Aurelio López-Malo<br />
98.0%, with only 13 misclassified predictions from a total of 648<br />
observations. In ten of these 13 discrepancies, growth was predicted<br />
when no growth was observed, and <strong>in</strong> the other three cases the model<br />
predicted no growth when growth occurred.<br />
Probabilistic microbial models based on logistic regression have been<br />
reported for Shigella flexneri (Ratkowsky and Ross, 1995), Escherichia<br />
coli (Presser et al., 1998), Saccharomyces cerevisiae (López-Malo et al.,<br />
2000), Listeria monocytogenes (Bolton and Frank, 1999) and<br />
Zygosaccharomyces bailii (Cole et al., 1987; López-Malo and Palou,<br />
2000a, b). These reports illustrate the flexibility of logistic regression <strong>in</strong><br />
construct<strong>in</strong>g the model, tak<strong>in</strong>g <strong>in</strong>to account square root type k<strong>in</strong>etic<br />
models (Ratkowsky and Ross, 1995; Presser et al., 1998) or polynomial<br />
type models (Cole et al., 1987; Bolton and Frank, 1999; López-Malo<br />
et al., 2000; López-Malo and Palou, 2000a, b). However, <strong>in</strong> every case<br />
growth/no growth observations were recorded at fixed storage times,<br />
which limited probabilistic models to predict microbial response dur<strong>in</strong>g<br />
that specific period of time. Polynomial type models do not contribute<br />
to the understand<strong>in</strong>g of the mechanism <strong>in</strong>volved <strong>in</strong> microbial<br />
growth <strong>in</strong>hibition. However, they are useful to determ<strong>in</strong>e <strong>in</strong>dependent<br />
variable effects and their <strong>in</strong>teractions. As reported for Z. bailii, the<br />
probability of growth depends on <strong>in</strong>dividual effects of pH, °Brix, sorbic<br />
acid, benzoic acid and sulfite concentrations as well as upon complex<br />
<strong>in</strong>teractions among preservation factors (Cole et al., 1987). For<br />
Saccharomyces cerevisiae, polynomial probabilistic models predicted<br />
the boundary between growth/no growth <strong>in</strong>terface of as a function of<br />
a w , pH and potassium sorbate concentration (López-Malo et al., 2000).<br />
The predicted probabilities of growth for a cocktail of Aspergillus<br />
flavus, A. ochraceus and A. parasiticus are given <strong>in</strong> Figures 1 and 2,<br />
and for Aspergillus flavus after one month of <strong>in</strong>cubation <strong>in</strong> Figures 3<br />
and 4. pH reduction gradually <strong>in</strong>creased the number of comb<strong>in</strong>ations<br />
of <strong>in</strong>cubation temperature and antimicrobial concentration with<br />
probabilities >0.05 for <strong>in</strong>hibition of mould growth. An important<br />
shift of probability of growth curves is also observed with <strong>in</strong>creas<strong>in</strong>g<br />
vanill<strong>in</strong> concentration (Figures 1 and 2). From Figures 3 and 4, the<br />
magnitude of the shift <strong>in</strong> the predicted boundary depends on the<br />
comb<strong>in</strong>ation of temperature, antimicrobial type and water activity<br />
considered.<br />
The transition from “likely to grow” conditions (p > 0.90, or > 90%<br />
likelihood of growth) to “unlikely to grow” conditions (p < 0.10, or <<br />
10% likelihood of growth), as predicted from the fitted models,<br />
was abrupt as can be seen graphically for comb<strong>in</strong>ations of pH and<br />
temperatures (Figures 1 and 2) as well as for pH and antimicrobial
Probabilistic Modell<strong>in</strong>g of Aspergillus Growth 299<br />
p<br />
1.0<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0.0<br />
2.7<br />
2.9<br />
3.1<br />
pH<br />
3.3<br />
3.5<br />
3.7<br />
3.9<br />
concentration (Figures 3 and 4). The abruptness of the transition<br />
between growth or no growth conditions <strong>in</strong>fluenced by pH can be as<br />
little as 0.1 to 0.2 pH units, which is close to the limit of reproducibility<br />
for pH measurements. For temperature the transition is much less<br />
abrupt, occurr<strong>in</strong>g over <strong>in</strong>crements of temperature that exceed that of<br />
measurement or experimental error. For 648 factor comb<strong>in</strong>ations for<br />
Aspergillus flavus, only 15 of these comb<strong>in</strong>ations gave a response different<br />
from “all grew” or “none grew”. Thus, the experimental data<br />
showed an abrupt transition between growth and no growth. This<br />
abruptness does <strong>in</strong>dicate a microbiological reality <strong>in</strong> which small<br />
changes <strong>in</strong> environmental factors with<strong>in</strong> an experiment may have a<br />
strong <strong>in</strong>fluence on the position of the <strong>in</strong>terface (Tienungoon et al.,<br />
2000; Masana and Baranyi, 2000a, b).<br />
An important feature of the generated probabilistic models is that<br />
the level of probability can be set, depend<strong>in</strong>g on the level of str<strong>in</strong>gency<br />
required, to calculate critical values of selected variables. To illustrate,<br />
three sets of model predictions us<strong>in</strong>g factorial design results were<br />
compared, p = 0.50, p = 0.10 and p = 0.05. More str<strong>in</strong>gent values<br />
4.1<br />
5<br />
10<br />
15<br />
20<br />
25<br />
30<br />
Temperature<br />
(�C)<br />
Figure 1. Aspergillus flavus, A. ochraceus and A. parasiticus cocktail probability of<br />
growth (p) <strong>in</strong> potato dextrose agar formulated at a w 0.98, selected pH values and 750<br />
ppm vanill<strong>in</strong> after one month of <strong>in</strong>cubation at different temperatures.
p<br />
1.0<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0.0<br />
2.7<br />
2.9<br />
3.1<br />
3.3<br />
pH<br />
3.5<br />
3.7<br />
3.9<br />
4.1<br />
5<br />
10<br />
15<br />
20<br />
25<br />
30<br />
Temperature<br />
(�C)<br />
Figure 2. Aspergillus flavus, A. ochraceus and A. parasiticus cocktail probability of<br />
growth (p) <strong>in</strong> potato dextrose agar formulated with a w 0.98, selected pH values<br />
and 1000 ppm vanill<strong>in</strong> concentration after one month of <strong>in</strong>cubation at different<br />
temperatures.<br />
p<br />
1<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
0<br />
100<br />
200<br />
300<br />
400<br />
500<br />
600<br />
concentration (ppm)<br />
700<br />
800<br />
900<br />
1000 3.0<br />
5.0<br />
4.6<br />
4.2<br />
3.8<br />
3.4<br />
Figure 3. Aspergillus flavus probability of growth (p) <strong>in</strong> potato dextrose agar formulated<br />
with selected pH, sodium benzoate concentration (ppm) and a w 0.94 after one<br />
month of <strong>in</strong>cubation at 25°C.<br />
pH
Probabilistic Modell<strong>in</strong>g of Aspergillus Growth 301<br />
p<br />
1<br />
0.9<br />
0.8<br />
0.7<br />
0.6<br />
0.5<br />
0.4<br />
0.3<br />
0.2<br />
0.1<br />
0<br />
0<br />
100<br />
200<br />
300<br />
400<br />
500<br />
(p = 0.01 or 0.001) may be necessary <strong>in</strong> some <strong>in</strong>stances. Critical<br />
antimicrobial concentration predictions (Tables 6 and 7) made at p =<br />
0.50 (50:50 chance that A. flavus will grow) represent a relatively conservative<br />
series of estimates, with the predicted response on the<br />
boundary be<strong>in</strong>g no better than a co<strong>in</strong> toss. Model predictions were<br />
made more str<strong>in</strong>gent by mak<strong>in</strong>g p = 0.10 or 0.05 (10 or 5% chance of<br />
a false prediction) which causes a shift <strong>in</strong> the predicted critical antimicrobial<br />
concentration or, <strong>in</strong> other words, <strong>in</strong> the growth/no growth<br />
boundary to a lower temperature, pH and water activity. As a w<br />
decreases from 0.98 (Table 6) to 0.94 (Table 7), the critical concentration<br />
of sodium benzoate or vanill<strong>in</strong> decreases. Also as <strong>in</strong>cubation temperature<br />
<strong>in</strong>creases higher antimicrobial concentrations are needed to<br />
achieve no growth results after one month of <strong>in</strong>cubation.<br />
4. CONCLUSIONS<br />
600<br />
Traditional preservation and storage procedures to produce safe<br />
and stable food product are generally based on microbial control. If<br />
700<br />
concentration (ppm)<br />
800<br />
900<br />
1000 3.0<br />
5.0<br />
4.6<br />
4.2<br />
3.8<br />
3.4<br />
Figure 4. Aspergillus flavus probability of growth (p) <strong>in</strong> potato dextrose agar formulated<br />
with selected pH, vanill<strong>in</strong> concentration (ppm) and a w 0.98 after one month of<br />
<strong>in</strong>cubation at 15°C.<br />
pH
302 Enrique Palou and Aurelio López-Malo<br />
Table 6. Critical antimicrobial concentrations (ppm) for selected probabilities (p) to<br />
<strong>in</strong>hibit Aspergillus flavus growth <strong>in</strong> laboratory media formulated at a w 0.98 and<br />
selected pH values dur<strong>in</strong>g one month of <strong>in</strong>cubation at various temperatures.<br />
Incubation Temperature (°C)<br />
p pH 15.0 17.5 20.0 22.5 25.0<br />
Sodium Benzoate<br />
0.05<br />
3.0 353 357 364 384 566<br />
3.5 360 365 372 393 656<br />
4.0 368 373 381 403 860<br />
4.5 377 381 390 415 > 1000<br />
5.0 385 389 399 427 > 1000<br />
0.10<br />
3.0 351 355 361 379 542<br />
3.5 359 363 370 388 623<br />
4.0 367 371 378 398 806<br />
4.5 375 379 387 409 > 1000<br />
5.0 383 387 396 422 > 1000<br />
0.50<br />
3.0 347 349 354 365 471<br />
3.5 355 357 362 374 525<br />
4.0 363 365 370 383 646<br />
4.5 371 374 379 393 > 1000<br />
5.0 379 382 388 405 > 1000<br />
Vanill<strong>in</strong><br />
0.05<br />
3.0 392 532 682 842 > 1000<br />
3.5 473 595 741 921 > 1000<br />
4.0 569 679 834 > 1000 > 1000<br />
4.5 685 799 1004 > 1000 > 1000<br />
5.0 829 984 > 1000 > 1000 > 1000<br />
0.10<br />
3.0 369 509 658 817 988<br />
3.5 448 567 711 888 > 1000<br />
4.0 542 647 796 > 1000 > 1000<br />
4.5 655 760 950 > 1000 > 1000<br />
5.0 795 934 > 1000 > 1000 > 1000<br />
0.50<br />
3.0 302 439 586 743 911<br />
3.5 375 488 624 790 998<br />
4.0 462 553 683 880 > 1000<br />
4.5 567 646 790 > 1000 > 1000<br />
5.0 696 790 > 1000 > 1000 > 1000
Probabilistic Modell<strong>in</strong>g of Aspergillus Growth 303<br />
Table 7. Critical antimicrobial concentrations (ppm) for selected probabilities (p) to<br />
<strong>in</strong>hibit Aspergillus flavus growth <strong>in</strong> laboratory media formulated at a w 0.94 and<br />
selected pH values dur<strong>in</strong>g one month of <strong>in</strong>cubation at various temperatures.<br />
Incubation Temperature (°C)<br />
p pH 15.0 17.5 20.0 22.5 25.0<br />
Sodium Benzoate<br />
0.05<br />
3.0 50 68 98 152 291<br />
3.5 79 94 120 173 341<br />
4.0 108 122 146 200 436<br />
4.5 139 151 175 236 701<br />
5.0 170 183 209 287 > 1000<br />
0.10<br />
3.0 49 67 96 150 287<br />
3.5 78 93 119 171 335<br />
4.0 107 120 144 197 428<br />
4.5 137 150 173 233 685<br />
5.0 169 182 207 282 > 1000<br />
0.50<br />
3.0 46 63 91 143 274<br />
3.5 75 89 113 163 318<br />
4.0 104 116 138 188 403<br />
4.5 134 146 167 222 638<br />
5.0 165 177 201 269 > 1000<br />
Vanill<strong>in</strong><br />
0.05<br />
3.0 232 410 599 799 > 1000<br />
3.5 233 383 558 767 > 1000<br />
4.0 235 349 502 714 > 1000<br />
4.5 236 307 417 614 > 1000<br />
5.0 238 251 275 345 > 1000<br />
0.10<br />
3.0 228 406 595 795 1007<br />
3.5 229 379 554 761 > 1000<br />
4.0 230 344 496 707 > 1000<br />
4.5 232 301 410 604 > 1000<br />
5.0 233 244 265 326 > 1000<br />
0.50<br />
3.0 217 394 582 782 994<br />
3.5 217 365 539 746 995<br />
4.0 217 330 479 687 997<br />
4.5 218 284 388 574 1001<br />
5.0 219 225 237 271 > 1000
304 Enrique Palou and Aurelio López-Malo<br />
the microbial hazard or spoilage cannot be totally elim<strong>in</strong>ated from the<br />
food, microbial growth and tox<strong>in</strong> production must be <strong>in</strong>hibited.<br />
Microbial growth can be <strong>in</strong>hibited by comb<strong>in</strong><strong>in</strong>g <strong>in</strong>tr<strong>in</strong>sic food characteristics<br />
with extr<strong>in</strong>sic storage and packag<strong>in</strong>g conditions. Accurate<br />
quantitative data about the effects of comb<strong>in</strong>ed factors on growth or<br />
survival of selected microorganisms are needed. Predictive models can<br />
provide decision support tools for the food <strong>in</strong>dustry. In many cases<br />
models are empirical, <strong>in</strong>terpret<strong>in</strong>g only the response of the microorganism<br />
without understand<strong>in</strong>g the mechanism of the response.<br />
However, if the models are used properly, predictive probabilistic<br />
models are helpful tools for evaluat<strong>in</strong>g microbial responses which can<br />
<strong>in</strong> turn identify potential problems for a product, process or storage<br />
conditions. Logistic regression is a useful tool for modell<strong>in</strong>g the<br />
boundary between growth and no growth. The probabilistic microbial<br />
modell<strong>in</strong>g approach can provide a practical means of evaluat<strong>in</strong>g<br />
the comb<strong>in</strong>ed effects of food formulation, process<strong>in</strong>g and storage<br />
conditions.<br />
5. ACKNOWLEDGMENTS<br />
We acknowledge f<strong>in</strong>ancial support from CONACyT -Mexico<br />
(Projects 32405-B and 44088), Universidad de las Américas-Puebla,<br />
and CYTED Program (Project XI.15).<br />
6. REFERENCES<br />
Alavi, S. H., Puri, V. M., Knabel, S. J., Mohtar, R. H., and Whit<strong>in</strong>g, R. C., 1999,<br />
Development and validation of a dynamic growth model for Listeria monocytogenes<br />
<strong>in</strong> fluid whole milk, J. <strong>Food</strong> Prot. 62:170-176.<br />
Alzamora, S.M., and López-Malo, A., 2002, Microbial behavior model<strong>in</strong>g as a tool <strong>in</strong><br />
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Quality and Stability, D. Simatos, and J. L. Multon, ed., Mart<strong>in</strong>us Nihof<br />
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Zygosaccharomyces bailii as a function of temperature, water activity, pH, potassium<br />
sorbate and sodium benzoate concentration, Presented at Predictive<br />
Model<strong>in</strong>g <strong>in</strong> <strong>Food</strong>s, Leuven, Belgium, September 12-15.<br />
López-Malo, A., and Palou E., 2000b, Model<strong>in</strong>g the growth/no growth <strong>in</strong>terface of<br />
Zygosaccharomyces bailii <strong>in</strong> mango puree, J. <strong>Food</strong> Sci. 65:516-520.<br />
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effects on mold growth, J. <strong>Food</strong> Sci. 63:143-146.<br />
López-Malo, A., Guerrero, S., and Alzamora, S. M., 2000, Probabilistic model<strong>in</strong>g of<br />
Saccharomyces cerevisiae <strong>in</strong>hibition under the effects of water activity, pH and<br />
potassium sorbate, J. <strong>Food</strong> Prot. 63:91-95.<br />
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con un equipo electrónico basado en el punto de rocío, Información Tecnológica.<br />
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effect on the risk of extrapolation, <strong>Food</strong> Microbiol. 17:367-374.<br />
Masana, M. O., and Baranyi, J., 2000b, Growth/no growth <strong>in</strong>terface of Brochothrix<br />
thermosphacta as a function of pH and water activity, <strong>Food</strong> Microbiol. 17:485-493.<br />
McMeek<strong>in</strong>, T. A., Olley, J., Ross, T., and Ratkowsky, D. A., 1993, Predictive<br />
Microbiology: Theory and Application, Research Studies Press, Tauton, UK.<br />
McMeek<strong>in</strong>, T. A., Presser, K., Ratkowsky, D. A., Ross, T., Salter, M., and<br />
Tienungoon, S., 2000, Quantify<strong>in</strong>g the hurdle concept by modell<strong>in</strong>g the growth/no<br />
growth <strong>in</strong>terface. A review, Int. J. <strong>Food</strong> Microbiol. 55:93-98.<br />
McMeek<strong>in</strong>, T. A., Ross, T., and Olley, J., 1992, Application of predictive microbiology<br />
to assure the quality and safety of fish and fish products, Int. J. <strong>Food</strong><br />
Microbiol. 15:13-32.<br />
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emerg<strong>in</strong>g technologies, <strong>in</strong>: Novel <strong>Food</strong> Process<strong>in</strong>g Technologies, G. V. Barbosa-<br />
Canovas, M. S. Tapia, and P. Cano, eds, Marcel Dekker, New York, pp. 629-651.<br />
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Suppl. 1989:1S-9S.<br />
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acid concentration, and water activity, Appl. Environ. Microbiol. 64:1773-1779.<br />
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Arrhenius-type and Belehradek-type models for prediction of bacterial growth <strong>in</strong><br />
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23:241-264.<br />
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comb<strong>in</strong>ed temperature and salt (NaCl) limits for growth of a pathogenic<br />
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Clostridium botul<strong>in</strong>um, <strong>Food</strong> Microbiol. 10:295-301.
ANTIFUNGAL ACTIVITY OF SOURDOUGH<br />
BREAD CULTURES<br />
Lloyd B. Bullerman, Marketa Giesova, Yousef Hassan,<br />
Dwayne Deibert and Doj<strong>in</strong> Ryu *<br />
1. INTRODUCTION<br />
Many strategies have been studied for control of mould growth<br />
and reduction <strong>in</strong> mycotox<strong>in</strong> production <strong>in</strong> foods. The most effective<br />
strategy for controll<strong>in</strong>g the presence of mycotox<strong>in</strong>s <strong>in</strong> foods is prevention<br />
of growth of the mycotox<strong>in</strong>-produc<strong>in</strong>g fungi <strong>in</strong> foods and field<br />
crops <strong>in</strong> the first place. Mycotox<strong>in</strong> contam<strong>in</strong>ation may occur prior to<br />
harvest of crops and is often the dom<strong>in</strong>ant reason for the occurrence<br />
of mycotox<strong>in</strong>s <strong>in</strong> foods and feeds. However, fungal growth on stored<br />
foods and commodities is also a serious and cont<strong>in</strong>u<strong>in</strong>g problem. In<br />
recent years <strong>in</strong>creased public concern over chemical food additives<br />
and fungicides <strong>in</strong> foods has prompted searches for safe naturally<br />
occurr<strong>in</strong>g biological agents with antifungal potential. One source of<br />
such compounds are the lactic acid bacteria.<br />
While only a relatively limited number of studies have reported the<br />
<strong>in</strong>hibitory effects of lactic acid bacteria on fungal growth and mycotox<strong>in</strong><br />
production, it is generally believed that it is safe for humans to<br />
consume lactic acid bacteria and has been known for many years that<br />
lactic acid bacteria may positively <strong>in</strong>fluence the gastro<strong>in</strong>test<strong>in</strong>al tract<br />
*Lloyd B. Bullerman, Yousef Hassan, Dwayne Deibert and Doj<strong>in</strong> Ryu, Department<br />
of <strong>Food</strong> Science and Technology, University of Nebraska, L<strong>in</strong>coln, NE 68583-0919,<br />
USA. Marketa Giesova, Department of Dairy and Fat Technology, Institute of<br />
Chemical Technology, Prague, Czech Republic. Correspondence to lbbuller@unlnotes.unl.edu<br />
307
308 Lloyd B. Bullerman et. al.<br />
of humans and other mammals (Sand<strong>in</strong>e, 1996). Lactic acid bacteria<br />
have been used to ferment foods for centuries, which suggests the nontoxic<br />
nature of metabolites produced by these bacteria (Garver and<br />
Muriana, 1993; Klaenhammer, 1998). Indigenous lactic acid bacteria<br />
are commonly found <strong>in</strong> retail foods, which suggests that the public<br />
consumes viable lactic acid bacteria <strong>in</strong> many ready-to-eat products<br />
(Garver and Muriana, 1993). Thus the metabolic activity of lactic acid<br />
bacteria that may contribute <strong>in</strong> a number of ways to the control of<br />
bacterial pathogens and might also have applications for prevent<strong>in</strong>g<br />
fungal growth (Gourama and Bullerman, 1995; Holzapfel et al., 1995;<br />
Klaenhammer, 1998).<br />
The potential of lactic acid bacteria for use as biological control<br />
agents of moulds <strong>in</strong> barley and thus improve the quality and safety of<br />
malt dur<strong>in</strong>g the malt<strong>in</strong>g process has been studied <strong>in</strong> F<strong>in</strong>land (Haikara<br />
et al., 1993; Haikara and Laitila, 1995). The latter authors found a<br />
group of lactic acid bacteria with antagonistic activities aga<strong>in</strong>st<br />
Fusarium. Lactobacillus planarum and Pediococcus pentosaceum <strong>in</strong>hibited<br />
Fusarium avenaceum obta<strong>in</strong>ed from barley kernels. The prelim<strong>in</strong>ary<br />
characterization of these starter cultures revealed new types of<br />
antimicrobial substances with low molecular mass and features not<br />
previously reported for lactic acid bacteria microbiocides (Haikara<br />
and Niku-Paavola, 1993; Niku-Paavola et al., 1999).<br />
Studies of Lactobacillus stra<strong>in</strong>s that possess antifungal properties<br />
carried out <strong>in</strong> the Czech Republic showed that Lactobacillus rhamnosus<br />
VT1 exhibited strong antifungal properties (Stiles et al., 1999;<br />
Plockova et al., 2000, 2001). Further research has shown that L. rhamnosus<br />
is capable of <strong>in</strong>hibit<strong>in</strong>g the growth of many spoilage and toxigenic<br />
fungi <strong>in</strong>clud<strong>in</strong>g species <strong>in</strong> the genera Aspergillus, Penicillium and<br />
Fusarium (Stiles et al., 2002).<br />
The use of sourdough bread cultures has been reported to <strong>in</strong>crease<br />
the shelf life of baked goods by delay<strong>in</strong>g mould growth due to the<br />
presence of lactic acid bacteria (Gobbetti, 1998). This activity has<br />
been attributed to the presence of organic acids, particularly lactic and<br />
acetic acids (Spicher, 1983; Rocken, 1996). Further studies have shown<br />
that although acetic acid contributes to the antifungal activity of lactic<br />
acid bacteria, other bacterial metabolites also have antifungal<br />
activity and may contribute to the <strong>in</strong>hibition of mould growth<br />
(Corsetti et al., 1998; Gourama, 1997; Niku-Paavola et al., 1999).<br />
Stra<strong>in</strong>s of Lactobacillus plantarum <strong>in</strong> particular seem to possess strong<br />
antifungal activity (Gobetti et al., 1994a,b; Karunaratne et al., 1990).<br />
Lavermicocca et al. (2000) found that L. plantarum from sourdough<br />
was fungicidal to F. gram<strong>in</strong>earum <strong>in</strong> wheat flour hydrolysate and
Antifungal Activity of Sourdough Bread Cultures 309<br />
reported the purification and characterization of novel antifungal<br />
compounds from this stra<strong>in</strong>. The compounds that they reported<br />
to have strong antifungal activity were phenyllactic acid and<br />
4-hydroxyphenyllactic acid. Two stra<strong>in</strong>s of L. plantarum isolated from<br />
sour-dough cultures produced these compounds and were <strong>in</strong>hibitory<br />
to a number of fungi isolated from baked products (Lavermicocca<br />
et al., 2003).<br />
The objective of this work was to test L. plantarum and L. paracasei<br />
isolated from sourdough bread cultures for antifungal activity<br />
aga<strong>in</strong>st several mycotoxigenic molds and to test the ability of an <strong>in</strong>tact<br />
sourdough bread culture to <strong>in</strong>hibit mold growth.<br />
2. MATERIALS AND METHODS<br />
The fungi used were as follows: Aspergillus flavus NRRL 1290,<br />
Aspergillus ochraceus NRRL 3174, Penicillium verrucosum NRRL<br />
846, Penicillium roqueforti NRRL 848, and Penicillium commune<br />
NRRL 1899. Bacterial cultures isolated and identified from the sourdough<br />
cultures <strong>in</strong>cluded, Lactobacillus plantarum 01 and 011,<br />
Lactobacillus paracasei 02, 03 and 05 and Lactobacillus paracasei SF1,<br />
SF2 and SF21. Isolates 01, 02, 03, 05 and 011 were obta<strong>in</strong>ed from an<br />
old orig<strong>in</strong>al household sourdough from West Texas, USA which has<br />
been kept active for about 100 years. Isolates SF1, SF2 and SF21 were<br />
obta<strong>in</strong>ed from a commercial San Francisco type sourdough culture.<br />
Sourdough starter cultures and other bacterial isolates were<br />
screened for antifungal activity us<strong>in</strong>g a dual agar plate assay <strong>in</strong> which<br />
1.0% of the activated sourdough culture or isolate was added to 15 ml<br />
of wheat flour hydrolysate agar (WFH) formulated accord<strong>in</strong>g to<br />
Gobbetti (et al., 1994b), commercial deMan-Rogosa-Sharpe (MRS)<br />
agar (Oxoid, Cat. CM0361) and modified MRS (mMRS) agar <strong>in</strong> Petri<br />
dishes. Modified MRS agar was produced by mak<strong>in</strong>g it without<br />
sodium acetate. The sourdough and bacterial isolate agar cultures<br />
were overlaid with soft (0.75% agar) yeast extract sucrose agar (YES)<br />
or potato dextrose agar (PDA). The centres of the YES or PDA agars<br />
were then <strong>in</strong>oculated at a s<strong>in</strong>gle po<strong>in</strong>t with a mould spore suspension<br />
conta<strong>in</strong><strong>in</strong>g 10 3 spores. The plates were <strong>in</strong>cubated at 30°C for 21 days.<br />
Colony diameters of the grow<strong>in</strong>g mould cultures were measured <strong>in</strong><br />
mm and recorded daily.<br />
After <strong>in</strong>itial studies the work concentrated on us<strong>in</strong>g L. plantarum 01<br />
and L. paracasei SF1 as they appeared to be the most active antifungal
310 Lloyd B. Bullerman et. al.<br />
cultures and were representative of the ma<strong>in</strong> isolates. These cultures<br />
were then grown and compared on MRS and mMRS agars with PDA<br />
overlay.<br />
3. RESULTS AND DISCUSSION<br />
The <strong>in</strong>fluence of bacterial cultures on fungal growth (colony diameter)<br />
was plotted as a functional of time (Figures 1-5). Fungal cultures<br />
grow<strong>in</strong>g on an underlay of un<strong>in</strong>oculated bacterial media were used as<br />
controls.<br />
Growth of Aspergillus flavus was delayed <strong>in</strong> the presence of L. paracasei<br />
SF1 and L. plantarum 01 (Figure 1). L. plantarum was more<br />
<strong>in</strong>hibitory than L. paracasei and caused a greater delay <strong>in</strong> growth.<br />
Both bacterial stra<strong>in</strong>s appeared to be more <strong>in</strong>hibitory when grown on<br />
MRS agar than mMRS agar, although there was no apparent difference<br />
<strong>in</strong> the growth of the control fungal cultures on the two media.<br />
Aspergillus ochraceus was <strong>in</strong>hibited to a greater degree than was<br />
A. flavus (Figure 2). Both L. plantarum and L. paracasei on MRS agar<br />
caused complete <strong>in</strong>hibition of growth of A. ochraceus. On mMRS<br />
Colony Diameter (mm)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 3 6 9 12 15<br />
Incubation Time (Days)<br />
Control (MRS) Lb. paracasei SF1 (MRS)<br />
Lb. plantarum O1(MRS) Control (mMRS)<br />
Lb.paracasei SF1 (mMRS) Lb. plantarum O1 (mMRS)<br />
Figure 1. Inhibitory effect of Lactobacillus plantarum O1 and Lactobacillus paracasei<br />
SF1 grown <strong>in</strong> MRS and mMRS agars on growth of Aspergillus flavus NRRL 1290 on<br />
a yeast extract sucrose agar overlay.
Antifungal Activity of Sourdough Bread Cultures 311<br />
Colony Diameter (mm)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 3 6 9<br />
Control (MRS)<br />
Incubation Time (Days)<br />
12 15<br />
Lb. paracasei SF1 (MRS)<br />
Lb. plantarum O1(MRS) Control (mMRS)<br />
Lb.paracasei SF1 (mMRS) Lb. plantarumO1 (mMRS)<br />
Figure 2. Inhibitory effect of Lactobacillus plantarum O1 and Lactobacillus paracasei<br />
SF1 grown <strong>in</strong> MRS and mMRS agars on growth of Aspergillus ochraceus NRRL 3174<br />
on a yeast extract sucrose agar overlay.<br />
Colony Diameter (mm)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 3 6 9 12 15<br />
Incubation Time (Days)<br />
Control (MRS) Lb. paracasei SF1 (MRS)<br />
Lb. plantarum O1(MRS) Control (mMRS)<br />
Lb.paracasei SF1(mMRS) Lb. plantarum O1 (mMRS)<br />
Figure 3. Inhibitory effect of Lactobacillus plantarum O1 and Lactobacillus paracasei<br />
SF1 grown <strong>in</strong> MRS and mMRS agars on growth of Penicillium verrucosum NRRL<br />
846 on a potato dextrose agar overlay.
312 Lloyd B. Bullerman et. al.<br />
agar limited growth of A. ochraceus occurred <strong>in</strong> the presence of<br />
L. plantarum, with growth be<strong>in</strong>g delayed until the fifth day of <strong>in</strong>cubation.<br />
In the presence of L. paracasei on mMRS growth of A. ochraceus<br />
began by the second day of <strong>in</strong>cubation, but growth was limited,<br />
though more growth occurred than <strong>in</strong> the presence of L. plantarum.<br />
Growth of Penicillium verrucosum was completely <strong>in</strong>hibited by<br />
L. plantarum on MRS agar and L. paracasei was also more <strong>in</strong>hibitory<br />
on MRS agar than on the mMRS agar (Figure 3). Growth occurred<br />
sooner on mMRS, but colonies were smaller than for the control.<br />
Penicillium roqueforti grew more rapidly <strong>in</strong> the presence of the two<br />
bacteria than the other mould species (Figure 4). L. plantarium<br />
showed a greater <strong>in</strong>hibitory effect on both MRS and mMRS. P. roquefortii<br />
was slightly <strong>in</strong>hibited by L. paracasei compared with the controls.<br />
Growth of P. commune was completely <strong>in</strong>hibited by L. plantarum<br />
on MRS medium and strongly <strong>in</strong>hibited by L. plantarum on mMRS<br />
agar (Figure 5). L. paracasei was less <strong>in</strong>hibitory than L. plantarum on<br />
both media but with no real difference <strong>in</strong> <strong>in</strong>hibitory effect between<br />
either medium.<br />
With all fungal species, differences <strong>in</strong> growth were seen due to the<br />
medium <strong>in</strong> which the Lactobacillus species were grown. The lactobacilli<br />
were more <strong>in</strong>hibitory when grown <strong>in</strong> MRS agar than <strong>in</strong> mMRS.<br />
The orig<strong>in</strong>al MRS medium and the Commercial MRS medium conta<strong>in</strong><br />
5 g of sodium acetate per litre (0.5%) of medium (de Man et al.,<br />
1960). Stiles et al. (2002) reported that L. rhamnosus had greater<br />
<strong>in</strong>hibitory action aga<strong>in</strong>st 40 different fungal stra<strong>in</strong>s when grown <strong>in</strong><br />
MRS medium than when grown <strong>in</strong> modified MRS <strong>in</strong> which the<br />
sodium acetate was omitted. They concluded that the acetate was also<br />
exert<strong>in</strong>g an <strong>in</strong>hibitory effect either <strong>in</strong>dependently or <strong>in</strong> addition to<br />
<strong>in</strong>hibitory substances produced by L. rhamnosus. P. roqueforti was<br />
least <strong>in</strong>hibited by either bacterium and did not seem to be <strong>in</strong>hibited to<br />
any greater degree on the MRS over the mMRS by either bacterium,<br />
although L. plantarum tended to be more <strong>in</strong>hibitory than L. paracasei.<br />
Penicillium roqueforti is known to have resistance to preservatives such<br />
as acetic acid (Gravesen et al., 1994).<br />
It appeared possible that MRS may be a better growth medium for<br />
lactobacilli from dairy environments, but sourdough cultures may<br />
grow better <strong>in</strong> a medium that has more cereal based <strong>in</strong>gredients.<br />
Therefore, it was decided to add a medium developed by Gobbetti<br />
et al. (1994b) called Wheat Flour Hydrolysate Agar (WFH). In this<br />
study a complete sourdough mixed culture, not <strong>in</strong>dividual bacterial<br />
isolates, was added to the base agar layers of MRS, mMRS and WFH<br />
<strong>in</strong> Petri dishes. Base agar layers were then overlaid with YES agar
Antifungal Activity of Sourdough Bread Cultures 313<br />
Colony Diameter (mm)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 3 6 9 12 15<br />
Incubation Time (Days)<br />
Control (MRS) Lb. paracasei SF1 (MRS)<br />
Lb. plantarumO1(MRS) Control (mMRS)<br />
Lb.paracasei SF1 (mMRS) Lb. plantarum O1 (mMRS)<br />
Figure 4. Inhibitory effect of Lactobacillus plantarum O1 and Lactobacillus paracasei<br />
SF1 grown <strong>in</strong> MRS and mMRS agars on growth of Penicillium roqueforti NRRL 848<br />
on a potato dextrose agar (PDA) overlay.<br />
Colony Diameter(mm)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 3 6 9 12 15<br />
Incubation Time (Days)<br />
Control (MRS) Lb. paracasei SF1 (MRS)<br />
Lb. plantarum O1(MRS) Control (mMRS)<br />
Lb.paracasei SF1 (mMRS) Lb. plantarum O1 (mMRS)<br />
Figure 5. Inhibitory effect of Lactobacillus plantarum O1 and Lactobacillus paracasei<br />
SF1 grown <strong>in</strong> MRS and mMRS agars on growth of Penicillium commune NRRL 1899<br />
on a potato dextrose agar overlay.
314 Lloyd B. Bullerman et. al.<br />
which was <strong>in</strong>oculated with a s<strong>in</strong>gle po<strong>in</strong>t of A. flavus NRRL 1290<br />
spores <strong>in</strong> the centre of the plate as previously described. The growth<br />
of A. flavus was delayed and reduced on all three media by the <strong>in</strong>tact<br />
or complete sourdough culture (Figure 6). Essentially no difference <strong>in</strong><br />
<strong>in</strong>hibition was observed <strong>in</strong> treatments where the sourdough culture<br />
was grown <strong>in</strong> the MRS, mMRS and WFH agars. Thus <strong>in</strong> this study<br />
with the complete sourdough culture there did not appear to be an<br />
effect from the medium <strong>in</strong> which the sourdough culture was grown as<br />
was observed with the <strong>in</strong>dividual bacterial cultures.<br />
Overall this study has shown that L. plantarum and L. paracasei isolated<br />
from sourdough bread cultures and an <strong>in</strong>tact sourdough bread<br />
culture were <strong>in</strong>hibitory to several species of mycotoxigenic fungi. The<br />
<strong>in</strong>hibition was manifested both as delay of growth and suppression of<br />
the growth rate. The <strong>in</strong>hibitory effects were <strong>in</strong>fluenced by the culture<br />
medium or substrate <strong>in</strong> which the <strong>in</strong>dividual sourdough bacteria, but<br />
not the complete sourdough culture were grown. The presence of<br />
0.5% sodium acetate <strong>in</strong> MRS medium resulted <strong>in</strong> a stronger <strong>in</strong>hibitory<br />
effect by L. plantarum and L. paracasei than mMRS medium from<br />
which the sodium acetate was removed. The <strong>in</strong>hibitory effect was<br />
about 20% less when sodium acetate was excluded from the medium.<br />
Additional studies are <strong>in</strong> progress to further evaluate the antifungal<br />
Colony Diameter (mm)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 3 6 9 12 15<br />
Incubation Time (Day)<br />
Control (MRS) Control (mMRS)<br />
Control (WFH) MRS<br />
mMRS WFH<br />
Figure 6. Inhibitory effect of an <strong>in</strong>tact sourdough bread culture grown <strong>in</strong> MRS,<br />
mMRS and WFH agars on growth of Aspergillus flavus NRRL 1290 on a yeast<br />
extract sucrose agar overlay.
Antifungal Activity of Sourdough Bread Cultures 315<br />
activities of the complete sourdough cultures, and various L. plantarum<br />
and L. paracasei isolates.<br />
4. ACKNOWLEDGEMENTS<br />
This manuscript is published as Paper No. 14941, Journal Series.<br />
This research was carried out under Project 16-097, Agricultural<br />
Research Division, University of Nebraska-L<strong>in</strong>coln and was supported<br />
<strong>in</strong> part by a research grant from the Anderson Research Fund<br />
of the NC-213 Multistate Research Project.<br />
5. REFERENCES<br />
Corsetti, A., Gobbetti M., and Damiani, P., 1998, Antimould activity of sourdough<br />
lactic acid bacteria: identification of a mixture of organic acids produced by<br />
Lactobacillus sanfrancisco CB1, Appl. Microbiol. Biotechnol. 50:253-256.<br />
deMan, J. C., Rogosa, M., and Sharpe, M. E., 1960, A medium for the cultivation of<br />
lactobacilli, J. Appl. Bacteriol. 23:130-135.<br />
Garver, K. I., and Muriana, P. M., 1993, Detection, identification and characterization<br />
of bacterioc<strong>in</strong>-produc<strong>in</strong>g lactic acid bacteria from retail food products, Int.<br />
J. <strong>Food</strong> Microbiol. 19:241-258.<br />
Graveson, S., Frisvad J. C., and Samson, R. A., 1994, Microfungi, Munksgaard.<br />
Copenhagen, Denmark.<br />
Gobbetti, M., 1998, The sourdough microflora: <strong>in</strong>teraction of lactic acid bacteria and<br />
yeasts, Trends <strong>Food</strong> Sci. Technol. 9:267-274.<br />
Gobbetti, M., Corsetti, A., and Rossi, I. 1994a, The sourdough microflora: <strong>in</strong>teractions<br />
between lactic acid bacteria and yeasts: metabolism of am<strong>in</strong>o acids, World<br />
J. Microbiol. Biotechnol. 10:275-279.<br />
Gobbetti, M., Corsetti, A. and Rossi, I., 1994b, The sourdough microflora: <strong>in</strong>teractions<br />
between lactic acid bacteria and yeasts: metabolism of carbohydrates, Appl.<br />
Microbiol. Biotechnol. 41:456-460.<br />
Gourama, H., 1997, Inhibition of growth and mycotox<strong>in</strong> production of Penicillium by<br />
Lactobacillus species, Lebensm. Wiss. Technol. 30:279-283.<br />
Gourama, H., and Bullerman, L. B., 1995, Antimycotic and antiaflatoxigenic effect of<br />
lactic acid bacteria. A Review, J. <strong>Food</strong> Prot. 57:1275-1280.<br />
Haikara, A., and Laitila, A., 1995, Influence of lactic acid starter cultures on the<br />
quality of malt and beer, <strong>in</strong>: Proceed<strong>in</strong>gs of the 26 th Congress of the European<br />
Brewers Convention, Brussels, IRL Press, Oxford, UK, pp. 249-256.<br />
Haikara, A., and Niku-Paavola, M., 1993, Fungicidic substances produced by lactic<br />
acid bacteria, FEMS Microbiol. Rev. 12:120.<br />
Haikara, A., Uljas, H., and Suurnakki, A., 1993, Lactic starter cultures <strong>in</strong> malt<strong>in</strong>g: a<br />
novel solution to gush<strong>in</strong>g problems, <strong>in</strong>: Proceed<strong>in</strong>gs of the 25 th Congress of the<br />
European Brewers Convention, Oslo, IRL Press, Oxford, UK, pp. 163-172.
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Holzapfel, W. H., Geisen, R., and Schill<strong>in</strong>ger, U., 1995, Biological preservation of<br />
foods with reference to protective cultures, bacterioc<strong>in</strong>s and food grade enzymes,<br />
Int. J. <strong>Food</strong> Microbiol. 24:343-362.<br />
Karunaratne, A., Wezenberg, E., and Bullerman, L. B., 1990, Inhibition of mold<br />
growth and aflatox<strong>in</strong> production by Lactobaciillus spp., J. <strong>Food</strong> Prot. 53:230-236.<br />
Klaenhammer, T. R., 1998, Functional activities of Lactobacillus probiotics: genetic<br />
mandate, Int. Dairy J. 8:497-505.<br />
Lavermicocca, P., Valerio, F., Evidente, A., Lazzaroni, S., Corsetti, A., and Gobbetti,<br />
M., 2000, Purification and characterization of novel antifungal compounds from<br />
the sourdough Lactobacillus plantarum Stra<strong>in</strong> 21B, Appl. Environ. Microbiol.<br />
66:4084-4090.<br />
Lavermicocca, P., Valerio, F., and Visconti, A., 2003, Antifungal activity of phenyllactic<br />
acid aga<strong>in</strong>st molds isolated from bakery products, Appl. Environ. Microbiol.<br />
69:634-640.<br />
Niku-Paavola, M.–L., Laitila, A., Mattila-Sandholm, T., and Haikara, A., 1999, New<br />
types of antimicrobial compounds produced by Lactobacillus plantarum, J. Appl.<br />
Microbiol. 86:29-35.<br />
Plockova, M., Stiles, J., and Chumchalova, J., 2000, Evaluation of antifungal activity<br />
of lactic acid bacteria by the milk agar plate method, Czech Dairy J. 62:19-19.<br />
Plockova, M., Stiles, J., Chumchalova, J., and Halfarova, R., 2001, Control of mould<br />
growth by Lactobacillus rhamnosus VT1 and Lactobacilus reuteri CCM 3625 on<br />
milk agar plates, Czech J. <strong>Food</strong> Sci. 19:46-50.<br />
Rocken, W., 1996, Applied aspects of sourdough fermentation, Adv. <strong>Food</strong> Sci. 18:<br />
212-216.<br />
Sand<strong>in</strong>e, W. E., 1996, Commercial production of dairy starter cultures, <strong>in</strong>: Dairy<br />
Starter Cultures, T. M. Cogan and J.-P. Accolas, eds., VCH Publishers, New York,<br />
pp. 191-206.<br />
Spicher, G., 1983, Baked goods, <strong>in</strong>: Biotechnology, Vol. 5: <strong>Food</strong> and Feed Production<br />
with Microorganisms, G. Reed, ed., Verlag Chemie, We<strong>in</strong>heim, Germany.<br />
Stiles, J., Plockova, M., Toth, V., and Chumchalova, J., 1999, Inhibition of Fusarium<br />
sp. DMF 0101 by Lactobacillus stra<strong>in</strong>s grown <strong>in</strong> MRS and Elliker broths, Adv.<br />
<strong>Food</strong> Sci. 21:117-121.<br />
Stiles, J., Penkar, S., Plockova, M., Chumchalova, J., and Bullerman, L. B., 2002,<br />
Antifungal activity of sodium acetate, a component of MRS medium, J. <strong>Food</strong><br />
Prot. 65:1188-1191.
PREVENTION OF OCHRATOXIN A IN<br />
CEREALS IN EUROPE<br />
Monica Olsen 1 , Nils Jonsson 2 , Naresh Magan 3 , John<br />
Banks 4 , Corrado Fanelli 5 , Aldo Rizzo 6 , Auli Haikara 7 , Alan<br />
Dobson 8 , Jens Frisvad 9 , Stephen Holmes 10 , Juhani Olkku 11 ,<br />
Sven-Johan Persson 12 and Thomas Börjesson 13<br />
1. INTRODUCTION<br />
This paper describes objectives and activities of a major European<br />
Community project (OTA PREV) aimed at understand<strong>in</strong>g sources of<br />
contam<strong>in</strong>ation of ochratox<strong>in</strong> A <strong>in</strong> European cereals and related foodstuffs,<br />
and the development of strategies to m<strong>in</strong>imise ochratox<strong>in</strong> A <strong>in</strong><br />
the food supply. The project ran from February 2000 to July 2003.<br />
1 National <strong>Food</strong> Adm<strong>in</strong>istration, PO Box 622, SE-751 26 Uppsala, Sweden<br />
2 Swedish Institute of Agricultural and Environmental Eng<strong>in</strong>eer<strong>in</strong>g, PO Box 7033,<br />
SE-750 07 Uppsala, Sweden<br />
3 Cranfield Biotechnology Centre, Cranfield University, Barton Road, Silsoe,<br />
Bedfordshire MK45 4DT, UK<br />
4 Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK<br />
5 Laboratorio di Micologia, Univerisità “La Sapienza”, Largo Crist<strong>in</strong>a di Svezia 24,<br />
I-00165 Roma, Italy<br />
6 National Veter<strong>in</strong>ary and <strong>Food</strong> Res. Inst., PO Box 45, FIN-00581, Hels<strong>in</strong>ki, F<strong>in</strong>land<br />
7 VTT Biotechnology, PO Box 1500, FIN-02044 Espoo, F<strong>in</strong>land<br />
8 Microbiology Department, University College Cork, Cork, Ireland<br />
9 BioCentrum-DTU, Build<strong>in</strong>g 221, DK-2800 Kgs. Lyngby, Denmark<br />
10 ADGEN Ltd, Nellies Gate, Auch<strong>in</strong>cruive, Ayr KA6 5HW, UK<br />
11 Oy Panimolaboratorio-Bryggerilaboratorium AB, P.O. Box 16, FIN-02150 Espoo,<br />
F<strong>in</strong>land<br />
12 Akron mask<strong>in</strong>er, SE-531 04 Järpås, Sweden<br />
13 Svenska Lantmännen, Östra hamnen, SE-531 87 Lidköp<strong>in</strong>g, Sweden.<br />
Correspondence to: mool@slv.se<br />
317
318 Monica Olsen et al.<br />
1.1. The Objectives<br />
The over-all objective for the OTA PREV project is the protection<br />
of the consumer’s health by describ<strong>in</strong>g measures for decreas<strong>in</strong>g the<br />
amount of ochratox<strong>in</strong> A <strong>in</strong> cereals produced <strong>in</strong> Europe. This has been<br />
achieved by identify<strong>in</strong>g the key elements <strong>in</strong> an effective HACCP programme<br />
for ochratox<strong>in</strong> A for cereals, and by provid<strong>in</strong>g tools for preventive<br />
and corrective actions. A summary of the tools provided by<br />
this project is presented <strong>in</strong> Table 1. The project <strong>in</strong>cluded 11 work packages<br />
cover<strong>in</strong>g the whole food cha<strong>in</strong> from primary production to the<br />
f<strong>in</strong>al processed food product (Table 2). The objectives and expected<br />
achievements were divided <strong>in</strong>to four tasks, all important steps <strong>in</strong><br />
a HACCP manag<strong>in</strong>g programme for ochratox<strong>in</strong> A <strong>in</strong> cereals:<br />
1. Identification of the critical control po<strong>in</strong>ts (CCP); 2. Establishment<br />
of critical limits for the critical control po<strong>in</strong>ts; 3. Develop<strong>in</strong>g rapid<br />
monitor<strong>in</strong>g methods, and 4. Establishment of corrective actions <strong>in</strong> the<br />
event of deviation of a critical limit. The outcome will serve as a pool<br />
of knowledge for HACCP-based management programmes, which<br />
will <strong>in</strong>crease food safety and support the European cereal <strong>in</strong>dustry.<br />
1.2. Why Ochratox<strong>in</strong> A?<br />
EC legislation and Codex Alimentarius are currently address<strong>in</strong>g<br />
the problem of ochratox<strong>in</strong> A <strong>in</strong> food commodities and raw materials.<br />
Ochratox<strong>in</strong> A can be found <strong>in</strong> cereals, w<strong>in</strong>e, grape juice, dried v<strong>in</strong>e<br />
fruits, coffee, spices, cocoa, and animal derived products such as pork<br />
products. The current EC legislation <strong>in</strong>cludes unprocessed cereals,<br />
cereal products and dried v<strong>in</strong>e fruits (Commission regulation<br />
472/2002) and limits for other commodities are be<strong>in</strong>g discussed,<br />
<strong>in</strong>clud<strong>in</strong>g baby food. JECFA (the FAO/WHO Jo<strong>in</strong>t Expert Committee<br />
on <strong>Food</strong> Additives) evaluated ochratox<strong>in</strong> A at its 56 th meet<strong>in</strong>g <strong>in</strong> 2001<br />
(JECFA, 2001). Ochratox<strong>in</strong> A is nephrotoxic <strong>in</strong> all tested animal<br />
species and may cause renal carc<strong>in</strong>ogenicity, but the mechanism of<br />
action is still be<strong>in</strong>g debated. Both genotoxic and non-genotoxic mechanisms<br />
have been proposed.<br />
JECFA reta<strong>in</strong>ed the previously established provisional tolerable<br />
weekly <strong>in</strong>take (PTWI) at 100 ng/kg bodyweight (b.w.), correspond<strong>in</strong>g<br />
to approximately 14 ng/kg b.w. per day. Estimates of tolerable daily<br />
<strong>in</strong>take for ochratox<strong>in</strong> A, based on non-threshold mathematical modell<strong>in</strong>g<br />
approaches or a safety factor/threshold approach, have ranged<br />
from 1.2 to 14 ng/kg b.w. per day. The Scientific Committee for <strong>Food</strong><br />
of the European Commission (SCF, 1998) considered that “it would
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 319<br />
Table 1. Summary of tools to prevent ochratox<strong>in</strong> A <strong>in</strong> the cereal production cha<strong>in</strong> as provided by the EC project known as OTA PREVa Control<br />
Site type Tools provided Comments (possible % reduction of OTA)<br />
Harvest GAP Recommendation: Keep mach<strong>in</strong>ery and areas <strong>in</strong> (% prevention not possible to estimate, but<br />
contact with the harvested gra<strong>in</strong>, clean. Remove old significant)<br />
gra<strong>in</strong> and dust. (WP1)<br />
Buffer storage CCP Mathematical model which can predict safe storage (up to 100 % prevention possible)<br />
before dry<strong>in</strong>g time (critical limits). (WP4) Monitor<strong>in</strong>g tools: LFDs and ELISAs.<br />
and dur<strong>in</strong>g Rapid monitor<strong>in</strong>g methods for OTA and produc<strong>in</strong>g<br />
dry<strong>in</strong>g (<strong>in</strong> fungi. (WP8)<br />
near-ambient Data on environmental conditions conducive to (% prevention not possible to estimate, but useful<br />
dryers) growth and OTA production. (WP3) tools <strong>in</strong> DSS)<br />
Storage GSP/ Recommendations on silo design and (% prevention not possible to estimate,<br />
CCP ma<strong>in</strong>tenance. (WP5) but significant)<br />
Critical limits for remoisten<strong>in</strong>g. (WP5) (up to 100 % prevention possible)<br />
<strong>Food</strong> grade antioxidants and natural control (>80 % prevention but not yet economically<br />
measures to prevent OTA formation <strong>in</strong> wet gra<strong>in</strong>. feasible)<br />
(WP 6)<br />
Intake at CCP Rapid monitor<strong>in</strong>g methods for OTA * and OTA LFD (with reader for ochratox<strong>in</strong> A) and ELISA<br />
cereal produc<strong>in</strong>g fungi <strong>in</strong> gra<strong>in</strong>. (WP8)<br />
process<strong>in</strong>g Critical limit: less than 1000 cfu/g P. verrucosum Indicat<strong>in</strong>g risk of OTA levels above 5 µg/kg<br />
<strong>in</strong>dustry <strong>in</strong> wheat. (WP4)<br />
Monitor<strong>in</strong>g method for P. verrucosum. (WP1, Monitor<strong>in</strong>g tools: DYSG, LFD, ELISA, and PCR<br />
WP8, and WP9)<br />
Mill<strong>in</strong>g <strong>in</strong>dustry GMP Reductive measures dur<strong>in</strong>g mill<strong>in</strong>g. (WP10) (clean<strong>in</strong>g 2-3%, scour<strong>in</strong>g 3-44%, mill<strong>in</strong>g up to 60%)<br />
Cont<strong>in</strong>ued
320 Monica Olsen et al.<br />
Table 1. Summary of tools to prevent ochratox<strong>in</strong> A <strong>in</strong> the cereal production cha<strong>in</strong> as provided by the EC project known as OTA PREVa— cont’d<br />
Control<br />
Site type Tools provided Comments (possible % reduction of OTA)<br />
Cereal process<strong>in</strong>g GMP Reductive measures dur<strong>in</strong>g extrusion and bak<strong>in</strong>g. (bak<strong>in</strong>g up to 5-10%, extrusion up to 40%)<br />
<strong>in</strong>dustry (WP10)<br />
Intake at malt<strong>in</strong>g CCP Critical limits:
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 321<br />
Table 2. Work packages <strong>in</strong>cluded <strong>in</strong> the OTA PREV project<br />
WP no. Work Package Title<br />
1 Which are the ochratox<strong>in</strong> A produc<strong>in</strong>g fungi and what are the sources<br />
of <strong>in</strong>oculum<br />
2 Differences depend<strong>in</strong>g on various cereal species, farm<strong>in</strong>g methods<br />
and climate.<br />
3 The effect of temporal environmental factors on fungal growth,<br />
patterns of colonisation and ochratox<strong>in</strong> production<br />
4 Modell<strong>in</strong>g the growth of Penicillium verrucosum <strong>in</strong> cereal gra<strong>in</strong> dur<strong>in</strong>g<br />
aerobic conditions<br />
5 Gra<strong>in</strong> silos – hygienic design<br />
6 Control of ochratox<strong>in</strong> production <strong>in</strong> cereals us<strong>in</strong>g food grade<br />
antioxidants and natural control measures to prevent entry <strong>in</strong>to the<br />
food cha<strong>in</strong><br />
7 Development of rapid biosensor for ochratox<strong>in</strong> detection us<strong>in</strong>g<br />
molecular impr<strong>in</strong>ted polymers<br />
8 Development of rapid ELISA system for ochratox<strong>in</strong> produc<strong>in</strong>g fungi<br />
9 Molecular characterisation of the ochratox<strong>in</strong> biosynthetic genes <strong>in</strong> the<br />
mycotoxigenic fungi Aspergillus ochraceus and Penicillium verrucosum<br />
10 Establish<strong>in</strong>g corrective actions dur<strong>in</strong>g mill<strong>in</strong>g and process<strong>in</strong>g<br />
11 Establish<strong>in</strong>g corrective actions dur<strong>in</strong>g malt<strong>in</strong>g and brew<strong>in</strong>g<br />
be prudent to reduce exposure to ochratox<strong>in</strong> A as much as possible,<br />
ensur<strong>in</strong>g that exposures are towards the lower end of the range of tolerable<br />
daily <strong>in</strong>takes of 1.2-14 ng/kg b.w. per day which have been estimated<br />
by other bodies, e.g. below 5 ng/kg b.w. per day”. In the most<br />
recent assessment of ochratox<strong>in</strong> A <strong>in</strong>take by European consumers<br />
(SCOOP, 2002) and <strong>in</strong> earlier <strong>in</strong>vestigations, cereals have been found<br />
to be the most important dietary source of ochratox<strong>in</strong> A, contribut<strong>in</strong>g<br />
from 50 to 80% of the <strong>in</strong>take. Consequently, prevention of ochratox<strong>in</strong><br />
A formation by specific moulds <strong>in</strong> cereals would have a significant<br />
impact on the consumer <strong>in</strong>take of ochratox<strong>in</strong> A <strong>in</strong> Europe.<br />
Maximum permissible levels of 5 µg/kg <strong>in</strong> raw cereals and 3 µg/kg<br />
<strong>in</strong> cereal products have been set for ochratox<strong>in</strong> A with<strong>in</strong> the EC.<br />
Surveys of stored cereals <strong>in</strong> Europe over many years (e.g. Olsen et al.<br />
1993; Scudamore et al., 1999; Wolff, 2000; Puntaric et al., 2001) show<br />
that samples exam<strong>in</strong>ed sometimes exceed this level. Even if the percentage<br />
of samples contam<strong>in</strong>ated at the statutory limits with<strong>in</strong> the<br />
Community were as low as 3% this would represent a large tonnage of<br />
gra<strong>in</strong> (approximately 6 million tonnes) and a potential serious economic<br />
loss. In monetary terms this would equate to a loss of 800-1000<br />
million euros assum<strong>in</strong>g that no alternative use for the gra<strong>in</strong> was available.<br />
In addition, the high cost of monitor<strong>in</strong>g programmes (roughly<br />
0.3 and 100 million euros for official and <strong>in</strong>ternal control, respectively)
322 Monica Olsen et al.<br />
for prevent<strong>in</strong>g contam<strong>in</strong>ated gra<strong>in</strong> enter<strong>in</strong>g the food cha<strong>in</strong> must also<br />
be considered.<br />
The food and brew<strong>in</strong>g <strong>in</strong>dustries are <strong>in</strong>creas<strong>in</strong>gly demand<strong>in</strong>g high<br />
quality cereals for food and dr<strong>in</strong>k products and require gra<strong>in</strong> at least<br />
conform<strong>in</strong>g to the statutory limits set for ochratox<strong>in</strong> A. Thus there is<br />
a major <strong>in</strong>centive for European cereal <strong>in</strong>dustry to m<strong>in</strong>imise ochratox<strong>in</strong><br />
A (and other mycotox<strong>in</strong>s) <strong>in</strong> gra<strong>in</strong> to enable it to rema<strong>in</strong> competitive<br />
worldwide and to reduce consumer risk as far as possible. Clearly,<br />
there is an urgent need to understand the factors that encourage<br />
mycotox<strong>in</strong> formation both pre and post harvest as this will assist <strong>in</strong><br />
develop<strong>in</strong>g strategies to m<strong>in</strong>imise mycotox<strong>in</strong> formation.<br />
Application of HACCP-like assessment of the cereal food cha<strong>in</strong> supports<br />
the established knowledge that dry<strong>in</strong>g gra<strong>in</strong> quickly at harvest is<br />
the most crucial factor <strong>in</strong> avoid<strong>in</strong>g mould and mycotox<strong>in</strong> formation<br />
dur<strong>in</strong>g subsequent storage. Thus, <strong>in</strong>stigation of a suitable monitor<strong>in</strong>g<br />
check system to ensure that gra<strong>in</strong> is dried rapidly to a safe moisture<br />
content, together with regular <strong>in</strong>spection and monitor<strong>in</strong>g of gra<strong>in</strong><br />
moisture, should elim<strong>in</strong>ate the risk of ochratox<strong>in</strong> A formation dur<strong>in</strong>g<br />
storage. In addition, compliance with Good Agricultural Practice<br />
(GAP) will assist <strong>in</strong> the production of good quality gra<strong>in</strong>. However, the<br />
cont<strong>in</strong>ued occurrence of ochratox<strong>in</strong> A <strong>in</strong> gra<strong>in</strong> shown by surveys suggests<br />
that either good practice is not or cannot always be fully followed<br />
or that all the factors <strong>in</strong>volved <strong>in</strong> the formation of ochratox<strong>in</strong> A are not<br />
completely understood. In other words, if gra<strong>in</strong> cannot always be dried<br />
quickly enough it becomes important that all of the factors that affect<br />
the potential for formation of ochratox<strong>in</strong> A are fully understood. Only<br />
by obta<strong>in</strong><strong>in</strong>g this <strong>in</strong>formation can sound and effective advice be made<br />
available <strong>in</strong> the field on how to m<strong>in</strong>imise this risk.<br />
One of the tasks with<strong>in</strong> this project (WP2; see Table 2), aims to<br />
establish how cereal production, harvest<strong>in</strong>g and storage procedures<br />
vary across the EC by compil<strong>in</strong>g a small data base from the <strong>in</strong>formation<br />
provided <strong>in</strong> a questionnaire sent to cereal experts. The cooperation<br />
of ‘grass root’ experts means that the <strong>in</strong>formation is their view<br />
and not necessarily an official or government view, especially where<br />
answers required to a particular question may be sensitive or subjective,<br />
e.g. ‘is ochratox<strong>in</strong> A considered a problem <strong>in</strong> your Country?’ The<br />
data and this appraisal are <strong>in</strong>tended to complement the results from<br />
the scientific studies of with<strong>in</strong> this project.<br />
1.3. Fungi Produc<strong>in</strong>g Ochratox<strong>in</strong> A<br />
Penicillium verrucosum has been found to be the ochratox<strong>in</strong> A<br />
producer <strong>in</strong> several national <strong>in</strong>vestigations of cereal gra<strong>in</strong> <strong>in</strong> Europe.
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 323<br />
Frisvad and Viuf (1986) <strong>in</strong>vestigated 70 samples of barley from<br />
Denmark conta<strong>in</strong><strong>in</strong>g up to 7400 µg/kg ochratox<strong>in</strong> A and all ochratox<strong>in</strong><br />
A producers from these samples were identified as Penicillium<br />
viridicatum II as def<strong>in</strong>ed by Ciegler et al. (1973). Taxonomists subsequently<br />
concluded that all known ochratox<strong>in</strong> A producers <strong>in</strong><br />
Penicillium belonged to P. verrucosum (Frisvad 1985; Pitt 1987;<br />
Frisvad 1989). Larsen et al. (2001) found that P. viridicatum III<br />
(Ciegler et al., 1973) was a dist<strong>in</strong>ct species, which was named<br />
Penicillium nordicum, and that it should be separated from P. verrucosum.<br />
P. nordicum was found to be associated with meat and cheese<br />
products while P. verrucosum isolates are usually derived from plants<br />
(Ciegler et al., 1973; Dragoni and Cantoni, 1979; Larsen et al.,<br />
2001).<br />
Several Aspergillus species and Aspergillus teleomorphs have been<br />
reported to produce ochratox<strong>in</strong> A <strong>in</strong>clud<strong>in</strong>g A. ochraceus, A. sulphureus,<br />
A. niger, A. carbonarius, Neopetromyces muricatus and<br />
Petromyces alliaceus (Frisvad and Samson, 2000). However, ochratox<strong>in</strong><br />
A produc<strong>in</strong>g isolates of Aspergillus species have never been<br />
found on European cereals. One of the objectives with this study was<br />
to <strong>in</strong>vestigate the occurrence of all potential ochratox<strong>in</strong> A producers<br />
<strong>in</strong> cereals produced <strong>in</strong> Europe (WP1).<br />
Dichloran Rose Bengal Yeast Extract Sucrose agar (DRYES,<br />
Frisvad et al., 1992) is recommended for the enumeration of P. verrucosum<br />
<strong>in</strong> food (NMKL Method no. 152, 1995), however, Dichloran<br />
Yeast extract Sucrose 18% Glycerol agar (DYSG, Frisvad et al., 1992)<br />
may be a more selective and better diagnostic medium. The efficiency<br />
of DYSG needs to be compared with DRYES and with DG18<br />
(Hock<strong>in</strong>g and Pitt, 1980), which is a general purpose medium suitable<br />
for detect<strong>in</strong>g P. verrucosum. The objective of the work, presented <strong>in</strong><br />
this report, was to exam<strong>in</strong>e whether the numbers of P. verrucosum,<br />
recovered on the different media are statistically different, by <strong>in</strong>vestigat<strong>in</strong>g<br />
a large number of cereal samples conta<strong>in</strong><strong>in</strong>g ochratox<strong>in</strong> A. In<br />
addition, the three different media were validated <strong>in</strong> a collaborative<br />
study (WP1).<br />
To be able to f<strong>in</strong>d contam<strong>in</strong>ation sources and critical control po<strong>in</strong>ts,<br />
new molecular f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g methods, such as AFLP (Vos et al.,<br />
1995; Jannsen et al., 1996), have been used with success <strong>in</strong> several studies<br />
of fungal populations and clones of different fungi (Majer et al.,<br />
1996; Arenal et al., 1999; Bakkeren et al., 2000; Tooley et al., 2000;<br />
Kothera, 2003; Kure et al., 2003; Lund et al., 2003; Schmidt et al.,<br />
2003). The AFLP method has been considered superior to RAPD f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g<br />
(Lund and Skouboe, 1998; Bakkeren et al., 2000). The aim<br />
of one study with<strong>in</strong> this project was to exam<strong>in</strong>e whether the AFLP
324 Monica Olsen et al.<br />
f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g method could be used to f<strong>in</strong>d critical control po<strong>in</strong>ts <strong>in</strong><br />
the process from soil to table <strong>in</strong> order to prevent or m<strong>in</strong>imize ochratox<strong>in</strong><br />
A formation (WP1).<br />
1.4. Critical Limits for Fungal Growth and Production of<br />
Ochratox<strong>in</strong> A<br />
Fungal growth and ochratox<strong>in</strong> A production dur<strong>in</strong>g gra<strong>in</strong> storage<br />
are <strong>in</strong>fluenced by a wide variety of complex <strong>in</strong>teractions between abiotic<br />
and biotic factors. Water availability and temperature are the two<br />
most important abiotic factors. They <strong>in</strong>teract to determ<strong>in</strong>e the range<br />
of microorganisms that can colonise a given substrate, their growth<br />
and contribution to spontaneous heat<strong>in</strong>g.<br />
Dur<strong>in</strong>g respiration of damp gra<strong>in</strong>, oxygen is utilized and carbon<br />
dioxide is produced. Concentrations of oxygen and carbon dioxide <strong>in</strong><br />
the <strong>in</strong>ter-granular atmosphere are important <strong>in</strong> determ<strong>in</strong><strong>in</strong>g the pattern<br />
of fungal colonization of gra<strong>in</strong> dur<strong>in</strong>g storage. As most moulds<br />
<strong>in</strong>clud<strong>in</strong>g P. verrucosum and A. ochraceus are considered obligate aerobes,<br />
<strong>in</strong>creased levels of CO 2 should <strong>in</strong>hibit their growth. However,<br />
relatively few studies have been carried out regard<strong>in</strong>g the relationship<br />
between various carbon dioxide levels, water activity (a w ) and temperatures<br />
on fungal growth, particularly P. verrucosum and A. ochraceus,<br />
the ma<strong>in</strong> ochratox<strong>in</strong> A producers dur<strong>in</strong>g wheat storage.<br />
Chang<strong>in</strong>g either temperature or a w affects growth and may affect the<br />
ability of species to compete (Magan and Lacey, 1985a,b) which could<br />
effect ochratox<strong>in</strong> A production (Ramakrishna et al., 1996). A range of<br />
<strong>in</strong>teractions can occur between species, which can be scored to compare<br />
competitiveness of different species <strong>in</strong> various culture conditions<br />
(Magan and Lacey, 1984). On maize gra<strong>in</strong>, <strong>in</strong>teractions and competition<br />
have been shown to have a marked <strong>in</strong>fluence on ochratox<strong>in</strong> A production<br />
by A. ochraceus (Ramakrishna et al., 1993, 1996; Mar<strong>in</strong> et al.,<br />
1998; Lee and Magan, 2000). There have been no previous studies to<br />
exam<strong>in</strong>e the competitiveness of P. verrucosum aga<strong>in</strong>st other fungi on<br />
wheat gra<strong>in</strong> and the <strong>in</strong>fluence these <strong>in</strong>teractions may have on ochratox<strong>in</strong><br />
A production. It has been suggested that the co-existence of<br />
microorganisms may be mediated via nutritional resources partition<strong>in</strong>g.<br />
Wilson and L<strong>in</strong>dow (1994a,b) determ<strong>in</strong>ed niche overlap <strong>in</strong>dices<br />
(NOI) for epiphytic bacteria and the level of ecological similarity for<br />
isolat<strong>in</strong>g effective bacterial control agents. Niche overlap values<br />
greater than 0.9 have been suggested to <strong>in</strong>dicate co-existence between<br />
species <strong>in</strong> an ecological niche, whereas scores less than 0.9 <strong>in</strong>dicate<br />
occupation of separate niches.
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 325<br />
In this project the effect of a w and temperature was exam<strong>in</strong>ed on 1)<br />
growth and ochratox<strong>in</strong> A production by Penicillium verrucosum over<br />
time on irradiated wheat gra<strong>in</strong> 2) growth and ochratox<strong>in</strong> A production<br />
at various gas compositions and 3) <strong>in</strong> vitro <strong>in</strong>teractions on growth and<br />
tox<strong>in</strong> production between an ochratox<strong>in</strong> A produc<strong>in</strong>g isolate of P. verrucosum<br />
and other wheat spoilage fungi. The effect of environmental<br />
factors on <strong>in</strong> vitro carbon source utilisation patterns and NOIs for the<br />
ochratox<strong>in</strong> A produc<strong>in</strong>g stra<strong>in</strong> of P. verrucosum <strong>in</strong> relation to all<br />
species was determ<strong>in</strong>ed (WP3).<br />
The m<strong>in</strong>imum moisture content (m.c.) <strong>in</strong> wheat that allows growth of<br />
P. verrucosum is about 16-17%, or approximately 0.80 a w (Northolt<br />
et al., 1979). To produce ochratox<strong>in</strong> A, the fungus probably needs about<br />
1% higher m.c. A study of the gra<strong>in</strong> quality on farms <strong>in</strong>dicates that the<br />
occurrence of ochratox<strong>in</strong> A <strong>in</strong> gra<strong>in</strong> is attributed to <strong>in</strong>sufficient dry<strong>in</strong>g<br />
or to excessively long pre-dry<strong>in</strong>g storage (Jonsson and Pettersson, 1992).<br />
Studies on the safe storage period for cereal gra<strong>in</strong>s are few, and are<br />
based on visible mould<strong>in</strong>g (Kreyger, 1972), dry-matter loss (Steel et al.,<br />
1969; White et al., 1982) or loss of seed germ<strong>in</strong>ation (Kreyger, 1972;<br />
White et al., 1982). For maize, maximum allowable storage time has<br />
been estimated based on the time before dry matter loss exceeds 0.5%<br />
(Steel et al., 1969). This loss is estimated to correspond to the loss of one<br />
US grade, which is based on visible <strong>in</strong>spection. Visible mould may be an<br />
unreliable criterion, because considerable losses can occur before<br />
mould<strong>in</strong>g is visible, depend<strong>in</strong>g on whether or not the conditions favour<br />
fungal growth and sporulation (Seitz et al., 1982).<br />
Measurement of respiration is a widely used method for estimat<strong>in</strong>g<br />
fungal growth, biomass and dry-matter losses. In soil the rate of CO 2<br />
production has been used to estimate total liv<strong>in</strong>g microbial biomass<br />
(Anderson and Domsch, 1975). CO 2 production was highly correlated<br />
with ergosterol (r = 0.98) content when Eurotium repens colonised<br />
maize (Mart<strong>in</strong> et al., 1989).<br />
The aim of one study <strong>in</strong> this project was to develop a mathematical<br />
model, which describes the effect of a w and temperature on safe storage<br />
time before obvious growth of P. verrucosum and formation of<br />
ochratox<strong>in</strong> A <strong>in</strong> cereal gra<strong>in</strong>. Data from a respirometer on CO 2 production<br />
dur<strong>in</strong>g the storage was compared with data on the growth of<br />
P. verrucosum and production of ochratox<strong>in</strong> A (WP4).<br />
1.5. Prevention of Ochratox<strong>in</strong> A Formation<br />
After cereal gra<strong>in</strong> is dried and cooled <strong>in</strong> a high temperature drier<br />
and placed <strong>in</strong>to storage at 13-14% moisture content, its temperature is
326 Monica Olsen et al.<br />
frequently well above the average ambient temperature <strong>in</strong> temperate<br />
climates (Brooker et al., 1992). Because dry gra<strong>in</strong> is an effective thermal<br />
<strong>in</strong>sulator, the periphery of the bulk changes temperature faster<br />
than the less exposed gra<strong>in</strong> <strong>in</strong> the centre. This is especially valid for<br />
gra<strong>in</strong> masses over 50 tons, which fail to cool or warm uniformly dur<strong>in</strong>g<br />
seasonal thermal changes (Foster and Tuite, 1992). The result<strong>in</strong>g<br />
temperature gradient causes moisture to move from warmer to colder<br />
parts of the gra<strong>in</strong> bulk. Moisture migration is particularly a problem<br />
<strong>in</strong> gra<strong>in</strong> with larger size, such as maize, stored <strong>in</strong> un<strong>in</strong>sulated outdoor<br />
steel silos <strong>in</strong> areas with large seasonal changes <strong>in</strong> air temperature.<br />
Factors responsible for heat and mass transfer dur<strong>in</strong>g storage without<br />
aeration are conduction, diffusion and natural convection.<br />
However, depend<strong>in</strong>g on the geometry of the storage structure, material<br />
properties of the gra<strong>in</strong> bulk and load<strong>in</strong>g practices, the effect of<br />
natural convection may be negligible (Smith and Sokhansanj, 1990;<br />
Maier and Montross, 1998). Small gra<strong>in</strong>, such as wheat, offers more<br />
resistance to air movement with<strong>in</strong> the gra<strong>in</strong> mass and is usually stored<br />
at a lower moisture content than maize (Foster and Tuite, 1992). It has<br />
been shown that mass transfer is not significant <strong>in</strong> wheat stored without<br />
aeration dur<strong>in</strong>g the summer <strong>in</strong> North Dakota, with a maximum<br />
<strong>in</strong>crease of the moisture content of 0.45 % just below the surface.<br />
(Hellevang and Hirn<strong>in</strong>g, 1988). Similar moisture <strong>in</strong>creases were measured<br />
<strong>in</strong> simulated tests <strong>in</strong> the laboratory. However, if the plenum open<strong>in</strong>gs<br />
are not fully sealed, w<strong>in</strong>d-<strong>in</strong>duced air currents may <strong>in</strong>crease the<br />
heat and mass transfer (Montross et al., 2002).<br />
Thermal properties of the construction material determ<strong>in</strong>e the<br />
extent and frequency of fluctuations of silo temperature, and research<br />
has shown that approximately 90 % of the environmental temperature<br />
changes were transmitted to the silo gas space with amplification of<br />
2.5-3.0 times <strong>in</strong> an un<strong>in</strong>sulated steel silo (Meier<strong>in</strong>g, 1986). This amplification<br />
is markedly reduced when the steel silo is placed <strong>in</strong>doors protected<br />
from direct solar radiation. A concrete silo does not follow the<br />
daily fluctuations of solar radiation and environmental air temperature<br />
due to its approximately 20 times higher resistance to heat flow,<br />
lower thermal conductivity and larger thermal <strong>in</strong>ertia.<br />
For smaller amounts of gra<strong>in</strong> stored <strong>in</strong> un<strong>in</strong>sulated steel silos outdoors,<br />
the headspace may be the most critical factor due to large diurnal<br />
temperature variations, caus<strong>in</strong>g water to condense on to the cold<br />
gra<strong>in</strong> (Bailey, 1992) and also on the <strong>in</strong>side of the roof, dripp<strong>in</strong>g back<br />
<strong>in</strong>to the gra<strong>in</strong>. The spoilage of gra<strong>in</strong> close to the headspace may go<br />
undetected because of mix<strong>in</strong>g that takes place when the silo is<br />
unloaded (Lund<strong>in</strong>, 1998). Un<strong>in</strong>sulated steel silos have become
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 327<br />
common for outdoor storage of dried cereal gra<strong>in</strong> <strong>in</strong> Europe because<br />
they require lower <strong>in</strong>vestment costs compared with those placed<br />
<strong>in</strong>doors, or re<strong>in</strong>forced concrete silos.<br />
Numerous <strong>in</strong>vestigators have developed mathematical models to<br />
describe the heat and mass transfer <strong>in</strong> stored bulk gra<strong>in</strong> although<br />
ma<strong>in</strong>ly for maize. Most models have only considered heat transfer<br />
dur<strong>in</strong>g storage but there are more comprehensive models which also<br />
<strong>in</strong>clude mass transfer (Metzger and Muir, 1983; Tanaka and Yoshida,<br />
1984; Smith and Sokhansanj, 1990; Maier, 1992; Casada and Young,<br />
1994; Chang et al., 1994; Khankari et al., 1995). Most numerical models<br />
assume overly simplistic boundary conditions and do not accurately<br />
model the headspace and plenum conditions dur<strong>in</strong>g storage<br />
(Maier and Montross, 1998). In a validation of a comprehensive<br />
model us<strong>in</strong>g realistic boundary conditions, it was concluded that the<br />
available literature values for a number of critical variables used to<br />
model headspace and plenum conditions proved to be <strong>in</strong>accurate and<br />
needed more research (Montross et al., 2002).<br />
The <strong>in</strong>fluence of different systems for air exchange between the<br />
headspace and the ambient air, on the temperature and relative<br />
humidity <strong>in</strong> the gra<strong>in</strong> close to the headspace were studied <strong>in</strong> this project.<br />
A mathematical model of dew po<strong>in</strong>t temperature and collected<br />
data were used to describe the risk of moisture condensation <strong>in</strong> the<br />
headspace of the silos dur<strong>in</strong>g the storage. Preventive strategies to<br />
avoid rehydration of the gra<strong>in</strong> and the risk for mould growth and<br />
production of ochratox<strong>in</strong> A were also suggested (WP5).<br />
There are two ma<strong>in</strong> strategies for reduc<strong>in</strong>g the occurrence of mycotox<strong>in</strong>s<br />
<strong>in</strong> cereals: prevention of contam<strong>in</strong>ation or detoxification of<br />
mycotox<strong>in</strong>s already present. One of the approaches <strong>in</strong> this project is<br />
based on the preventive strategy dur<strong>in</strong>g post harvest storage. The use<br />
of gra<strong>in</strong> protectant chemicals has been widely applied dur<strong>in</strong>g the last<br />
few years. This has achieved good results <strong>in</strong> the short-term, but the<br />
widespread use of chemical compounds has resulted <strong>in</strong> resistance <strong>in</strong><br />
the target organisms.<br />
Storage fungi are commonly controlled us<strong>in</strong>g preservatives such as<br />
organic acids, however the use of preservatives has met with <strong>in</strong>creas<strong>in</strong>g<br />
consumer resistance <strong>in</strong> recent years (Foeged<strong>in</strong>g and Busta, 1991;<br />
Basilico and Basilico, 1999). In the past decade research has focused<br />
on the use of natural preservatives (Foeged<strong>in</strong>g and Busta, 1991),<br />
which are perceived as rais<strong>in</strong>g few concerns among consumers, regulatory<br />
agencies or with<strong>in</strong> the food <strong>in</strong>dustry (Dillon and Board, 1994;<br />
Nychas, 1995; Lopez-Malo et al., 1997; Basilico and Basilico, 1999;<br />
Etcheverry et al., 2002).
328 Monica Olsen et al.<br />
Essential oils are volatile products of plant secondary metabolism<br />
which <strong>in</strong> many cases are biologically active, with antimicrobial,<br />
antioxidant and bioregulatory properties (French, 1985; Caccioni<br />
et al., 1998). Essential oils of oregano, thyme, basil, garlic, onion and<br />
c<strong>in</strong>namon have been reported hav<strong>in</strong>g the greatest antimicrobial effectiveness<br />
(Paster et al., 1995; Basilico and Basilico, 1999; Cosent<strong>in</strong>o<br />
et al., 1999; Y<strong>in</strong> and Tsao, 1999). More specifically, cloves and c<strong>in</strong>namon<br />
have been found to be strong antifungal agents aga<strong>in</strong>st<br />
Penicillium and Aspergillus species. Many of the essential oils that<br />
have been tested have been shown to have an antagonistic effect<br />
aga<strong>in</strong>st aflatoxigenic Aspergillus spp. However, few studies have <strong>in</strong>vestigated<br />
the effects of essential oils on growth and ochratox<strong>in</strong> A production<br />
by ochratoxigenic stra<strong>in</strong>s of A. ochraceus (Basilico and<br />
Basilico, 1999). Furthermore, many studies have not taken account<br />
of different environmental factors such as temperature and a w on<br />
the effectiveness of essential oils <strong>in</strong> control of fungal growth and<br />
ochratox<strong>in</strong> A production.<br />
Resveratrol is a polyphenolic compound, which exhibits antimicrobial<br />
and antioxidant properties. It is a phytoalex<strong>in</strong> <strong>in</strong> grapes<br />
and is produced <strong>in</strong> response to various k<strong>in</strong>ds of stress <strong>in</strong>clud<strong>in</strong>g<br />
attack by fungal pathogens. Resveratrol also has anticancer<br />
activity (Pervaiz, 2001) and antimicrobial activity aga<strong>in</strong>st some<br />
dermatophytes (Man-Y<strong>in</strong>g, 2002). The effect of resveratrol <strong>in</strong> comb<strong>in</strong>ation<br />
with environmental variables on growth and ochratox<strong>in</strong> A<br />
production by P. verrucosum and A. ochraceus has not been<br />
<strong>in</strong>vestigated.<br />
Lactic acid bacteria have been used as biocontrol agents <strong>in</strong> malt<strong>in</strong>g<br />
trials (Haikara et al., 1993). Lactobacillus plantarum (VTT E-78076)<br />
was reported to have a fungistatic effect aga<strong>in</strong>st Fusarium species<br />
<strong>in</strong> vitro and <strong>in</strong> laboratory-scale malt<strong>in</strong>g (Laitila et al., 1997, 2002).<br />
One of the objectives <strong>in</strong> the present <strong>in</strong>vestigation was to determ<strong>in</strong>e<br />
the effect of a range of food-grade essential oils, antioxidants and<br />
resveratrol under different <strong>in</strong>teract<strong>in</strong>g a w and temperature regimens on<br />
(a) growth rate, (b) ochratox<strong>in</strong> A production by P. verrucosum and<br />
A. ochraceus isolates from wheat gra<strong>in</strong> and (c) the concentration of<br />
essential oil needed for control of P. verrucosum. In addition, for barley<br />
and malt<strong>in</strong>g, the aims were to first <strong>in</strong>vestigate the effectiveness of<br />
lactic acid bacteria aga<strong>in</strong>st the growth of P. verrucosum and subsequent<br />
production of ochratox<strong>in</strong> A <strong>in</strong> vitro and <strong>in</strong> a m<strong>in</strong>i-scale storage<br />
experiment. A pilot-scale experiment was also carried out to study<br />
mould growth and ochratox<strong>in</strong> A formation dur<strong>in</strong>g storage of barley at<br />
different moisture levels (WP6).
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 329<br />
1.6. Monitor<strong>in</strong>g Methods<br />
Establishment of regulatory limits for ochratox<strong>in</strong> A <strong>in</strong> Europe has<br />
led to the requirement for rapid monitor<strong>in</strong>g methods for the presence<br />
of ochratox<strong>in</strong> A. Conventional analysis of ochratox<strong>in</strong> A by HPLC is<br />
expensive and does not offer a turn round time that is compatible with<br />
the needs of the <strong>in</strong>dustry. However, rapid immunodiagnostic methods<br />
offer a real alternative.<br />
The concept of us<strong>in</strong>g immunological systems to detect mycotox<strong>in</strong>s<br />
is not new and was first reported by Aalund et al. (1975). Commercial<br />
kits to detect ochratox<strong>in</strong> A were first <strong>in</strong>troduced <strong>in</strong> the late 1980s and<br />
clearly demonstrate that assays can be made user friendly and rapid<br />
(around 20 m<strong>in</strong>utes). At the start of the OTA PREV project, currently<br />
available immunoassays had generally been optimised to meet the<br />
USA requirements of around 20 ppb and at best were only sensitive<br />
down to about 8 ppb, which far exceeds the EU limits
330 Monica Olsen et al.<br />
A has not yet been completely established; however, the isocoumar<strong>in</strong><br />
group is a pentaketide skeleton formed from acetate and malonate via<br />
a polyketide synthesis pathway with the L-phenylalan<strong>in</strong>e be<strong>in</strong>g<br />
derived from the shikimic acid pathway (Moss, 1996, 1998). No <strong>in</strong>formation<br />
currently is available on the enzymes or the genes responsible<br />
for any of these biosynthetic steps. In this project two studies have<br />
been performed with the objectives to clone and characterise genes<br />
<strong>in</strong>volved <strong>in</strong> the biosynthesis of ochratox<strong>in</strong> A and to develop nucleic<br />
acid sequence based methods to detect the presence of A. ochraceus<br />
and P. verrucosum. As ochratox<strong>in</strong> A has a polyketide backbone, a<br />
polyketide synthase gene was targeted (WP9).<br />
1.7. Reductive Measures Dur<strong>in</strong>g Process<strong>in</strong>g<br />
Dry<strong>in</strong>g and subsequently keep<strong>in</strong>g gra<strong>in</strong> under safe storage conditions<br />
should reduce or elim<strong>in</strong>ate the risk of occurrence of ochratox<strong>in</strong><br />
A. However, this has often been found difficult to achieve <strong>in</strong> practice<br />
and surveys cont<strong>in</strong>ue to f<strong>in</strong>d contam<strong>in</strong>ated gra<strong>in</strong> and cereal food<br />
products (e.g. Scudamore et al., 1999; Wolff, 2000). The extent to<br />
which ochratox<strong>in</strong> A is degraded or lost dur<strong>in</strong>g process<strong>in</strong>g and<br />
whether it can be, at least partially, removed dur<strong>in</strong>g the preparation<br />
of cereal products such as bread and breakfast cereals is uncerta<strong>in</strong>.<br />
Relatively few studies have been carried out on the reduction of<br />
ochratox<strong>in</strong> A dur<strong>in</strong>g process<strong>in</strong>g. Cook<strong>in</strong>g of polished wheat us<strong>in</strong>g a<br />
procedure common <strong>in</strong> Egypt only removed 6% of ochratox<strong>in</strong> A (El-<br />
Banna and Scott, 1984). A similar result was shown by Osborne et al.<br />
(1996) when whole wheat (both hard and soft) conta<strong>in</strong><strong>in</strong>g ochratox<strong>in</strong><br />
A at about 60 µg/kg was milled. There was a reduction <strong>in</strong> the ochratox<strong>in</strong><br />
A content <strong>in</strong> the white flour compared with the orig<strong>in</strong>al gra<strong>in</strong>,<br />
although subsequently only a small further reduction occurred when<br />
this was baked <strong>in</strong>to bread. The fate of ochratox<strong>in</strong> A dur<strong>in</strong>g bread<br />
mak<strong>in</strong>g (Subirade, 1996), malt<strong>in</strong>g and brew<strong>in</strong>g (Baxter, 1996), the<br />
effects of process<strong>in</strong>g on the occurrence of ochratox<strong>in</strong> A <strong>in</strong> cereals<br />
(Alldrick, 1996), and animal feed (Scudamore, 1996) and effects of<br />
process<strong>in</strong>g and detoxification treatments on ochratox<strong>in</strong> A (Scott,<br />
1996a) have all been reported.<br />
Ochratox<strong>in</strong> A is relatively stable once formed but, some breakdown<br />
occurs under high temperature, acid or alkal<strong>in</strong>e conditions or <strong>in</strong> the<br />
presence of enzymes. Ochratox<strong>in</strong> A tends to be concentrated <strong>in</strong><br />
the outer bran layers of cereals, so that both reduction and <strong>in</strong>crease <strong>in</strong><br />
concentration can occur depend<strong>in</strong>g on the milled fraction exam<strong>in</strong>ed<br />
(Osborne et al., 1996). However, <strong>in</strong> contrast, studies <strong>in</strong> Poland showed
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 331<br />
that clean<strong>in</strong>g and mill<strong>in</strong>g wheat and barley did not remove ochratox<strong>in</strong><br />
A <strong>in</strong> naturally contam<strong>in</strong>ated samples and levels <strong>in</strong> flour and bran were<br />
the same as <strong>in</strong> the whole gra<strong>in</strong>s (Chelkowski et al., 1981).<br />
Extrusion process<strong>in</strong>g is used for produc<strong>in</strong>g a range of cereal products<br />
such as breakfast foods, snacks and animal feedstuffs.<br />
Commodities such as wholemeal flour are forced through a die under<br />
pressure, and the process causes mechanical shear<strong>in</strong>g stresses, elevated<br />
temperatures and rapid expansions (Riaz, 2000; Guy, 2001). Dur<strong>in</strong>g<br />
this process, chemical bonds <strong>in</strong> polymers may break and free radicals<br />
may form. Therefore, a contam<strong>in</strong>ant such as ochratox<strong>in</strong> A may be<br />
subjected both to high temperatures (up to 200°C) and to chemical<br />
reactions by free radical mechanisms. Information about the destruction<br />
of ochratox<strong>in</strong> A dur<strong>in</strong>g extrusion may assist <strong>in</strong> develop<strong>in</strong>g strategies<br />
for reduc<strong>in</strong>g consumer exposure to this mycotox<strong>in</strong>.<br />
Boudra et al. (1995) studied the decomposition of ochratox<strong>in</strong> A<br />
under different moisture and temperature conditions. In the presence<br />
of 50% water, ochratox<strong>in</strong> A loss <strong>in</strong>creased at 100°C and 150°C <strong>in</strong> comparison<br />
to drier gra<strong>in</strong>, but decreased at 200°C. Little work has been<br />
published on the effect of extrusion on ochratox<strong>in</strong> A <strong>in</strong> wheat<br />
although unpublished studies <strong>in</strong> the authors’ laboratory suggested<br />
that loss below 150°C is relatively small. Extrusion process<strong>in</strong>g has<br />
been shown to have variable effects on other mycotox<strong>in</strong>s. For example,<br />
approximately 15% of aflatox<strong>in</strong>s B 1 and B 2 survived extrusion at<br />
150°C when spiked maize dough was treated (Mart<strong>in</strong>ez and Monsalve,<br />
1989) although <strong>in</strong> a study by Cazzaniga et al., (2001) <strong>in</strong> which the<br />
effects of flour moisture content, temperature and addition of sodium<br />
metabisulphite were exam<strong>in</strong>ed, the reduction of aflatox<strong>in</strong> B 1 was only<br />
between 10 and 25%.<br />
A part of this project was carried out to confirm and clarify these<br />
earlier results, to establish the change <strong>in</strong> concentration of ochratox<strong>in</strong><br />
A dur<strong>in</strong>g mill<strong>in</strong>g, abrasive scour<strong>in</strong>g of the gra<strong>in</strong> and bak<strong>in</strong>g and to<br />
assess the pattern of the distribution of ochratox<strong>in</strong> A <strong>in</strong> all milled<br />
fractions. In addition, the stability of ochratox<strong>in</strong> A was exam<strong>in</strong>ed<br />
dur<strong>in</strong>g extrusion of wheat under a range of extrusion cooker<br />
conditions (WP10).<br />
Malt is produced from barley by germ<strong>in</strong>at<strong>in</strong>g the seed under regulated<br />
conditions of moisture and temperature. The barley moisture<br />
content is raised from 14-16% (storage level) to roughly 45% by steep<strong>in</strong>g<br />
for 1-2 days at 12-20°C. The seed is then germ<strong>in</strong>ated at 15-20°C for<br />
4-6 days. The germ<strong>in</strong>ated barley, or green malt, is dried to about 4-5%<br />
moisture <strong>in</strong> a kiln<strong>in</strong>g step where the temperature is <strong>in</strong>creased to 85°C<br />
with<strong>in</strong> 21 h.
332 Monica Olsen et al.<br />
The malt<strong>in</strong>g process can provide conditions favourable for the<br />
growth of toxigenic fungi. Moist conditions dur<strong>in</strong>g germ<strong>in</strong>ation and<br />
the <strong>in</strong>itial stages of kiln<strong>in</strong>g are conducive to the growth of many fungi.<br />
The extent of the development of mycotox<strong>in</strong> produc<strong>in</strong>g fungi depends<br />
largely on the <strong>in</strong>itial contam<strong>in</strong>ation of the barley and on the vitality of<br />
the organisms present. Moreover problems of mould growth dur<strong>in</strong>g<br />
malt<strong>in</strong>g have often been associated with elevated temperatures<br />
(Flannigan, 1996). If P. verrucosum is present on barley it could proliferate<br />
dur<strong>in</strong>g malt<strong>in</strong>g and produce ochratox<strong>in</strong> A especially at temperatures<br />
near 20°C. However, no published data is available on the<br />
formation of ochratox<strong>in</strong> A dur<strong>in</strong>g the malt<strong>in</strong>g process. The European<br />
malt<strong>in</strong>g <strong>in</strong>dustry is now fac<strong>in</strong>g the new challenges caused by the established<br />
legislation for maximum levels of ochratox<strong>in</strong> A for raw cereal<br />
gra<strong>in</strong>s and products derived from cereals.<br />
There is little data available on the fate of ochratox<strong>in</strong> A dur<strong>in</strong>g the<br />
brew<strong>in</strong>g process. In experiments where ochratox<strong>in</strong> A was added at various<br />
stages dur<strong>in</strong>g malt<strong>in</strong>g and brew<strong>in</strong>g or malt conta<strong>in</strong><strong>in</strong>g high levels<br />
of ochratox<strong>in</strong> A was used, reduced amounts (13-32%) were always<br />
found <strong>in</strong> beer (Chu et al., 1975; Nip et al., 1975; Baxter et al., 2001).<br />
Losses to spent gra<strong>in</strong>s, uptake by the yeast and degradation, especially<br />
dur<strong>in</strong>g mash<strong>in</strong>g, were the ma<strong>in</strong> reasons for reduced amounts recovered<br />
<strong>in</strong> beer (Baxter et al.; 2001). Due to the thermo stable nature of<br />
ochratox<strong>in</strong> A, it is not destroyed to any significant extent dur<strong>in</strong>g kiln<strong>in</strong>g<br />
or wort boil<strong>in</strong>g (Scott, 1996b; Baxter et al.; 2001). Surveys of<br />
ochratox<strong>in</strong> A <strong>in</strong> commercial beer are regularly carried out <strong>in</strong> several<br />
countries. Very low concentrations of ochratox<strong>in</strong> A have ever been<br />
detected, the maximum levels rang<strong>in</strong>g from 0.026 to 0.33 µg/l (Vanne<br />
and Haikara, 2001). The f<strong>in</strong>al part of this project aimed at study<strong>in</strong>g<br />
the formation of ochratox<strong>in</strong> A dur<strong>in</strong>g malt<strong>in</strong>g and the subsequent fate<br />
dur<strong>in</strong>g the brew<strong>in</strong>g process (WP11).<br />
2. SUMMARY OF RESULTS FROM THE OTA<br />
PREV PROJECT<br />
2.1. Determ<strong>in</strong>ation of the Critical Control Po<strong>in</strong>ts<br />
Investigation of gra<strong>in</strong> samples has revealed that Penicillium verrucosum<br />
is the ma<strong>in</strong>, if not the only, producer of ochratox<strong>in</strong> A <strong>in</strong><br />
European cereals (Lund and Frisvad, 2003). It was concluded that<br />
P. verrucosum <strong>in</strong>fection was best detected on DYSG media after seven
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 333<br />
days at 20°C. Numbers of P. verrucosum found on DYSG and ochratox<strong>in</strong><br />
A content <strong>in</strong> cereals were correlated. Kernel <strong>in</strong>fection with<br />
P. verrucosum of more than 7% <strong>in</strong>dicated likely ochratox<strong>in</strong> A contam<strong>in</strong>ation.<br />
In the action to identify critical control po<strong>in</strong>ts for <strong>in</strong>fection, it<br />
was found that the AFLP f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g technique developed did not<br />
generate additional important <strong>in</strong>formation over that ga<strong>in</strong>ed by the<br />
detection of P. verrucosum at species level by traditional taxonomic<br />
methods (Frisvad et al., 2005).<br />
The sources of <strong>in</strong>fection of the gra<strong>in</strong> were the contam<strong>in</strong>ated environments<br />
of comb<strong>in</strong>es, dryers, and silos. Prompt and effective dry<strong>in</strong>g<br />
of cereals at harvest is the major CCP for prevent<strong>in</strong>g the formation of<br />
ochratox<strong>in</strong> A. In regions of Europe where the cereal harvest is at<br />
greatest risk, measures to avoid mould and tox<strong>in</strong> problems are often<br />
most effective, while areas normally at less risk may not be the best<br />
prepared to avoid storage problems when unusual conditions occur. It<br />
may not be economic to have expensive dry<strong>in</strong>g mach<strong>in</strong>ery idle some<br />
years while <strong>in</strong> others the supply of damp gra<strong>in</strong> may exceed the dry<strong>in</strong>g<br />
capacity available. Delays <strong>in</strong> dry<strong>in</strong>g may then put the gra<strong>in</strong> at risk.<br />
Another problem arises when the <strong>in</strong>frastructure is such that sufficient<br />
funds and expertise are unavailable to advise on and ensure best<br />
storage practice (Scudamore, 2003).<br />
2.2. Specification of Critical Limits and Establish<strong>in</strong>g<br />
Preventive Actions<br />
The studies of the effect of temporal environmental factors on fungal<br />
growth, patterns of colonisation and ochratox<strong>in</strong> A production<br />
revealed <strong>in</strong>terest<strong>in</strong>g characteristics, which may expla<strong>in</strong> why P. verrucosum<br />
is the ma<strong>in</strong> ochratox<strong>in</strong> A producer <strong>in</strong> cereal gra<strong>in</strong> <strong>in</strong> Europe.<br />
Generally, P. verrucosum was dom<strong>in</strong>ant at lower a w and 15°C, whereas<br />
Aspergillus ochraceus was dom<strong>in</strong>ant at higher a w at 25°C.<br />
Furthermore, results <strong>in</strong>dicated that P. verrucosum was less sensitive to<br />
higher concentrations of CO 2 than A. ochraceus, which may also be a<br />
competitive advantage dur<strong>in</strong>g storage (Cairns et al., 2003). A mathematical<br />
model for safe storage time before onset of significant growth<br />
of P. verrucosum and ochratox<strong>in</strong> A production has been developed,<br />
describ<strong>in</strong>g the effect of water activity and temperature on the rate of<br />
growth of P. verrucosum <strong>in</strong> cereal gra<strong>in</strong>. The model is valid for aerobic<br />
conditions, for <strong>in</strong>stance when dry<strong>in</strong>g gra<strong>in</strong> <strong>in</strong> near-ambient dryers or<br />
cool<strong>in</strong>g gra<strong>in</strong> by aeration prior to high-temperature dry<strong>in</strong>g. The<br />
model is described <strong>in</strong> the f<strong>in</strong>al report for the OTA PREV project,<br />
which is available at www.slv.se/otaprev.
334 Monica Olsen et al.<br />
The probability, of ochratox<strong>in</strong> A levels above the EC maximum<br />
limit of 5 µg/kg at different concentration of P. verrucosum <strong>in</strong> the<br />
gra<strong>in</strong>, clearly <strong>in</strong>creased when the levels of P. verrucosum were above<br />
1000 colony form<strong>in</strong>g units/gram (L<strong>in</strong>dblad et al., 2004). A mathematical<br />
model was developed which describes the risk for condensation <strong>in</strong><br />
the headspace of a silo dur<strong>in</strong>g storage of cereal gra<strong>in</strong>. The model has<br />
been used to identify the conditions which cause moisten<strong>in</strong>g of the<br />
gra<strong>in</strong>, and to develop control strategies to reduce this and the risk for<br />
mould growth and ochratox<strong>in</strong> A production (see www.slv.se/otaprev).<br />
Essential oils, resveratrol and lactic acid bacteria (LAB) can control<br />
growth and ochratox<strong>in</strong> A production by both P. verrucosum and<br />
A. ochraceus on gra<strong>in</strong> (Ricelli et al., 2002; Cairns and Magan, 2003;<br />
Fanelli et al., 2003). However, <strong>in</strong> small scale storage experiments and<br />
experimental malt<strong>in</strong>gs, the <strong>in</strong>hibitory effect of the selected LAB stra<strong>in</strong><br />
could not be shown clearly. Of twenty four essential oils tested the<br />
most effective were found to be thyme, c<strong>in</strong>namon leaf and clove bud.<br />
2.3. Establish<strong>in</strong>g Monitor<strong>in</strong>g Systems<br />
New diagnostic tools have become available that will provide the<br />
means for rapid determ<strong>in</strong>ation of ochratox<strong>in</strong> A <strong>in</strong> cereals. This will<br />
enable the effective implementation of the European legislation and<br />
facilitate future <strong>in</strong>ternal control and scientific studies. Immunoassays<br />
<strong>in</strong> ELISA format, sensitive enough to meet the EU legislation for<br />
ochratox<strong>in</strong> A, have been developed where hundreds of samples can be<br />
analysed <strong>in</strong> a few hours. A lateral flow device (LFD) tak<strong>in</strong>g less than<br />
five m<strong>in</strong>utes to perform, which can be used on-site, has also been<br />
developed (Danks et al., 2003, and unpublished results). These assays<br />
are <strong>in</strong> the prototype stage but it is anticipated that a full validation and<br />
comparison with CEN or the criteria mentioned <strong>in</strong> directive<br />
2002/26/EC can be carried out <strong>in</strong> the near future. However, it has been<br />
shown that these techniques are sensitive enough for the EU legislation<br />
for ochratox<strong>in</strong> A.<br />
A number of genes <strong>in</strong>volved <strong>in</strong> ochratox<strong>in</strong> A biosynthesis have been<br />
cloned, among them a polyketide synthase gene. PCR primer<br />
pairs have been developed which appear to be highly specific for<br />
A. ochraceus and P. verrucosum (O’Callaghan et al., 2003). The<br />
primers may f<strong>in</strong>d use <strong>in</strong> the development of rapid identification<br />
protocols for ochratoxigenic fungi.<br />
Several advances have been made towards a molecularly impr<strong>in</strong>ted<br />
polymer specific for ochratox<strong>in</strong> A and its <strong>in</strong>tegration <strong>in</strong>to a solid phase<br />
extraction (SPE) and sensor systems. Several polymers have been
Prevention of Ochratox<strong>in</strong> A <strong>in</strong> Cereals 335<br />
designed us<strong>in</strong>g a computational method and tested us<strong>in</strong>g SPE. The<br />
materials demonstrate a high aff<strong>in</strong>ity and specificity for the target<br />
molecule <strong>in</strong> aqueous model samples, however <strong>in</strong>tegration <strong>in</strong> real<br />
samples with complex biological matrices (gra<strong>in</strong> samples) has proved<br />
difficult as <strong>in</strong>terfer<strong>in</strong>g compounds affect b<strong>in</strong>d<strong>in</strong>g and measurements<br />
of ochratox<strong>in</strong> A. Attempts to isolate and remove these <strong>in</strong>terfer<strong>in</strong>g<br />
materials were unsuccessful and consequently the detection limits<br />
were not at the level required to meet the legislative requirements<br />
(Turner et al., 2003).<br />
2.4. Establish<strong>in</strong>g Corrective or Reductive Measures<br />
This project (OTA PREV) has contributed tools and recommendations<br />
for the cereal process<strong>in</strong>g <strong>in</strong>dustry. These will facilitate decisions<br />
to be made to enable the dual maximum levels for ochratox<strong>in</strong> A<br />
described <strong>in</strong> the Commission Regulation (EC) No 472/2002 of 12<br />
March 2002 sett<strong>in</strong>g maximum levels for ochratox<strong>in</strong> A <strong>in</strong> foodstuffs to<br />
be followed.<br />
Exam<strong>in</strong><strong>in</strong>g the fate of ochratox<strong>in</strong> A dur<strong>in</strong>g mill<strong>in</strong>g revealed white<br />
flour hav<strong>in</strong>g the most significant reduction of ochratox<strong>in</strong> A of about<br />
50%. An <strong>in</strong>itial clean<strong>in</strong>g stage and scour<strong>in</strong>g (1-2%) prior to mill<strong>in</strong>g,<br />
removed small amounts of ochratox<strong>in</strong> A. Bak<strong>in</strong>g resulted <strong>in</strong> only a<br />
small fall <strong>in</strong> concentration. However, an overall reduction of about<br />
80% is achievable for white bread with scour<strong>in</strong>g <strong>in</strong>cluded and up to<br />
35% for wholemeal bread (Scudamore et al., 2003; 2004).<br />
The <strong>in</strong>crease of ochratox<strong>in</strong> A concentration dur<strong>in</strong>g malt<strong>in</strong>g was<br />
2-4-fold <strong>in</strong> 75 % of the samples studied and process temperature had<br />
a pronounced effect. At the higher temperatures of 16-18°C ochratox<strong>in</strong><br />
A formation was 20-fold compared to 5-fold at the temperatures<br />
of 12-14°C. Dur<strong>in</strong>g the brew<strong>in</strong>g process approximately 20% of the<br />
orig<strong>in</strong>al ochratox<strong>in</strong> A from the malt rema<strong>in</strong>ed <strong>in</strong> the beer (Lehtonen<br />
and Haikara, 2002; Haikara et al., 2003).<br />
3. ACKNOWLEDGEMENT<br />
This work was supported by the European Commission, Quality of<br />
Life and Management of Liv<strong>in</strong>g Resources Programme (contract<br />
no. QLK1-CT-1999-00433). The authors wish to thank all Project<br />
Participants and the Scientific Officer Achim Boenke for their work<br />
and constructive criticism dur<strong>in</strong>g the progress of project. The help<br />
provided and valuable discussion with Claud<strong>in</strong>e Vandemeulebrouke
336 Monica Olsen et al.<br />
(EUROMALT), Guisla<strong>in</strong>e Veron Delor (IRTAC), Hans de Keijzer<br />
(GAM/COCERAL), Esko Pajunen (European Brewery Convention)<br />
and Roger Williams (Home Grown Cereals Authority, UK) have been<br />
highly appreciated. The contributions and support of Keith<br />
Scudamore (KAS Mycotox<strong>in</strong>s), who has been subcontracted to this<br />
project, have been very valuable.<br />
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Baxter, E. D., Slaid<strong>in</strong>g, I. R., and Kelly, B., 2001, Behavior of ochratox<strong>in</strong> A <strong>in</strong> brew<strong>in</strong>g,<br />
J. Am. Soc. Brew. Chem. 59:98-100.<br />
Boudra, H., Le Bars, P., and Le Bars, J., 1995, Thermostability of ochratox<strong>in</strong> A <strong>in</strong><br />
wheat under two moisture conditions, Appl. Environ. Microbiol. 61:1156-1158.<br />
Brooker D. B. F. W., Bakker-Arkema, and Hall C. W., 1992, Dry<strong>in</strong>g and Storage of<br />
Gra<strong>in</strong>s and Oilseeds, Van Nostrand Re<strong>in</strong>hold, New York.<br />
Caccioni, D. R. L., Guizzardi, M., Biondi, D. M., Renda, A., and Ruberto, G., 1998,<br />
Relationships between volatile components of citrus fruits, essential oils and<br />
antimicrobial action on Penicillium digitatum and P. italicum, Int. J. <strong>Food</strong><br />
Microbiol. 43:73-79.<br />
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Scott, P. M., 1996a, Effects of process<strong>in</strong>g and detoxification treatments on ochratox<strong>in</strong><br />
A, <strong>Food</strong> Addit. Contam. 13 (suppl):19-22<br />
Scott, P. M., 1996b, Mycotox<strong>in</strong>s transmitted <strong>in</strong>to beer from contam<strong>in</strong>ated gra<strong>in</strong>s dur<strong>in</strong>g<br />
brew<strong>in</strong>g, J. AOAC Int., 79:875-882.<br />
Scudamore, K. A., 1996, Ochratox<strong>in</strong> A <strong>in</strong> animal feed-effects of process<strong>in</strong>g, <strong>Food</strong><br />
Addit. Contam. 13 (suppl.): 39-42.<br />
Scudamore, K. A., 2003, Report on questionnaire replies from cereal experts <strong>in</strong><br />
Europe. Report available at www.slv.se/otaprev<br />
Scudamore, K. A., Banks, J. N., and MacDonald, S. J., 2003, Fate of ochratox<strong>in</strong> A <strong>in</strong><br />
the process<strong>in</strong>g of whole wheat gra<strong>in</strong>s dur<strong>in</strong>g mill<strong>in</strong>g and bread production, <strong>Food</strong><br />
Addit. Contam. 20:1153-1163.<br />
Scudamore, K. A., Banks, J. N., and MacDonald, S. J., 2004, Fate of ochratox<strong>in</strong> A<br />
<strong>in</strong> the process<strong>in</strong>g of whole wheat gra<strong>in</strong> dur<strong>in</strong>g extrusion, <strong>Food</strong> Addit. Contam.<br />
21:488-497.<br />
Scudamore, K. A., Patel, S., and Breeze, V., 1999, Surveillance of stored gra<strong>in</strong> from<br />
the 1997 harvest <strong>in</strong> the United K<strong>in</strong>gdom for ochratox<strong>in</strong> A, <strong>Food</strong> Addit. Contam.<br />
16:281-290.
342 Monica Olsen et al.<br />
Seitz, L. M., Sauer, D. B., and Mohr, H. E., 1982, Storage of high-moisture corn: fungal<br />
growth and dry matter loss, Cereal Chem. 59:100-105.<br />
Smith, E. A., and Sokhansanj, S., 1990, Moisture transport caused by agricultural<br />
convection <strong>in</strong> gra<strong>in</strong> stores, J. of Agric. Engng Res. 47:23-34.<br />
Steel, I. F., Saul, R. A., and Hukill, W.V., 1969, Deterioration rate of shelled corn as<br />
measured by carbon dioxide production, Trans. ASAE 12:685-689.<br />
Subirade, I., 1996, Fate of ochratox<strong>in</strong> A dur<strong>in</strong>g breadmak<strong>in</strong>g, <strong>Food</strong> Addit. Contam.<br />
13(suppl.):25-26.<br />
Tanaka, H., and Yoshida, K., 1984, Heat and mass transfer mechanisms <strong>in</strong> a gra<strong>in</strong><br />
storage silo, <strong>in</strong>: Eng<strong>in</strong>eer<strong>in</strong>g and <strong>Food</strong>, B. M. McKenna, ed., Elsevier, New York,<br />
NY.<br />
Tooley, P. W., O’Neill, N. R., Goley, E. D., and Carras, M. M., 2000, Assessment of<br />
diversity <strong>in</strong> Claviceps africana and other Claviceps species by RAM and AFLP<br />
analysis, Phytopathology 90:1126-1130.<br />
Turner N. W., Piletska, E. V., Karim, K., Whitcombe, M., Malecha, M., Magan N.,<br />
Baggiani C., and Piletsky, S. A., 2004, Effect of the solvent on recognition properties<br />
of molecularly impr<strong>in</strong>ted polymer specific for ochratox<strong>in</strong> A, Biosen.<br />
Bioelectron. 20:1060-1067.<br />
Vanne, L., and Haikara, A., 2001, Mycotox<strong>in</strong>s <strong>in</strong> the total cha<strong>in</strong> from barley to beer,<br />
Proc. 28th Congr. Eur. Brew. Conv., Budapest 2001, CD-ROM. pp. 839-848.<br />
Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes., M., Frijters, A.,<br />
Pot, J., Peleman, J., Kuiper, M., and Zabeau, M., 1995, AFLP: a new technique for<br />
DNA f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g, Nucl. Acids Res. 23: 4407-4414.<br />
Wilson, M., and L<strong>in</strong>dow, S. E., 1994a, Co-existence among epiphytic bacterial populations<br />
mediated through nutritional resource partition<strong>in</strong>g, Appl. Environ.<br />
Microbiol. 60:4468-4477.<br />
Wilson, M., and L<strong>in</strong>dow S E., 1994b, Ecological similarity and coexistence of epiphytic<br />
ice-nucleat<strong>in</strong>g (Ice +) Pseudomonas syr<strong>in</strong>gae stra<strong>in</strong>s and a non-ice-nucleat<strong>in</strong>g<br />
(Ice −) biological control agent, Appl. Environ. Microbiol. 60:3128-3137.<br />
White, N. D. G., S<strong>in</strong>ha, R. N., and Muir, W. E., 1982, Intergranular carbon dioxide as<br />
an <strong>in</strong>dicator of biological activity associated with the spoilage of stored wheat,<br />
Can. Agric. Eng. 24:35-42.<br />
Wolff, J., 2000, Ochratox<strong>in</strong> A <strong>in</strong> cereals and cereal products, Arch. Lebensmittelhygiene<br />
51:85-88.<br />
Y<strong>in</strong>, M. C., and Tsao, S. M., 1999, Inhibitory effect of seven Allium plants upon three<br />
Aspergillus species, Int. J. <strong>Food</strong> Microbiol. 49:49-56.
RECOMMENDED METHODS FOR FOOD<br />
MYCOLOGY<br />
In the op<strong>in</strong>ion of the International Commission on <strong>Food</strong><br />
<strong>Mycology</strong> (ICFM), the follow<strong>in</strong>g methods are the most satisfactory<br />
currently available for the mycological exam<strong>in</strong>ation of foods. The<br />
methods outl<strong>in</strong>ed below are based on those published by Pitt et al.<br />
(1992) and have been developed after numerous collaborative studies<br />
carried out by ICFM members s<strong>in</strong>ce its <strong>in</strong>ception. Formulations for<br />
all media discussed appear <strong>in</strong> the Media Appendix.<br />
1. GENERAL PURPOSE ENUMERATION<br />
METHODS AND MEDIA<br />
1.1. Methods<br />
1.1.1. Dilution plat<strong>in</strong>g<br />
Dilution plat<strong>in</strong>g is recommended for liquid foods and powders,<br />
and also for particulate foods where the total mycoflora is of importance,<br />
as for example <strong>in</strong> gra<strong>in</strong>s <strong>in</strong>tended for manufacture of flour.<br />
Samples: Samples should be as representative as possible. For suitable<br />
microbiological sampl<strong>in</strong>g procedures, refer to the publication by<br />
the International Commission on Microbiological Specifications for<br />
<strong>Food</strong>s (ICMSF, 1986).<br />
The sample size should be as large as possible, consistent with<br />
equipment used for homogenisation. If a Stomacher 400 is used, 10 to<br />
40 g samples are suitable.<br />
343
344 Recommended Methods for <strong>Food</strong> <strong>Mycology</strong><br />
Diluents and dilution: The recommended diluent for fungi <strong>in</strong>clud<strong>in</strong>g<br />
yeasts is 0.1% aqueous peptone. Dilution should be 1:10 (1+9).<br />
Homogenisation: A Coleworth Stomacher or equivalent Bag Mixer<br />
is the preferred type of homogeniser, used for 2 m<strong>in</strong>utes per sample.<br />
Blend<strong>in</strong>g for 30-60 sec or shak<strong>in</strong>g <strong>in</strong> a closed bottle with glass beads<br />
for 2-5 m<strong>in</strong> are less preferable alternatives.<br />
Plat<strong>in</strong>g: For mycological studies, spread plates are recommended;<br />
pour plates are less effective. Inocula should be 0.1 ml per plate, spread<br />
with a sterile, bent glass rod.<br />
Incubation: For general purpose enumeration, 5 days <strong>in</strong>cubation as<br />
25°C is recommended. Plates should be <strong>in</strong>cubated upright. A higher<br />
temperature, e.g. 30°C, is suitable <strong>in</strong> tropical regions.<br />
1.1.2. Direct plat<strong>in</strong>g<br />
Direct plat<strong>in</strong>g is considered to be the more effective technique for<br />
mycological exam<strong>in</strong>ation of particulate foods such as gra<strong>in</strong>s and nuts.<br />
In most situations, surface dis<strong>in</strong>fection (also commonly referred to as<br />
surface sterilisation) before direct plat<strong>in</strong>g is considered essential, to<br />
permit detection and enumeration of fungi actually <strong>in</strong>vad<strong>in</strong>g the food.<br />
An exception is to be made for cases where surface contam<strong>in</strong>ants<br />
become part of the downstream mycoflora, e.g. wheat gra<strong>in</strong>s to be<br />
used <strong>in</strong> flour manufacture. In such cases, gra<strong>in</strong>s should not be surface<br />
dis<strong>in</strong>fected.<br />
Surface dis<strong>in</strong>fection: Surface dis<strong>in</strong>fect food particles by immersion <strong>in</strong><br />
0.4% chlor<strong>in</strong>e solution (household bleach, diluted 1:10) for 2 m<strong>in</strong>utes.<br />
A m<strong>in</strong>imum of 50 particles should be dis<strong>in</strong>fected and plated. The<br />
chlor<strong>in</strong>e solution should be used only once.<br />
R<strong>in</strong>se: After pour<strong>in</strong>g off chlor<strong>in</strong>e solution, r<strong>in</strong>se once <strong>in</strong> sterilised<br />
distilled or deionised water. Note: this step has not been shown to be<br />
essential, but is generally recommended.<br />
Surface plate: As quickly as possible, transfer food particles with<br />
sterile forceps to previously poured and set plates, at the rate of 5-10<br />
particles per plate, depend<strong>in</strong>g on the size of the particles.<br />
Incubation: The standard <strong>in</strong>cubation regimen for general purpose<br />
enumeration is 25°C for 5 days. A higher temperature (30°C) may be<br />
used <strong>in</strong> the tropics. Plates should be <strong>in</strong>cubated upright.<br />
Results: Express results as per cent of particles <strong>in</strong>fected by fungi.<br />
Differential count<strong>in</strong>g of a variety of genera is possible us<strong>in</strong>g a stereomicroscope.
Recommended Methods for <strong>Food</strong> <strong>Mycology</strong> 345<br />
1.2. Media<br />
Dichloran Rose Bengal Chloramphenicol agar (DRBC; K<strong>in</strong>g et al.,<br />
1979; Pitt and Hock<strong>in</strong>g 1997) and Dichloran 18% Glycerol agar<br />
(DG18; Hock<strong>in</strong>g and Pitt, 1980; Pitt and Hock<strong>in</strong>g, 1997) (see Media<br />
Appendix) are recommended as general purpose isolation and enumeration<br />
media.<br />
DRBC is recommended for fresh foods, <strong>in</strong>clud<strong>in</strong>g fruits and vegetables,<br />
meats and dairy products. Note that media conta<strong>in</strong><strong>in</strong>g rose bengal<br />
are sensitive to light. Inhibitory compounds are produced after<br />
relatively short exposures to light. Prepared media should be stored<br />
protected from light until used.<br />
For foods of reduced water activity, i.e. less than 0.95 a w , DG18 is<br />
preferred. Although orig<strong>in</strong>ally formulated for enumeration of<br />
xerophilic fungi, DG18 is now widely used as an effective generally<br />
purpose medium, both for foods and for sampl<strong>in</strong>g of <strong>in</strong>door air<br />
(Hoekstra et al., 2000). Its water activity (0.955) reduces <strong>in</strong>terference<br />
from both bacteria and rapidly grow<strong>in</strong>g fungi.<br />
Chloramphenicol is the antibacterial agent of choice as it is heat stable<br />
and can be autoclaved after <strong>in</strong>corporation <strong>in</strong>to the agar. It should<br />
generally be <strong>in</strong>corporated at a concentration of 100 mg/kg. If a high<br />
bacterial population is expected (as <strong>in</strong> soil or fresh meat), the concentration<br />
of chloramphenicol may be doubled, or an equal concentration<br />
of a second antibiotic such as oxytetracycl<strong>in</strong>e can be added<br />
aseptically after autoclav<strong>in</strong>g.<br />
2. SELECTIVE MEDIA<br />
2.1. Media for xerophilic fungi<br />
Dichloran 18% Glycerol agar (DG18; Hock<strong>in</strong>g and Pitt 1980; Pitt<br />
and Hock<strong>in</strong>g 1997) is the recommended medium for enumeration of<br />
common xerophilic fungi <strong>in</strong> foods. Incubation at 25°C for up to 7 days<br />
is recommended. Growth of Eurotium species on DG18 is rather<br />
rapid, and colonies do not have discrete marg<strong>in</strong>s.<br />
For isolation of extreme xerophiles, e.g. Xeromyces bisporus,<br />
Eremascus and xerophilic Chrysosporium species, Malt Yeast 50%<br />
Glucose agar (MY50G; Pitt and Hock<strong>in</strong>g, 1997) is recommended.<br />
Direct plat<strong>in</strong>g should be used. Incubate plates at 25°C and exam<strong>in</strong>e after
346 Recommended Methods for <strong>Food</strong> <strong>Mycology</strong><br />
7 days and, if no growth appears, after longer periods, up to 21 days.<br />
Plates should be <strong>in</strong>cubated <strong>in</strong> polyethylene bags to prevent desiccation.<br />
2.2. Media for Fusarium species<br />
The most effective medium for isolation of Fusarium species from<br />
foods is Czapek Iprodione Dichloran agar (CZID; Albidgren et al.,<br />
1987). Dichloran Chloramphenicol Peptone agar (DCPA; Andrews<br />
and Pitt, 1986) can also be used, but is less selective.<br />
2.3. Media for toxigenic Penicillium species<br />
Dichloran Rose bengal Yeast Extract Sucrose agar (DRYES;<br />
Frisvad, 1983; Samson et al., 2004) is useful for detect<strong>in</strong>g and dist<strong>in</strong>guish<strong>in</strong>g<br />
Penicillium verrucosum and P. viridicatum, particularly <strong>in</strong><br />
gra<strong>in</strong>s from cool climates.<br />
2.4. Media for species produc<strong>in</strong>g aflatox<strong>in</strong>s<br />
Aspergillus Flavus and Parasiticus Agar (AFPA; Pitt et al., 1983;<br />
Pitt and Hock<strong>in</strong>g, 1997) is effective for detect<strong>in</strong>g and enumerat<strong>in</strong>g<br />
Aspergillus flavus and A. parasiticus and closely related species. Plates<br />
should be <strong>in</strong>cubated at 30°C for 2-3 days to allow development of<br />
orange reverse colours <strong>in</strong> colonies of these species.<br />
3. METHODS FOR YEASTS<br />
3.1. Diluents<br />
For enumeration of yeasts <strong>in</strong> high a w foods and beverages, the recommended<br />
diluent is 0.1% aqueous peptone. For concentrates, syrups<br />
and other low a w samples, 20 to 30% glucose (w/v) <strong>in</strong> 0.1% aqueous<br />
peptone is recommended.<br />
3.2. General purpose media<br />
For products such as beverages, where yeasts usually predom<strong>in</strong>ate,<br />
nonselective media such as Malt Extract Agar (Pitt and Hock<strong>in</strong>g,<br />
1997) or Tryptone Glucose Yeast extract agar (TGY; Pitt and
Recommended Methods for <strong>Food</strong> <strong>Mycology</strong> 347<br />
Hock<strong>in</strong>g, 1997) plus chloramphenicol or oxytetracycl<strong>in</strong>e (100 mg/kg)<br />
are recommended.<br />
For products where yeasts must be enumerated <strong>in</strong> the presence of<br />
moulds, DRBC is recommended.<br />
For products where bacteria may be encountered, or are suspected<br />
(e.g. cultured dairy products, fresh meat products) exam<strong>in</strong>ation of<br />
representative colonies under the microscope is important.<br />
3.3. Detection of Low Numbers of Yeasts<br />
For detection of low numbers of yeasts <strong>in</strong> liquid products, membrane<br />
filtration is recommended. If the substrate is not filterable,<br />
enrichment techniques are available but are not quantitative.<br />
3.4. Preservative Resistant Yeasts<br />
An effective medium for detection of preservative resistant yeasts<br />
is Tryptone Glucose Yeast extract agar with 0.5% acetic acid (TGYA)<br />
(Hock<strong>in</strong>g, 1996). Malt Extract Agar with 0.5% acetic acid (MAc) may<br />
also be used, but the pH is lower than TGYA, and the medium may be<br />
too <strong>in</strong>hibitory for recovery of some <strong>in</strong>jured cells.<br />
4. PREPARATION OF DRIED SAMPLES FOR<br />
DILUTION PLATING<br />
Where dried particulate foods such as cereal gra<strong>in</strong>s are to be dilution<br />
plated, soak<strong>in</strong>g for 30 m<strong>in</strong>utes <strong>in</strong> 0.1% aqueous peptone solution<br />
before homogenis<strong>in</strong>g is recommended.<br />
5. ENUMERATION OF HEAT RESISTANT<br />
FUNGI<br />
The method for detection of heat resistant fungi detailed <strong>in</strong> an earlier<br />
chapter of this volume (Houbraken and Samson, 2006) is based<br />
on the method previously endorsed by ICFM. Note that very acid<br />
samples should be adjusted to pH 3.5-4.0 with NaOH before heat<strong>in</strong>g.<br />
Care should be taken to avoid contam<strong>in</strong>ation dur<strong>in</strong>g pour<strong>in</strong>g of
348 Recommended Methods for <strong>Food</strong> <strong>Mycology</strong><br />
plates.. Heavily sporulat<strong>in</strong>g colonies of, for example, Penicillium and<br />
Aspergillus species generally <strong>in</strong>dicate contam<strong>in</strong>ation. Such colonies<br />
should be ignored.<br />
6. REFERENCES<br />
Albidgren, M. P., Lunf, F., Thrane, U., and Elmholt, S., 1987, Czapek-Dox agar conta<strong>in</strong><strong>in</strong>g<br />
iprodione and dicloran as a selective medium for the isolation of Fusarium<br />
species, Lett. Appl. Microbiol., 5:83-86.<br />
Andrews, S., and Pitt, J. I., 1986, Selective medium for isolation of Fusarium<br />
species and dematiaceous hyphomycetes from cereals, Appl. Environ. Microbiol.<br />
51:1235-1238.<br />
Hock<strong>in</strong>g, A. D., 1996, Media for preservative resistant yeasts: a collaborative study,<br />
Int. J. <strong>Food</strong> Microbiol. 29:167-175.<br />
Hoekstra, E. S., Samson, R. A., and Summerbell, R. C., 2000, Methods for the detections<br />
and isolation of fungi <strong>in</strong> the <strong>in</strong>door environment, <strong>in</strong>: Introduction to <strong>Food</strong>and<br />
Airborne Fungi, 6th edition, R. A. Samson, E. S. Hoekstra, J. C. Frisvad and<br />
O. Filtenborg, eds, Centraalbureau voor Schimmelcultures, Utrecht, Netherlands,<br />
pp. 298-305.<br />
Houbraken, J., and Samson, R. A., 2006, Standardization of methods for detection<br />
of heat resistant fungi, <strong>in</strong>: <strong>Advances</strong> <strong>in</strong> <strong>Food</strong> <strong>Mycology</strong>, A. D. Hock<strong>in</strong>g, J. I. Pitt,<br />
R. A. Samson and U. Thrane, eds, Spr<strong>in</strong>ger, New York. pp. 107-111.<br />
ICMSF (International Commission on Microbiological Specifications for <strong>Food</strong>s),<br />
1986, Sampl<strong>in</strong>g for Microbiological Analysis: Pr<strong>in</strong>ciples and Specific Applications,<br />
2nd edition, University of Toronto Press, Toronto.<br />
K<strong>in</strong>g, A. D., Hock<strong>in</strong>g, A. D., and Pitt, J. I., 1979, Dichloran-rose bengal medium for<br />
enumeration of molds from foods, Appl. Environ. Microbiol. 37:959-964.<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, 2nd edition, Blackie<br />
Academic and Professional, London.<br />
Pitt, J. I., Hock<strong>in</strong>g, A. D., and Glenn, D. R., 1983, An improved medium for the<br />
detection of Aspergillus flavus and A. parasiticus, J. Appl. Bacteriol. 54:109-114.<br />
Pitt, J. I., Hock<strong>in</strong>g, A. D., Samson, R. A., and K<strong>in</strong>g, A. D., 1992, Recommended<br />
methods for the mycological exam<strong>in</strong>ation of foods, 1992, <strong>in</strong>: Modern Methods <strong>in</strong><br />
<strong>Food</strong> <strong>Mycology</strong>, R. A. Samson, A. D. Hock<strong>in</strong>g, J. I. Pitt and A. D. K<strong>in</strong>g, eds,<br />
Elsevier, Amsterdam, pp. 365-368.<br />
Samson, R. A., Hoekstra, E. S., and Frisvad, J. C., eds, 2004, Introduction to <strong>Food</strong>and<br />
Airborne Fungi, 7th edition, Centraalbureau voor Schimmelcultures, Utrecht,<br />
Netherlands, 389 pp.
APPENDIX 1 – MEDIA<br />
All media are sterilized by autoclav<strong>in</strong>g at 121°C for 15 m<strong>in</strong> unless<br />
otherwise specified. Czapek agars may be made from basic <strong>in</strong>gredients,<br />
or their production can be simplified by the use of Czapek concentrate<br />
and Czapek trace metal solution which conta<strong>in</strong> the required<br />
additional salts <strong>in</strong> the specified concentrations. Both options are provided<br />
<strong>in</strong> this appendix.<br />
AFPA: Aspergillus flavus and parasiticus agar (Pitt et al., 1983; Pitt<br />
and Hock<strong>in</strong>g 1997)<br />
Peptone 10 g<br />
Yeast extract 20 g<br />
Ferric ammonium citrate 0.5 g<br />
Chloramphenicol 100 mg<br />
Agar 15 g<br />
Dichloran 2 mg<br />
(0.2% w/v <strong>in</strong> ethanol, 1 ml)<br />
Water, distilled 1 litre<br />
After addition of all <strong>in</strong>gredients, sterilise by autoclav<strong>in</strong>g at 121°C<br />
for 15 m<strong>in</strong>. F<strong>in</strong>al pH 6.0-6.5.<br />
CMA: Corn Meal agar (Samson et al., 2004).<br />
Add 60 g freshly ground cornmeal to 1 litre water. Heat to boil<strong>in</strong>g<br />
and simmer gently for 1 h. Stra<strong>in</strong> through cloth. Make volume up to<br />
1 litre with water, add 15 g agar, heat to dissolve agar, then autoclave<br />
for 15 m<strong>in</strong> at 121°C. Also available commercially.<br />
349
350 Appendix 1 – Media<br />
CREA: Creat<strong>in</strong>e Sucrose agar (Samson et al., 2004).<br />
Creat<strong>in</strong>e.1H 2 O 3.0 g<br />
Sucrose 30 g<br />
KCl 0.5 g<br />
MgSO 4 .7H 2 O 0.5 g<br />
FeSO 4 .7H 2 O 0.01 g<br />
K 2 HPO 4 .3H 2 O 1.3 g<br />
Bromocresol purple 0.05 g<br />
Agar 15 g<br />
Distilled water 1 litre<br />
F<strong>in</strong>al pH 8.0 ± 0.2 (adjust after medium is autoclaved)<br />
Czapek concentrate (Pitt and Hock<strong>in</strong>g, 1997)<br />
NaNO3 KCl<br />
30 g<br />
5 g<br />
MgSO .7H O 4 2<br />
FeSO .7H O 4 2<br />
Water, distilled<br />
5 g<br />
0.1 g<br />
100 ml<br />
Czapek concentrate and trace metal solution (below) do not require<br />
sterilisation. The precipitate of Fe(OH) 3 which forms <strong>in</strong> time can be<br />
resuspended by shak<strong>in</strong>g before use.<br />
Czapek trace metal solution (Pitt and Hock<strong>in</strong>g, 1997)<br />
CuSO 4 .5H 2 O 0.5 g<br />
ZnSO 4 .7H 2 O 1 g<br />
Water, distilled 100 ml<br />
Cz: Czapek Dox agar (Samson et al., 2004)<br />
NaNO 3<br />
K HPO 2 4<br />
KCl<br />
MgSO z 4<br />
3 g<br />
1 g<br />
0.5 g<br />
. 7H O 2<br />
FeSO z 4<br />
0.5 g<br />
. 7H O 2<br />
ZnSO z 4<br />
0.01 g<br />
. 7H O 2<br />
CuSO z 4<br />
0.01 g<br />
. 5H O 2<br />
Sucrose<br />
0.005 g<br />
30 g<br />
Agar 20 g<br />
Water, distilled l litre<br />
F<strong>in</strong>al pH 6.3 ± 0.2
Appendix 1 – Media 351<br />
CYA (1): Czapek Yeast Autolysate agar (Samson et al., 2004)<br />
NaNO3 K HPO 2 4<br />
KCl<br />
MgSO c 4<br />
3 g<br />
1 g<br />
0.5 g<br />
. 7H O 2<br />
FeSO c 4<br />
0.5 g<br />
. 7H O 2<br />
ZnSO c 4<br />
0.01 g<br />
. 7H O 2<br />
CuSO c 4<br />
0.01 g<br />
. 5H O 2<br />
Yeast extract (Difco)<br />
0.005 g<br />
5 g<br />
Sucrose 30 g<br />
Agar 20 g<br />
Distilled water l litre<br />
This formulation should be used to prepare CYA from base <strong>in</strong>gredients,<br />
without the use of Czapek concentrate or trace metal solutions.<br />
CYA (2): Czapek Yeast extract agar (Pitt, 1979; Pitt and Hock<strong>in</strong>g,<br />
1997)<br />
K HPO 2 4<br />
Czapek concentrate<br />
1 g<br />
10 ml<br />
Trace metal solution 1 ml<br />
Yeast extract, powdered 5 g<br />
Sucrose 30 g<br />
Agar 15 g<br />
Water, distilled 1 litre<br />
Prepared us<strong>in</strong>g Czapek concentrate and Czapek trace metal solution.<br />
Table grade sucrose is satisfactory for use provided it is free from<br />
sulphur dioxide. F<strong>in</strong>al pH 6.7.<br />
CZID: Czapek iprodione dichloran agar (Albidgren et al., 1987; Pitt<br />
and Hock<strong>in</strong>g 1997)<br />
Sucrose 30 g<br />
Yeast extract 5 g<br />
Chloramphenicol 100 mg<br />
Dichloran 2 mg<br />
(0.2% <strong>in</strong> ethanol, 1 ml)<br />
Czapek concentrate 10 ml<br />
Trace metal solution 1 ml<br />
Agar 15 g<br />
Water, distilled 1 litre<br />
Iprodione (suspension) 1 ml
352 Appendix 1 – Media<br />
Add iprodione suspension [0.3 g Roval 50WP (Rhone-Poulenc<br />
Agro-Chemie, Lyon, France) <strong>in</strong> 50 ml sterile water, shaken before<br />
addition to medium] after autoclav<strong>in</strong>g at 121°C for 15 m<strong>in</strong>.<br />
CY20S: Czapek Yeast Extract agar with 20% Sucrose (Pitt and<br />
Hock<strong>in</strong>g, 1997; Samson et al., 2004)<br />
K HPO 2 4<br />
Czapek concentrate<br />
1 g<br />
10 ml<br />
Yeast extract 5 g<br />
Sucrose 200 g<br />
Agar 15 g<br />
Water, distilled 1 litre<br />
F<strong>in</strong>al pH 5.2.<br />
DCPA: Dichloran chloramphenicol peptone agar (Andrews and Pitt,<br />
1986; Pitt and Hock<strong>in</strong>g 1997)<br />
Peptone 15 g<br />
KH PO 1 g<br />
2 4<br />
MgSO .7H O 0.5 g<br />
4 2<br />
Chloramphenicol 100 mg<br />
Agar 15 g<br />
Dichloran 2 mg<br />
(0.2% w/v <strong>in</strong> ethanol, 1 ml)<br />
Water, distilled 1 litre<br />
After addition of all <strong>in</strong>gredients, sterilise by autoclav<strong>in</strong>g at 121°C<br />
for 15 m<strong>in</strong>. F<strong>in</strong>al pH 5.5.-6.0.<br />
DG18: Dichloran 18% Glycerol agar (Hock<strong>in</strong>g and Pitt, 1980; Pitt<br />
and Hock<strong>in</strong>g 1997)<br />
Glucose 10 g<br />
Peptone 5 g<br />
KH PO 2 4<br />
MgSO .7H O 4 2<br />
Glycerol, A.R.<br />
1 g<br />
0.5 g<br />
220 g<br />
Agar 15 g<br />
Dichloran 2 mg<br />
(0.2% w/v <strong>in</strong> ethanol, 1 ml)<br />
Chloramphenicol 100 mg<br />
Water, distilled 1 litre<br />
Add m<strong>in</strong>or <strong>in</strong>gredients and agar to ca 800 ml distilled water. Steam<br />
to dissolve agar, then make to 1 litre with distilled water. Add glycerol:
Appendix 1 – Media 353<br />
note that the f<strong>in</strong>al concentration is 18% weight <strong>in</strong> weight, not weight<br />
<strong>in</strong> volume. Sterilise by autoclav<strong>in</strong>g at 121°C for 15 m<strong>in</strong>. F<strong>in</strong>al a w 0.955,<br />
pH 5.5 to 5.8.<br />
DRBC: Dichloran Rose Bengal Chloramphenicol agar (Pitt and<br />
Hock<strong>in</strong>g, 1997)<br />
Glucose 10 g<br />
Peptone, bacteriological 5 g<br />
KH PO 2 4<br />
MgSO .7H O 4 2<br />
Agar<br />
1 g<br />
0.5 g<br />
15 g<br />
Rose bengal 25 mg<br />
Dichloran<br />
(5% w/v <strong>in</strong> water, 0.5 ml)<br />
2 g<br />
(0.2% w/v <strong>in</strong> ethanol, 1 ml)<br />
Chloramphenicol 100 mg<br />
Water, distilled 1 litre<br />
F<strong>in</strong>al pH 5.5 – 5.8. Store prepared media away from light; photoproducts<br />
of rose bengal are highly <strong>in</strong>hibitory to some fungi, especially<br />
yeasts. In the dark, the medium is stable for at least one month at<br />
1–4°C. The stock solutions of rose bengal and dichloran need no sterilisation,<br />
and are also stable for very long periods.<br />
DRYES: Dichloran Rose Bengal Yeast Extract Sucrose agar<br />
(Frisvad, 1983; Samson et al., 2004)<br />
Yeast extract 20 g<br />
Sucrose 150 g<br />
Dichloran 2 mg<br />
(0.2% <strong>in</strong> ethanol, 1 ml)<br />
Rose bengal 25 mg<br />
(5% w/v <strong>in</strong> water, 0.5 ml)<br />
Agar 20 g<br />
Chloramphenicol 0.1 g<br />
Water, distilled to 1 litre<br />
F<strong>in</strong>al pH 5.6 (adjusted after medium is autoclaved). This medium<br />
detects P. verrucosum and P. viridicatum by production of a purple<br />
reverse colour. Can be modified by add<strong>in</strong>g 0.5 g MgSO 4 .7H 2 O.<br />
HAY: Hay Infusion agar (Samson et al., 2004)<br />
Sterilise 50 g hay <strong>in</strong> one litre of water at 121°C for 30 m<strong>in</strong>. Stra<strong>in</strong><br />
through cloth and make volume up to 1 litre. Adjust pH to 6.2 with<br />
K 2 HPO 4 and add 15 g agar. Autoclave for 15 m<strong>in</strong> at 121°C.
354 Appendix 1 – Media<br />
MEA: Malt extract agar (Pitt and Hock<strong>in</strong>g, 1997; Samson et al.,<br />
2004)<br />
Malt extract, powdered 20 g<br />
Peptone l g<br />
Glucose 20 g<br />
Agar 20 g<br />
Water, distilled l litre<br />
Commercial malt extract used for home brew<strong>in</strong>g is satisfactory for<br />
use <strong>in</strong> MEA, as is bacteriological peptone. Do not sterilise for longer<br />
than 15 m<strong>in</strong> at 121°C, as this medium will become soft on prolonged<br />
or repeated heat<strong>in</strong>g. F<strong>in</strong>al pH 5.6.<br />
MY50G: Malt extract yeast extract 50% glucose agar (Pitt and<br />
Hock<strong>in</strong>g, 1997)<br />
Malt extract 10 g<br />
Yeast extract 2.5 g<br />
Agar 10 g<br />
Water, distilled, to 500 g<br />
Glucose, A.R. 500 g<br />
Add the m<strong>in</strong>or constituents and agar to ca 450 ml distilled water<br />
and steam to dissolve the agar. Immediately make up to 500 g with distilled<br />
water. While the solution is still hot, add the glucose all at once<br />
and stir rapidly to prevent the formation of hard lumps of glucose<br />
monohydrate. If lumps do form, dissolve them by steam<strong>in</strong>g for a few<br />
m<strong>in</strong>utes. Sterilise by steam<strong>in</strong>g for 30 m<strong>in</strong>; note that this medium is of<br />
a sufficiently low a w not to require autoclav<strong>in</strong>g. <strong>Food</strong> grade glucose<br />
monohydrate (dextrose) may be used <strong>in</strong> this medium <strong>in</strong>stead of analytical<br />
reagent grade glucose, but allowance must be made for the<br />
additional water present. Use 550 g of C 6 H 12 0 6 .H 2 0, and 450 g of the<br />
basal medium. F<strong>in</strong>al a w 0.89, f<strong>in</strong>al pH 5.3.<br />
MME: Mercks Malt Extract agar (El-Banna and Leistner, 1988)<br />
F<strong>in</strong>al pH 5.6<br />
Malt extract 30 g<br />
Soy peptone 3 g<br />
ZnSO 4 c . 7H 2 O 0.01 g<br />
CuSO 4 c . 5H 2 O 0.005 g<br />
Agar 20 g<br />
Distilled water l litre
Appendix 1 – Media 355<br />
PCA: Potato Carrot Agar (Simmons, 1992; Samson et al., 2004)<br />
Carrots 40 g<br />
Potatoes 40 g<br />
Wash, peel and chop carrots and potatoes. Boil carrots and potatoes<br />
separately <strong>in</strong> 1 litre water each for 5 m<strong>in</strong> then filter off. Sterilise filtrates<br />
(121°C, 15 m<strong>in</strong>), add 250 ml potato extract and 250 ml carrot<br />
extract to 500 ml distilled water, add 15 g agar and sterilise at 121°C<br />
for 15 m<strong>in</strong>.<br />
PDA: Potato Dextrose agar (Pitt and Hock<strong>in</strong>g 1997; Samson et al.,<br />
2004),<br />
Potatoes 250 g<br />
Glucose 20 g<br />
Agar 15 g<br />
Water, distilled to 1 litre<br />
PDA prepared from raw <strong>in</strong>gredients is superior to commercially prepared<br />
media. Wash the potatoes, which should not be of a red sk<strong>in</strong>ned<br />
variety, and dice or slice, unpeeled, <strong>in</strong>to 500 ml of water. Steam or boil<br />
for 30 to 45 m<strong>in</strong>. At the same time, melt the agar <strong>in</strong> 500 ml of water.<br />
Stra<strong>in</strong> the potato through several layers of cheese cloth <strong>in</strong>to the flask<br />
conta<strong>in</strong><strong>in</strong>g the melted agar. Squeeze some potato pulp through also.<br />
Add the glucose, mix thoroughly, and make up to 1 litre with water if<br />
necessary. Autoclave at 121°C for 15 m<strong>in</strong>. F<strong>in</strong>al pH 5.6 ± 0.1.<br />
SNA: Synthetischer nährstoffarmer agar (Nirenberg, 1976; Samson<br />
et al., 2004)<br />
KH PO 2 4<br />
KNO3 MgSO c 4<br />
1.0 g<br />
1.0 g<br />
. 7H O 2<br />
KCl<br />
0.5 g<br />
0.5 g<br />
Glucose 0.2 g<br />
Sucrose 0.2 g<br />
Agar 20 g<br />
Water, distilled 1 litre<br />
Note: pieces of sterile filter paper may be placed on the agar.<br />
Recommended for cultivation of Fusarium, but also for poorly<br />
sporulat<strong>in</strong>g Deuteromycetes.
356 Appendix 1 – Media<br />
V8: V8 juice agar (Simmons, 1992; Samson et al., 2004)<br />
V-8 juice 200 ml<br />
CaCO3 Water, distilled<br />
3 g<br />
1 litre<br />
V-8 juice is vegetable juice, available commercially (Campbell’s Soup<br />
Company). Add <strong>in</strong>gredients, mix well and autoclave at 110°C for 30<br />
m<strong>in</strong>.<br />
YES: Yeast Extract Sucrose agar (YES) (Frisvad and Filtenborg,<br />
1983)<br />
Yeast extract (Difco) 20 g<br />
Sucrose 150 g<br />
MgSO 4 c . 7H 2 O 0.5 g<br />
ZnSO 4 c . 7H 2 O 0.01 g<br />
CuSO 4 c . 5H 2 O 0.005 g<br />
Agar 20 g<br />
Distilled water l litre<br />
Recommended for secondary metabolite analysis. Can be modified<br />
by add<strong>in</strong>g 0.5 g MgSO 4 .7H 2 O.<br />
REFERENCES<br />
Abildgren, M. P., Lund, F., Thrane, U., and Elmholt, S., 1987, Czapek-Dox agar conta<strong>in</strong><strong>in</strong>g<br />
iprodione and dicloran as a selective medium for the isolation of Fusarium<br />
species, Lett. Appl. Microbiol. 5:83-86.<br />
Andrews, S., and Pitt, J. I., 1986, Selective medium for isolation of Fusarium<br />
species and dematiaceous hyphomycetes from cereals, Appl. Environ. Microbiol.<br />
51:1235-1238.<br />
El-Banna, A. A., and Leistner, L., 1988, Production of penitrem A by Penicillium<br />
crustosum isolated from foodstuffs, Int. J. <strong>Food</strong> Microbiol. 7: 9-17.<br />
Frisvad, J. C., 1983, A selective and <strong>in</strong>dicative medium for groups of Penicillium viridicatum<br />
produc<strong>in</strong>g different mycotox<strong>in</strong>s on cereals, J. Appl. Bacteriol. 54:409-416.<br />
Frisvad, J. C., and Filtenborg, O., 1983, Classification of terverticillate Penicillia<br />
based on profiles of mycotox<strong>in</strong>s and other secondary metabolites, Appl. Environ.<br />
Microbiol. 46: 1301-1310.<br />
Hock<strong>in</strong>g, A. D., and Pitt, J. I., 1980, Dichloran-glycerol medium for enumeration of<br />
xerophilic fungi from low moisture foods, Appl. Environ. Microbiol. 39:488-492.<br />
Nirenberg, H., 1976, Untersuchungen über die morphologische und biologische<br />
Differenzierung <strong>in</strong> der Fusarium-Sektion Liseola, Mitteilungen aus der Biologische<br />
Bundesanstalt für Land-und Forstwirtschaft. Berl<strong>in</strong>-Dahlem 169:1-117.<br />
Pitt, J. I., 1979, The Genus Penicillium and its Teleomorphic States Eupenicillium and<br />
Talaromyces, Academic Press, London.
Appendix 1 – Media 357<br />
Pitt, J. I., and Hock<strong>in</strong>g, A. D., 1997, Fungi and <strong>Food</strong> Spoilage, 2nd edition, Blackie<br />
Academic and Professional, London.<br />
Pitt, J. I., Hock<strong>in</strong>g, A. D., and Glenn, D. R., 1983, An improved medium for the<br />
detection of Aspergillus flavus and A. parasiticus, J. Appl. Bacteriol. 54:109-114.<br />
Samson, R. A., Hoekstra, E. S., and Frisvad, J. C., eds, 2004, Introduction to <strong>Food</strong>and<br />
Airborne Fungi, 7th edition, Centraalbureau voor Schimmelcultures, Utrecht,<br />
Netherlands, 389 pp.<br />
Simmons, E. G., 1992, Alternaria taxonomy: current status, viewpo<strong>in</strong>t, challenge, <strong>in</strong>:<br />
Alternaria Biology, Plant Diseases and Metabolites, J. Chelkowski and A. Visconti,<br />
eds, Elsevier, Amsterdam, pp. 1-35.
APPENDIX 2 – INTERNATIONAL<br />
COMMISSION ON FOOD MYCOLOGY<br />
Aims<br />
The aims of the Commission are:<br />
(1) to improve and standardise methods for isolation, enumeration<br />
and identification of fungi <strong>in</strong> foods;<br />
(2) to promote studies of the mycological ecology of foods and<br />
commodities;<br />
(3) to <strong>in</strong>teract with regulatory bodies, both national and <strong>in</strong>ternational,<br />
concern<strong>in</strong>g standards for mycological quality <strong>in</strong> foods and<br />
commodities;<br />
(4) to support regional <strong>in</strong>itiatives <strong>in</strong> this area.<br />
The Commission further aims to extend understand<strong>in</strong>g of the pr<strong>in</strong>ciples<br />
and methodology of food mycology <strong>in</strong> the scientific community<br />
by publish<strong>in</strong>g its f<strong>in</strong>d<strong>in</strong>gs, and by sponsor<strong>in</strong>g meet<strong>in</strong>gs, specialist<br />
workshops, courses and sessions deal<strong>in</strong>g with aspects of its work.<br />
Members, 2003<br />
Chairman<br />
Dr Ailsa D. Hock<strong>in</strong>g, <strong>Food</strong> Science Australia, PO Box 52, North<br />
Ryde, NSW 1670, Australia<br />
Secretary<br />
Dr John I. Pitt, <strong>Food</strong> Science Australia, PO Box 52, North Ryde,<br />
NSW 1670, Australia<br />
Treasurer<br />
Dr Robert A. Samson, Centraalbureau voor Schimmelcultures, P.O.<br />
Box 85167, 3508 AG Utrecht, Netherlands<br />
358
Appendix 2 – International Commission on <strong>Food</strong> <strong>Mycology</strong> 359<br />
Members<br />
Dr Larry R. Beuchat, Center for <strong>Food</strong> Safety, University of<br />
Georgia, Griff<strong>in</strong>, GA 30223-1797, USA<br />
Dr Lloyd B. Bullerman, Dept of <strong>Food</strong> Science, University of<br />
Nebraska, L<strong>in</strong>coln, NE 68583-0919, USA<br />
Dr Maribeth A. Cous<strong>in</strong>, <strong>Food</strong> Science Department, Purdue<br />
University, West Lafayette, IN 47907-1160, USA<br />
Dr Tibor Deak, Dept of Microbiology, University of Horticulture,<br />
Somloi ut 14-16, Budapest, Hungary<br />
Dr Ole Filtenborg, Biocentrum-DTU, Technical University of<br />
Denmark, 2800 Lyngby, Denmark<br />
Dr Graham H. Fleet, <strong>Food</strong> Science and Technology, School of<br />
Chemical Eng<strong>in</strong>eer<strong>in</strong>g and Industrial Chemistry, University of New<br />
South Wales, Sydney, NSW 2052, Australia<br />
Dr Jens C. Frisvad, Biocentrum-DTU, Technical University of<br />
Denmark, 2800 Lyngby, Denmark<br />
Dr Narash Magan, Institute of Bioscience and Technology,<br />
Cranfield University, Silsoe, Bedfordshire MK45 4DT, United<br />
K<strong>in</strong>gdom<br />
Dr Ludwig Neissen, Inst. für Technische Mikrobiologie, Technische<br />
Univ. München, 85350 Freis<strong>in</strong>g, Germany<br />
Dr Monica Olsen, National <strong>Food</strong> Authority, P.O. Box 622, 751 26<br />
Uppsala, Sweden<br />
Dr Emilia Rico-Munoz, BCN Research Laboratories, P.O. Box<br />
50305, Knoxville, TN 37950, U.S.A.<br />
Dr Johan Schnürer, Department of Microbiology, Swedish<br />
University of Agricultural Sciences, 750 07 Uppsala, Sweden<br />
Dr Marta Taniwaki, Instituto de Tecnologia de Alimentos, C.P. 138,<br />
Camp<strong>in</strong>as, SP 13073-001, Brazil<br />
Dr Ulf Thrane, Biocentrum-DTU, Technical University of<br />
Denmark, 2800 Lyngby, Denmark<br />
Dr Bennie C. Viljoen, Dept Microbiology and Biochemistry, Univ.<br />
of the Orange Free State, P.O. Box 339, Bloemfonte<strong>in</strong> 9300, South<br />
Africa<br />
Mr Tony P. Williams, Williams and Neaves, 28 Randalls Road,<br />
Leatherhead, Surrey KT22 7 TQ, United K<strong>in</strong>gdom
INDEX<br />
Aflatox<strong>in</strong>s, 5, 6, 33–35, 40, 130, 146<br />
analysis, 228<br />
co-occurrence with cyclopiazonic<br />
acid, 225, 226<br />
effects of a w on production, 226–233<br />
effects of temperature on production,<br />
226–233<br />
<strong>in</strong> olives, 203, 209<br />
<strong>in</strong> peanuts, 225<br />
<strong>in</strong>correct species, 34, 35<br />
species produc<strong>in</strong>g, 36<br />
AFLP, for yeast differentiation, 81, 85<br />
AFPA, 346<br />
Age of colony, effect on growth, 56<br />
Alkaloid agar, 212<br />
Allspice, antimicrobial activity, 263<br />
Altenuene, 146–148<br />
Alternaria spp., 138, 141–150<br />
<strong>in</strong> apples, 141<br />
<strong>in</strong> cherries, 144<br />
<strong>in</strong> fruit and gra<strong>in</strong>, 141–150<br />
tox<strong>in</strong>s, 22, 232<br />
alternata, 22<br />
arborescens, 142, 144, 145, 147<br />
citri, 22<br />
<strong>in</strong>fectoria, 143, 145, 149<br />
Alternaria spp. (Cont<strong>in</strong>ued)<br />
japonica, 22<br />
kikuchiana, 22<br />
longipes, 22<br />
mali, 22<br />
oryzae, 22<br />
solani, 22<br />
tenuissima, 22, 142, 144, 145, 147,<br />
149<br />
Alternariols, 146, 147, 148, 150<br />
Altertox<strong>in</strong>, 147<br />
Altranones, 23<br />
Andropogon sorghum, 8<br />
Antibiotic Y, 10–11, 146–148, 150<br />
Antifungal agents, 261–286<br />
activity, 266–268<br />
additive, 266–268<br />
antagonistic, 266–268<br />
synergistic, 266–268<br />
b<strong>in</strong>ary mixtures, 268–277<br />
chemicals from plants, 262<br />
comb<strong>in</strong>ations, 266<br />
effects on aflatox<strong>in</strong> formation, 263<br />
eugenol, from plants and<br />
herbs, 263<br />
factors affect<strong>in</strong>g activity, 265<br />
361
362 Index<br />
Antifungal agents (Cont<strong>in</strong>ued)<br />
from plants, 263<br />
mode of action, 264<br />
phenolic compounds, 264<br />
of sourdough bread cultures,<br />
307–315<br />
of lactic acid bacteria, 307–315<br />
natural, 261–262<br />
phenolics, naturally occurr<strong>in</strong>g, 263<br />
test<strong>in</strong>g activity, 266<br />
aga<strong>in</strong>st Aspergillus flavus, 268–276<br />
ternary mixtures, 269, 277–281<br />
traditional, 261<br />
Apple flowers, fungi <strong>in</strong>, 139<br />
Apple juice, patul<strong>in</strong> <strong>in</strong>, 18<br />
Apples, Alternaria tox<strong>in</strong>s <strong>in</strong>, 22<br />
Apples, Antibiotic Y <strong>in</strong>, 10<br />
Apples, spoilage fungi, 138, 139,<br />
142–144, 149–150<br />
Apricots, dried, fungi <strong>in</strong>, 182, 184<br />
Apricots, dried, ochratox<strong>in</strong> A <strong>in</strong>, 184<br />
Arthr<strong>in</strong>ium,<br />
aureum, 8<br />
phaeospermum, 8<br />
sacchari, 8<br />
saccharicola, 8<br />
sereanis, 8<br />
term<strong>in</strong>alis, 8<br />
Ascladiol, 148<br />
Ascospores, activation of, 253–258<br />
dormancy, 256–258<br />
effects of heat and pressure, 253–256<br />
germ<strong>in</strong>ation process, 251<br />
Aspergillic acid, 146, 147<br />
Aspergillus flavus and parasiticus agar<br />
(AFPA), 346, 349<br />
Aspergillus spp.,<br />
<strong>in</strong> barley and wheat, 145<br />
section Circumdati, 9, 19, 38<br />
section Nigri, 153, 167, 174<br />
tox<strong>in</strong>s, 5–10<br />
aculeatus, 21, 154, 176<br />
alliaceus, 176, 178<br />
bombycis, 6<br />
caespitosus, 10<br />
candidus, 8, 145, 147<br />
carbonarius<br />
<strong>in</strong> air, 156, 157, 163<br />
<strong>in</strong> coffee, 190, 193, 194<br />
Aspergillus spp. (Cont<strong>in</strong>ued)<br />
<strong>in</strong> dried fruit, 181, 182, 184–186<br />
<strong>in</strong> soil, 156–157, 161–163<br />
<strong>in</strong> v<strong>in</strong>eyards, 153–154, 161–162,<br />
167<br />
ochratox<strong>in</strong> production, 8–9, 178,<br />
321<br />
on grapes, 153–158, 165–168,<br />
174–178, 181, 186<br />
survival on grapes, 157,<br />
carneus, 17<br />
clavatus, 7,19<br />
cretensis, 9<br />
flavipes, 176<br />
flavus, 130<br />
aflatox<strong>in</strong>s, 5–6, 147<br />
cyclopiazonic acid, 5–7, 147<br />
effect of antifungals, 268–276,<br />
309–314<br />
growth and tox<strong>in</strong> production,<br />
226, 231<br />
growth, measurement, 51, 52, 56,<br />
58–61, 65<br />
growth, predictive modell<strong>in</strong>g,<br />
287–306<br />
<strong>in</strong> gra<strong>in</strong>, 145<br />
<strong>in</strong> grapes, 176<br />
<strong>in</strong> olives, 203<br />
tox<strong>in</strong>s, 8<br />
floccosus, 9<br />
fumigatus, 7, 10, 176,<br />
giganteus, 19<br />
lactocoffeatus, 9<br />
longivesica, 19<br />
melleus, 9, 176, 178<br />
niger,<br />
<strong>in</strong> cereals, 145, 147,<br />
<strong>in</strong> coffee, 190, 193<br />
<strong>in</strong> dried fruit, 181, 184, 186<br />
<strong>in</strong> grapes, 154, 175, 177, 186<br />
ochratox<strong>in</strong> production, 8–9, 154,<br />
178, 322–324<br />
nomius, 5,6<br />
ochraceoroseus, 6<br />
ochraceus<br />
effect of antifungals, 309–312<br />
growth, predictive modell<strong>in</strong>g,<br />
287–306<br />
<strong>in</strong> coffee, 190, 193, 194
Index 363<br />
Aspergillus spp. (Cont<strong>in</strong>ued)<br />
<strong>in</strong> dried fruit, 181–182, 184–187<br />
<strong>in</strong> grapes, 176<br />
mycotox<strong>in</strong>s, 22<br />
ochratox<strong>in</strong> production, 8–9, 17,<br />
18, 173, 194, 322–324<br />
physiology and ochratox<strong>in</strong> production,<br />
324–325<br />
oryzae, cyclopiazonic acid production,<br />
7<br />
β-nitropropionic acid production,<br />
7<br />
ostianus, 9, 176, 178<br />
parasiticus, 5–7, 176, 203<br />
growth, predictive modell<strong>in</strong>g,<br />
287–306<br />
parvisclerotigenus, 5, 6<br />
persii, 9<br />
pseudoelegans, 9<br />
pseudotamarii, 7<br />
rambellii, 6<br />
roseoglobulosus, 9<br />
sclerotioniger, 9<br />
sclerotiorum, 9<br />
steynii, 22<br />
sulphureus, 9<br />
ochratox<strong>in</strong> production by, 322<br />
tamarii, 7, 176<br />
terreus, 17, 19, 176, 204<br />
toxicarius, 6<br />
ustus, 176<br />
versicolor, 10, 176, 206, 207<br />
wentii, 8, 176<br />
westerdijkiae, 8,22<br />
Aureobasidium pullulans, 81<br />
Aurofusar<strong>in</strong>, 147, 148, 150<br />
a w see Water activity<br />
Barley, cyctochalas<strong>in</strong> <strong>in</strong>, 7<br />
Barley, surface dis<strong>in</strong>fection, 140<br />
Beauvaric<strong>in</strong>, 12, 146, 147<br />
Beauveria bassiana, 12<br />
Beta-nitropropionic acid (BNP), 7<br />
Bipolaris sorok<strong>in</strong>iana, 143, 145, 147<br />
Bipolaris spp., <strong>in</strong> gra<strong>in</strong>, 143<br />
BNP, 7<br />
Botrytis<br />
<strong>in</strong> apples, 141, 144<br />
<strong>in</strong> cherries, 142, 144<br />
<strong>in</strong> fruit and gra<strong>in</strong>, 141–145, 149<br />
Brettanomyces, 73, 79<br />
Butenolide, 11<br />
Byssochlamic acid, 147, 211, 219<br />
Byssochlamys spp., 110, 144, 147,<br />
211–224, 247<br />
heat resistance of, 215, 219–220<br />
morphological characteristics, 216<br />
multivariate analysis of, 215<br />
mycotox<strong>in</strong> production, 219<br />
secondary metabolites of, 215<br />
taxonomy, 212–224<br />
divaricatum, 213, 214, 217, 218, 219,<br />
221, 222<br />
fulva<br />
effect of high pressure process<strong>in</strong>g,<br />
240–241, 243–245<br />
heat resistance, 248<br />
measurement of growth, 51–59<br />
method for detection, 107<br />
taxonomy, 212, 213, 216–221<br />
lagunculariae, 213, 217–221<br />
nivea, 51–54, 56, 58–61, 107, 211,<br />
213, 216–221, 253<br />
heat resistance, 248<br />
mycophenolic acid, 18, 219<br />
patul<strong>in</strong> production, 19, 219, 221<br />
pressure resistance, 253<br />
spectabilis, 213, 217–222<br />
heat resistance, 221–223, 247, 248<br />
verrucosa, 214, 216, 217–221<br />
zollneriae, 212, 214, 216–221<br />
Byssochlamysol, 211<br />
Byssotox<strong>in</strong> A, 211<br />
Candida spp., 73, 78, 81<br />
albicans, 78, 81,<br />
boid<strong>in</strong>ii, 83<br />
catenulata, 81<br />
dubl<strong>in</strong>ensis, 81<br />
krusei, 78, 79<br />
lambica, 82<br />
mesenterica, 83<br />
parapsilosis, 78<br />
sake, 83<br />
stellata, 77, 82, 83<br />
tropicalis, 78<br />
v<strong>in</strong>i, 81<br />
zelanoides, 82<br />
Carvacrol, antimicrobial activity,<br />
269–282
364 Index<br />
Cellulase, from F. culmorum, 133–134<br />
Cereals<br />
citr<strong>in</strong><strong>in</strong> <strong>in</strong>, 17<br />
cyclopiazonic acid <strong>in</strong>, 17<br />
fungi <strong>in</strong>, 137–150<br />
Fusarium tox<strong>in</strong>s <strong>in</strong>, 30<br />
mycotox<strong>in</strong>s <strong>in</strong>, 23<br />
ochratox<strong>in</strong> A <strong>in</strong>, 9<br />
Chaetoglobos<strong>in</strong>s, 16, 146–148<br />
Chaetomium globosum, 16<br />
Chardonnay grapes, Aspergillus carbonarius<br />
on, 163–165<br />
Cheese, mycophenolic acid <strong>in</strong>, 18<br />
Chemilum<strong>in</strong>escent <strong>in</strong> situ hybridisation,<br />
75, 78<br />
Cherries,<br />
Antibiotic Y <strong>in</strong>, 11<br />
fungi <strong>in</strong>, 137, 143<br />
Cherry flowers, fungi <strong>in</strong>, 137<br />
Chit<strong>in</strong>, 50<br />
C<strong>in</strong>namon, antimicrobial activity, 263,<br />
267–282<br />
effect on aflatox<strong>in</strong> production, 263<br />
CISH, 75, 78<br />
Citeromyces spp., 73<br />
Citr<strong>in</strong><strong>in</strong>, 17, 23, 40, 146, 147, 203–208<br />
analysis, 205–206<br />
<strong>in</strong> olives, 204–209<br />
<strong>in</strong>correct species, 36<br />
LD 50 , 204<br />
species produc<strong>in</strong>g, 36<br />
Citroevirid<strong>in</strong>, 16–17<br />
Cladosporium, <strong>in</strong> fruit and gra<strong>in</strong>,<br />
141–145, 149<br />
Claviceps spp.,<br />
tox<strong>in</strong>s, 22<br />
paspali, 22<br />
purpurea, 21, 22<br />
Clavispora opuntiae, 74<br />
CMA, 212, 349<br />
Coconut cream agar (CCA), for ochratox<strong>in</strong><br />
screen<strong>in</strong>g, 158<br />
Coffee beans, mycotoxigenic fungi <strong>in</strong>,<br />
22<br />
Coffee<br />
control of ochratox<strong>in</strong> A <strong>in</strong>, 199<br />
dehull<strong>in</strong>g, 195<br />
dry<strong>in</strong>g, 194–195<br />
ochratoxigenic fungi <strong>in</strong>, 190<br />
ochratox<strong>in</strong> A <strong>in</strong>, 9, 189–202<br />
Coffee (Cont<strong>in</strong>ued)<br />
penicillic acid <strong>in</strong>, 28<br />
production and process<strong>in</strong>g, 193–198<br />
roast<strong>in</strong>g, 197<br />
effect on ochratox<strong>in</strong> A, 197–198<br />
storage, 196<br />
Colony diameter, 50, 51, 52, 56,<br />
57, 59<br />
Communes<strong>in</strong>s, 147<br />
Cornmeal agar (CMA), 212, 349<br />
Cottonseed, aflatox<strong>in</strong>s <strong>in</strong>, 5<br />
CPA, see Cyclopiazonic acid<br />
Creat<strong>in</strong>e sucrose agar (CREA), 139,<br />
212, 350<br />
Cryptococcus<br />
laurentii, 81<br />
neoformans, 79, 81<br />
Culmor<strong>in</strong>, 11, 146, 147<br />
Culture collections, 41<br />
CY20S, see Czapek Yeast Extract 20%<br />
sucrose agar<br />
Cyclic peptides, 12<br />
Cyclic pept<strong>in</strong>es, 17<br />
Cyclic trichothecenes, 23<br />
Cyclochlorot<strong>in</strong>e, 17<br />
Cyclopiazonic acid, 5–6, 17, 40, 146,<br />
147, 225–233<br />
analysis, 228<br />
co-occurrence with aflatox<strong>in</strong>, 225,<br />
226<br />
effects of a w on production, 225–233<br />
effects of temperature on production,<br />
225–233<br />
<strong>in</strong> peanuts, 225–233<br />
<strong>in</strong>correct species produc<strong>in</strong>g, 37, 38<br />
production by Aspergillus flavus,<br />
225–233<br />
production by Penicillium commune,<br />
230<br />
species produc<strong>in</strong>g, 36<br />
Cytochalas<strong>in</strong> E, 7<br />
Czapek concentrate, 350<br />
Czapek Dox agar, 139, 350<br />
Czapek Iprodione Dichloran agar<br />
(CZID), 138, 346, 351– 352<br />
Czapek trace metal solution, 350<br />
Czapek Yeast Autolysate agar (CYA),<br />
42, 212, 351<br />
Czapek Yeast Extract 20% sucrose<br />
agar (CY20S), 51
Index 365<br />
Czapek Yeast Extract agar (CYA), 51,<br />
139, 175<br />
D1/D2 doma<strong>in</strong>, 71, 73<br />
DAS, 15<br />
Debaryomyces, 73<br />
hansenii, 78, 81, 82<br />
Decimal reduction time,<br />
247<br />
Dekkera, 73, 79<br />
anomala, 83<br />
bruxellensis, 78, 83<br />
Deoxynivalenol, 14, 128, 130,<br />
132–133, 150<br />
analysis for, 126<br />
production by Fusarium culmorum,<br />
128, 131, 132–133<br />
DG18, see Dichloran 18% glycerol<br />
agar<br />
DGGE, for yeast, 86–90<br />
Diacetoxyscirpenol, 12, 15<br />
Dichloran 18% glycerol agar (DG18),<br />
138, 182, 345<br />
Dichloran Chloramphenicol Peptone<br />
agar (DCPA), 346<br />
Dichloran Rose Bengal<br />
Chloramphenicol agar (DRBC),<br />
174, 175<br />
Dichloran Rose Bengal Yeast Extract<br />
sucrose agar (DRYES), 138–139,<br />
346<br />
Diluents, 344, 346<br />
Dilution plat<strong>in</strong>g, 343–347<br />
Direct plat<strong>in</strong>g, 344<br />
Dot blot, 75, 78<br />
DRBC, see Dichloran Rose Bengal<br />
Chloramphenicol agar<br />
DRYES, see Dichloran Rose Bengal<br />
Yeast Extract sucrose agar<br />
Ear blight, Fusarium, 123, 124<br />
Emericella<br />
astellata, 6, 176<br />
nidulans, 176<br />
variecolor, 176<br />
venezeulensis, 6<br />
Emod<strong>in</strong>, 219<br />
Enniat<strong>in</strong>s, 12, 146, 147<br />
Enzyme activity, 126<br />
Enzymes,<br />
from Fusarium culmorum, 132–134<br />
fungal, 124<br />
analysis for, 126–127<br />
Epicoccum nigrum, 144, 145<br />
Equ<strong>in</strong>e mycotoxicoses, 23<br />
Ergosterol, 50, 51, 57, 58, 62–65<br />
and hyphal length, 51, 62–65<br />
and mycelium dry weight, 61, 62–65<br />
assay, 52–53<br />
validation, 55<br />
Ergot alkaloids, 22<br />
Essential oils, effect on fumonis<strong>in</strong> production,<br />
118<br />
Eugenol, antimicrobial activity, 263<br />
aga<strong>in</strong>st Aspergillus flavus, 269–276<br />
Eupenicillium spp., 110<br />
c<strong>in</strong>namopurpureum, 17<br />
Eurotium<br />
<strong>in</strong> barley and wheat, 142–146<br />
amstelodami, 176<br />
chevalieri, 51, 52, 54–56, 58–61, 64,<br />
248<br />
heat resistance, 248<br />
Eurotium herbariorum, heat resistance,<br />
248<br />
Eurotium repens, ascospore activation,<br />
255<br />
Facial eczema, 23<br />
Fescue foot, 11<br />
Figs<br />
fungi <strong>in</strong> dried, 182, 184<br />
ochratox<strong>in</strong> A <strong>in</strong>, 9, 184, 185<br />
F<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g, 85–86<br />
Fruit, fungi <strong>in</strong>, 137–150<br />
Fumitremorg<strong>in</strong>s, 10<br />
Fumonis<strong>in</strong>s, 13, 115, 128, 150<br />
<strong>in</strong> maize, 116<br />
Fungal growth<br />
colony form<strong>in</strong>g units, 50<br />
comparison of methods, 57–58, 59<br />
measurement of, 65<br />
Fungicides<br />
for Fusarium control, 124<br />
on apples and cherries, 138,<br />
142, 149<br />
on grapes, 168<br />
Fusaprolifer<strong>in</strong>, 13
366 Index<br />
Fusarenon X, 15<br />
Fusar<strong>in</strong> C, 146, 147<br />
Fusarium<br />
ear blight, 123<br />
<strong>in</strong> fruit and gra<strong>in</strong>, 141–150<br />
section Liseola, 113–122<br />
tox<strong>in</strong>s, 10–16<br />
acum<strong>in</strong>atum, 12, 13<br />
anthophilum, 13<br />
avenaceum, 10–12, 14, 142–145, 147<br />
chlamydosporum, 11<br />
crookwellense, 11, 15, 16<br />
culmorum, 11, 14–16, 123–136, 144,<br />
145, 147<br />
a w effects, 123, 128–129<br />
deoxynivalenol production, 128,<br />
130,<br />
enzyme production, 124, 133–134<br />
growth temperatures, 123,<br />
128–129<br />
nivalenol production, 128,<br />
dlam<strong>in</strong>ii, 12<br />
equiseti, 12, 15, 16, 144, 145, 147<br />
globosus, 13<br />
gram<strong>in</strong>earum, 11, 14–16, 145, 147<br />
guttiforme, 13<br />
langsethiae, 11, 13, 15, 145, 147<br />
lateritium, 11, 13, 142, 145, 147<br />
longipes, 12<br />
moniliforme, 13<br />
napiforme, 13, 14<br />
nivale, 11<br />
nygamai, 12–14<br />
oxysporum, 12, 14, 51, 52, 54,<br />
55–60, 62, 65, 107, 240–243<br />
effect of high pressure process<strong>in</strong>g,<br />
240–243<br />
poae, 11, 13, 15, 144, 145, 147<br />
proliferatum, 12–14, 116–119, 124,<br />
128, 135<br />
effect of compet<strong>in</strong>g species,<br />
118–119<br />
effect of substrate, 116<br />
effect of temperature, 116–117,<br />
119<br />
effect of water activity, 116–117,<br />
119<br />
pseudocirc<strong>in</strong>atum, 13<br />
pseudogram<strong>in</strong>earum, 14<br />
pseudonygami, 13<br />
Fusarium (Cont<strong>in</strong>ued)<br />
sambuc<strong>in</strong>um, 11, 12, 15<br />
sporotrichioides, 11, 13, 15, 145, 147<br />
subglut<strong>in</strong>ans, 12–14<br />
thaps<strong>in</strong>um, 13, 14<br />
tric<strong>in</strong>ctum, 10, 11, 14, 144, 145, 147<br />
venenatum, 11, 15<br />
verticillioides, 12–14, 116–120, 124,<br />
128, 135<br />
effect of compet<strong>in</strong>g species,<br />
118–119<br />
effect of substrate, 116<br />
effect of temperature, 116–117, 119<br />
effect of water activity, 116–117,<br />
119<br />
Fusarochromanone, 146, 147<br />
Geotrichum candidum, 81, 82<br />
Gibberella fujikuroi complex, 12<br />
G<strong>in</strong>ger, mycophenolic acid <strong>in</strong>, 18<br />
Glassy state, <strong>in</strong> ascospores, 256<br />
Gliocladium virens, 7<br />
Gliotox<strong>in</strong>, 7<br />
Gra<strong>in</strong>, fungi <strong>in</strong>, 137–150<br />
Grapes, fungi <strong>in</strong>, 153–168, 174–179,<br />
ochratox<strong>in</strong> A <strong>in</strong>, 8, 154–156<br />
Growth, predictive modell<strong>in</strong>g, 287–306<br />
Halosarpeia sp., 12<br />
Ham, ochratox<strong>in</strong> A <strong>in</strong>, 9, 18<br />
Hamigera, 110<br />
Hanseniaspora, 76, 82<br />
uvarum, 83<br />
Hay <strong>in</strong>fusion agar (HAY), 212, 214,<br />
353<br />
Heat resistance, of<br />
Byssochlamys species, 215, 248<br />
Byssochlamys spectabilis, 220–222,<br />
247, 248<br />
Eurotium chevalieri, 248<br />
Eurotium herbariorum, 248<br />
Monascus ruber, 247<br />
Neosartorya fischeri, 248<br />
Neosartorya pseudofischeri, 248<br />
Talaromyces flavus, 249<br />
Talaromyces helicus, 249<br />
Talaromyces macrosporus, 248–250<br />
Talaromyces stipitatus, 249<br />
Talaromyces trachyspermus, 249<br />
Xeromyces bisporus, 249
Index 367<br />
Heat resistant fungi, 211–224<br />
<strong>in</strong>cubation time, 110<br />
methods for, 107–111, 346–348<br />
High pressure process<strong>in</strong>g, effect on<br />
ascospores, 251–256<br />
effect on fungi, 239–246, 251–254<br />
Homogenisation, 344<br />
Horses, mycotoxicoses, 23<br />
Humicola fuscoatra, 10<br />
Hybridisation, 75<br />
Hyphal length, 49, 53, 57–59, 62–63<br />
ICFM, 343<br />
IGS region, of yeasts, 71, 73, 74<br />
International Commission on <strong>Food</strong><br />
<strong>Mycology</strong>, 343, 356–357<br />
Isaria fumorosea, 12<br />
Islanditox<strong>in</strong>, 17<br />
Isofumigaclav<strong>in</strong>, 147<br />
Issatchenkia<br />
orientalis, 81, 83<br />
terricola, 83<br />
ITS region, of yeasts, 71, 73, 74<br />
Katsuobushi, β-nitropropionic acid <strong>in</strong>,<br />
7<br />
Kloeckera, 76<br />
apiculata, 77, 82<br />
Kluveromyces spp., 73<br />
lactis, 81, 82<br />
marxianus, 73, 81, 82<br />
Kojic acid 7<br />
Lactobacillus spp., 307–315<br />
antifungal effects, 307–315<br />
on Aspergillus flavus, 309–314<br />
on Aspergillus ochraceus, 309–311<br />
on Penicillium commune, 309–313<br />
on Penicillium roqueforti, 309–313<br />
on Penicillium verrucosum, 309–312<br />
Lup<strong>in</strong>osis tox<strong>in</strong>, 22<br />
Lup<strong>in</strong>s, tox<strong>in</strong>s <strong>in</strong>, 23<br />
Luteoskyr<strong>in</strong>, 17<br />
Maize<br />
aflatox<strong>in</strong>s <strong>in</strong>, 5<br />
fumonis<strong>in</strong>s <strong>in</strong>, 13, 116<br />
temperature effect, 117–118<br />
water activity effect, 116–118<br />
Malform<strong>in</strong>s, 147<br />
Malt Extract agar (MEA), 51, 139,<br />
174, 182, 205, 212, 346, 354<br />
Malt Extract Yeast Extract 50% glucose<br />
agar (MY50G), 51, 345, 354<br />
MAS 15<br />
MEA, 51, 139, 174, 175, 182, 205,<br />
212, 346, 354<br />
Media recommended for food mycology,<br />
345–346,<br />
Media for<br />
aflatoxigenic fungi, 346, 349<br />
Fusarium, 346, 351–352<br />
toxigenic Penicillium species, 346<br />
xerophilic fungi, 345–346, 352, 354<br />
yeasts, 346–347<br />
Media, formulations, 349–356<br />
Media, general purpose, 345<br />
Merck’s Malt Extract Agar (MME),<br />
42, 354<br />
Metabolite profil<strong>in</strong>g, of fungi, 140–141<br />
Methods for food mycology, 343–348<br />
Methods for yeasts, 346–347<br />
Metschnikowia spp., 81<br />
Metschnikowia pulcherrima, 77, 82, 83,<br />
90<br />
Microsatellites, <strong>in</strong> yeasts, 81–84, 85<br />
Miso, β-nitropropionic acid <strong>in</strong>, 7<br />
MME, see Merck’s Malt Extract Agar<br />
Molecular methods for yeasts, 69–106<br />
factors affect<strong>in</strong>g performance,<br />
91–94<br />
standardisation, 94–95<br />
Monascus ruber, 23, 204, 247<br />
heat resistance of, 247<br />
Monascus tox<strong>in</strong>s, 23, 204<br />
Monilia, 144<br />
Moniliform<strong>in</strong>, 13–14, 146, 147<br />
Monoacetyoxscirpenol, 15<br />
Monocillium nord<strong>in</strong>ii, 10<br />
Mrakia spp., 74<br />
Mucor plumbeus, 51, 52, 56–62, 245<br />
Multivariate analysis of Byssochlamys,<br />
215<br />
MY50G, 51, 345, 354<br />
Mycelium dry weight and hyphal<br />
length, 60<br />
Mycelium dry weight, 50, 52, 57, 59,<br />
60, 62
368 Index<br />
Mycophenolic acid, 18, 219, 222<br />
Mycotoxigenic fungi<br />
correct identification, 40<br />
reference cultures, 40<br />
Mycotox<strong>in</strong>s<br />
confirmation procedures, 41–42<br />
<strong>in</strong> apples and cherries, 147<br />
<strong>in</strong> cereals, 146<br />
production conditions, 41<br />
significant, 23<br />
Naphtho-γ-pyrones, 147<br />
Neopetromyces muricatus, ochratox<strong>in</strong><br />
production by, 322–324<br />
Neosartorya spp., 107, 110, 247<br />
fischeri, 10, 240–241, 243–245, 258<br />
heat activation, 257-258<br />
heat resistance, 248<br />
<strong>in</strong>activation by high pressure,<br />
241, 243–245<br />
pseudofischeri, heat resistance, 248<br />
Nephrotoxic glycopeptodes, 146, 147<br />
Nivalenol, 14, 15, 128, 131<br />
analysis for, 126<br />
production by Fusarium culmorum,<br />
129, 133<br />
Ochratox<strong>in</strong> A, 8–9, 18, 40, 146, 147,<br />
150, 154, 160, 165– 167, 173, 174,<br />
181–188<br />
analysis, 160–161, 182–183<br />
control <strong>in</strong> coffee, 199<br />
effect of w<strong>in</strong>emak<strong>in</strong>g, 159–160,<br />
165–167<br />
fungi produc<strong>in</strong>g, 34, 179,<br />
322–324<br />
<strong>in</strong> barley, 142<br />
<strong>in</strong> cereals and products<br />
monitor<strong>in</strong>g, 329–330<br />
prevention, 317–322, 325–328<br />
reduction dur<strong>in</strong>g process<strong>in</strong>g,<br />
330–332<br />
<strong>in</strong> coffee, 189–202<br />
<strong>in</strong> dried fruit, 181–188<br />
<strong>in</strong> grapes, 154–155<br />
<strong>in</strong> raw coffee, 189<br />
<strong>in</strong> roasted coffee, 199<br />
<strong>in</strong> salami, 9, 18<br />
<strong>in</strong> soluble coffee, 193<br />
<strong>in</strong>correct species, 36<br />
Ochratox<strong>in</strong> (Cont<strong>in</strong>ued)<br />
<strong>in</strong>take from coffee, 198–199<br />
legislation <strong>in</strong> EC, 318<br />
limits for, <strong>in</strong> coffee, 189<br />
provisional tolerable daily <strong>in</strong>take,<br />
198–199<br />
removal, 155<br />
Olives<br />
aflatox<strong>in</strong>s <strong>in</strong>, 203–204<br />
citr<strong>in</strong><strong>in</strong> <strong>in</strong>, 204<br />
fungi <strong>in</strong>, 203–210<br />
mycotox<strong>in</strong>s <strong>in</strong>, 203–210<br />
production of, 204–205<br />
Onyalai 22<br />
Paecilomyces<br />
dactyloerythromorphus, 214, 217,<br />
218, 220–222<br />
fumoroseus, 12<br />
maximus, 214, 220, 222<br />
variotii, 107, 218, 222<br />
taxonomy of, 152<br />
Patul<strong>in</strong>, 18–19, 40, 137, 146–148, 150,<br />
207, 219<br />
<strong>in</strong> apple juice, 137<br />
<strong>in</strong> olives, 207<br />
<strong>in</strong>correct species produc<strong>in</strong>g, 37<br />
species produc<strong>in</strong>g, 37<br />
PCR, <strong>in</strong> yeast identification, 71–95<br />
PCR, real time, 79<br />
PCR-based f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g, for yeasts,<br />
80–86<br />
PCR-ELISA, 78<br />
PDA, 51, 212 355<br />
Peanuts,<br />
aflatox<strong>in</strong>s <strong>in</strong>, 5, 225<br />
cyclopiazonic acid <strong>in</strong>, 225<br />
Penicillic acid, 19, 38, 40, 146, 147,<br />
207<br />
<strong>in</strong> olives, 207<br />
Penicill<strong>in</strong>, 33<br />
Penicillium<br />
<strong>in</strong> apples, 142<br />
<strong>in</strong> barley seed, 142<br />
series Viridicata, 19, 21<br />
tox<strong>in</strong>s, 16–22<br />
albocoremium, 20<br />
allii, 20<br />
atramentosum, 20<br />
atrovenetum, 8
Index 369<br />
Penicillium (Cont<strong>in</strong>ued)<br />
aurantiogriseum, 19, 21, 143, 145,<br />
147, 149<br />
brasilianum, 10<br />
brevicompactum, 18, 176, 207<br />
camemberti, 17<br />
carneum, 18, 19, 20, 147<br />
casei, 9,18<br />
chrysogenum, 8, 20, 149, 176<br />
citreonigrum, 17<br />
citr<strong>in</strong>um, 17, 176, 204, 207, 208<br />
clavigerum, 19, 20<br />
commune, 17, 51, 52, 54, 56, 58, 59,<br />
230<br />
effect of antifungals, 309–313<br />
concentricum, 19, 21<br />
confertum, 21<br />
coprobium, 19, 21<br />
coprophilum, 21<br />
crateriforme, 21<br />
crustosum, 20, 21, 142, 144, 147,<br />
206, 207<br />
cyclopium, 8, 19, 22, 143, 145, 149<br />
digitatum, 207<br />
dipodomycola, 17<br />
discolour, 16<br />
expansum, 16–18, 20, 142, 144, 147,<br />
150, 204<br />
effect of high pressure process<strong>in</strong>g,<br />
240–243, 245<br />
fagi, 18<br />
flavigerum, 21<br />
formosanum, 19<br />
freii, 22, 145, 147, 149<br />
glabrum, 176<br />
glandicola, 19–21<br />
griseofulvum, 17, 19, 20<br />
hirsutum, 20<br />
hordei, 20, 143, 145, 147<br />
islandicum, 17<br />
janczewskii, 20<br />
janth<strong>in</strong>ellum, 22<br />
lilac<strong>in</strong>oech<strong>in</strong>ulatum, 7<br />
mang<strong>in</strong>ii, 17<br />
mariaecrucis, 22<br />
mar<strong>in</strong>um, 16, 19, 21<br />
melanoconidium, 19–22, 145, 147<br />
mel<strong>in</strong>ii, 19<br />
miczynskii, 17<br />
mononematosum, 10<br />
Penicillium (Cont<strong>in</strong>ued)<br />
nordicum<br />
ochratox<strong>in</strong> production by, 9, 18,<br />
322–324<br />
novae-zeelandiae, 19<br />
odoratum, 17<br />
oxalicum, 21<br />
palitans, 17<br />
paneum, 19, 20<br />
persic<strong>in</strong>um, 21<br />
polonicum, 19, 21, 143, 144, 145,<br />
147, 149<br />
radicicola, 17, 19, 20<br />
roqueforti, 206, 207<br />
effect of antifungals, 309–313<br />
<strong>in</strong> olives, 206–207<br />
measurement of growth, 51, 52,<br />
54, 56, 57, 59–61,<br />
mycophenolic acid, 18<br />
PR tox<strong>in</strong>, 20<br />
roquefot<strong>in</strong>e C, 20<br />
sclerotigenum, 20<br />
sclerotiorum, 176<br />
smithii, 17<br />
solitum, 144, 207<br />
thomii, 176<br />
tricolor, 22<br />
tulipae, 19, 20<br />
venetum, 20<br />
verrucosum, 130, 176<br />
citr<strong>in</strong><strong>in</strong> production by, 17, 204,<br />
effect of antifungals, 309–312<br />
<strong>in</strong> cereals, 143, 145, 147, 149, 173,<br />
ochratox<strong>in</strong> production by, 9, 18,<br />
181, 322–324<br />
physiology and ochratox<strong>in</strong> production,<br />
324–325<br />
selective medium for, 346, 353<br />
viridicatum, 22, 145, 147, 206, 206,<br />
346<br />
selective medium for, 346<br />
vulp<strong>in</strong>um, 19, 21<br />
westl<strong>in</strong>gii, 17<br />
Penitrem A, 20, 40, 146, 147<br />
<strong>in</strong>correct species produc<strong>in</strong>g, 37<br />
species produc<strong>in</strong>g, 37<br />
Petromyces albertensis, 9<br />
Petromyces alliaceus, 9<br />
ochratox<strong>in</strong> production by, 322–324<br />
PFGE, 80
370 Index<br />
Phaffia, 74<br />
Phoma<br />
tenuazonic acid <strong>in</strong>, 22<br />
tox<strong>in</strong>s, 22–23<br />
sorgh<strong>in</strong>a, 22<br />
terrestris, 21<br />
Phomops<strong>in</strong>, 22<br />
Phomopsis tox<strong>in</strong>s, 22–23<br />
Phompsis leptostromiformis, 22<br />
Pichia spp., 73<br />
anomala, 78, 81, 240, 242–243, 245<br />
effect of high pressure process<strong>in</strong>g,<br />
240–243, 245<br />
galeiformis, 82<br />
guillermondii, 78<br />
kluyveri, 90<br />
membranifaciens, 81–83<br />
Pigs, mycotoxicosis <strong>in</strong>, 21<br />
Pithomyces chartarum, 23<br />
Pithomyces tox<strong>in</strong>s, 23<br />
Plat<strong>in</strong>g methods, for fungi, 343–344<br />
Plums<br />
fungi <strong>in</strong> dried, 182, 184<br />
ochratox<strong>in</strong> A <strong>in</strong> dried, 185<br />
Polymerase cha<strong>in</strong> reaction, see PCR<br />
Potato Carrot Agar (PCA), 139,<br />
355<br />
Potato Dextrose Agar (PDA), 51, 139,<br />
212, 355<br />
Potatoes, diacetoxyscirpenol <strong>in</strong>, 15<br />
PR tox<strong>in</strong>, 20<br />
Predictive modell<strong>in</strong>g, fungal growth,<br />
288–306<br />
Preservative resistant yeasts, methods<br />
for, 347<br />
Preservatives<br />
effect on fumonis<strong>in</strong> production,<br />
117–118<br />
effect on Fusarium growth, 117–118<br />
Primers, species-specific, for yeasts,<br />
75, 79<br />
Probes, nucleic acid, for yeasts, 75<br />
Provisional tolerable daily <strong>in</strong>take, for<br />
ochratox<strong>in</strong> A, 198–199<br />
Pulsed field gel electrophoresis<br />
(PFGE), 80<br />
RAPD, for yeast differentiation,<br />
81–83, 85<br />
RC, see Rice powder corn steep agar<br />
rDNA, 71<br />
Restriction enzymes, 74<br />
Restriction length fragment polymorphism,<br />
see RFLP, 71<br />
Resveratrol, and fumonis<strong>in</strong>s production,<br />
118<br />
RFLP, 72, 75–79, 80<br />
for yeast stra<strong>in</strong> differentiation, 80<br />
Rhodosporidium<br />
diobovatum, 78<br />
sphaerocarpum, 78<br />
Rhodotorula<br />
glut<strong>in</strong>is, 79, 82<br />
mucilag<strong>in</strong>osa, 79, 81<br />
rubra, 81<br />
Rhubarb w<strong>in</strong>e, rubratox<strong>in</strong> <strong>in</strong>, 21<br />
Ribosomal DNA sequenc<strong>in</strong>g, 71<br />
Rice powder corn steep agar (RC), 42<br />
Rice, citreovirid<strong>in</strong> <strong>in</strong>, 16<br />
Roquefort<strong>in</strong>e C, 20, 40, 146, 147<br />
Rosell<strong>in</strong>ia necatrrix, 7<br />
Rubratox<strong>in</strong>, 21, 39, 40<br />
Rugulos<strong>in</strong>, 17<br />
Rye, ergot alkaloids <strong>in</strong>, 22<br />
Saccharomyces, 73, 74, 76, 81, 83<br />
bayanus, 76, 77, 79, 81, 83, 89<br />
boulardii, 74<br />
brasiliensis, 77<br />
carlsbergensis, 77<br />
cerevisiae, 74, 76–79, 81–84, 89,<br />
240–242, 245<br />
effect of high pressure process<strong>in</strong>g,<br />
240–242, 245<br />
exiguus, 77, 81<br />
paradoxus, 76, 77, 89<br />
pastorianus, 77, 79, 81, 89<br />
uvarum, 77<br />
willianus, 81<br />
Saccharomycodes ludwigii, 83<br />
Salami, ochratox<strong>in</strong> A <strong>in</strong>, 9, 18<br />
Satratox<strong>in</strong>, 23<br />
Schizosaccharomyces pombe, 77, 82, 83<br />
Secalonic acid D, 21<br />
Secondary metabolites, of<br />
Byssochlamys, 215<br />
Sequenc<strong>in</strong>g of genes, 72<br />
Sheep, mycotoxicoses, 22, 23<br />
Shiraz grapes, Aspergillus carbonarius<br />
on, 163–165<br />
Shoyu, β-nitropropionic acid <strong>in</strong>, 7<br />
SNA, 355
Index 371<br />
Species-mycotox<strong>in</strong> associations, 3–24,<br />
<strong>in</strong>correct, 33–42<br />
Sporodesm<strong>in</strong>, 23<br />
Stachybotrys<br />
chartarum, 23<br />
chlorohalonata, 23<br />
tox<strong>in</strong>s, 23<br />
Stemphylium, 144, 145, 146<br />
Sterigmatocyst<strong>in</strong>, 9–10, 40, 147<br />
species produc<strong>in</strong>g, 35<br />
<strong>in</strong>correct species, 35<br />
Sugar cane, β-nitropropionic acid <strong>in</strong>,<br />
7<br />
Sultanas<br />
fungi <strong>in</strong>, 182, 184<br />
ochratox<strong>in</strong> A <strong>in</strong>, 184, 185<br />
Surface dis<strong>in</strong>fection, 344<br />
Synthetischer nährstoffarmer agar<br />
(SNA), 355<br />
T-2 tox<strong>in</strong>, 15<br />
Talaromyces<br />
avellaneus, 245<br />
flavus, heat resistance, 249<br />
helicus, 247<br />
heat resistance, 249<br />
macrosporus, 248–253, 255–256<br />
ascospore activation, 255, 258<br />
heat resistance, 248–250<br />
spectabilis, 215, 216<br />
stipitatus, 247<br />
heat resistance, 249<br />
trachyspermus, 107, 249<br />
heat resistance, 249<br />
Tenuazonic acid, 22, 146, 147<br />
Terphenyll<strong>in</strong>, 146, 147<br />
Tetrapisispora fleetii, 73<br />
TGGE, for yeast, 86–90<br />
TGY, 346<br />
Thymol, antimicrobial activity, 264<br />
aga<strong>in</strong>st Aspergillus flavus, 269–276<br />
Tomatoes, Alternaria tox<strong>in</strong>s <strong>in</strong>, 22<br />
Torulaspora, 73, 77<br />
Torulaspora delbrueckii, 79, 81–83<br />
Trehalose, <strong>in</strong> ascospores, 250, 256<br />
Trichosporon cutaneum, 79<br />
Trichothecenes, 14–15, 39, 146, 147<br />
Tryptone Glucose yeast extract agar<br />
(TGY), 346<br />
Turkey X disease, 225<br />
V8 juice agar, 138, 356<br />
Vanill<strong>in</strong>, antimicrobial activity, 263,<br />
268–282<br />
effect on growth of Aspergillus<br />
flavus, 280–304<br />
Verrucologen, 10<br />
Verrucosid<strong>in</strong>, 21, 40, 146, 147<br />
Verticllium hemipterigenum, 12<br />
Viomelle<strong>in</strong>, 19, 21–22, 38, 146, 147<br />
Vioxanth<strong>in</strong>, 19, 21–22, 38, 146, 147<br />
Viridic acid, 146, 147<br />
Viriditox<strong>in</strong>, 219<br />
Water activity, 51, 116–119, 183–184,<br />
effect on growth, 196<br />
effect on mycotox<strong>in</strong> production, 120<br />
Wheat, Antibiotic Y <strong>in</strong>, 11<br />
surface dis<strong>in</strong>fection, 140<br />
Whole cell hybridisation, fluorescent,<br />
78<br />
W<strong>in</strong>e fermentation, yeasts <strong>in</strong>, 77, 83,<br />
84, 88–89<br />
W<strong>in</strong>emak<strong>in</strong>g, effect on ochratox<strong>in</strong> A,<br />
159, 160, 165–167<br />
Xanthoasc<strong>in</strong>, 146, 147<br />
Xanthomegn<strong>in</strong>, 19, 21–22, 38, 40, 146,<br />
147<br />
Xeromyces bisporus, 51, 52, 55, 56, 59<br />
Xeromyces bisporus, heat<br />
resistance, 249<br />
Yarrowia lipolytica, 81, 82<br />
Yeast Extract Sucrose agar (YES), 42,<br />
139, 175, 214, 356<br />
Yeast<br />
f<strong>in</strong>gerpr<strong>in</strong>t<strong>in</strong>g, 85–86<br />
<strong>in</strong>activation by high pressure,<br />
242–243<br />
molecular identification, 96–106<br />
stra<strong>in</strong> differentiation, 80–84<br />
Yellow rice, 16<br />
YES, 42, 139, 175, 214, 356<br />
Zearalenone, 16, 146–148, 150<br />
Zygomycetes, 144, 146<br />
Zygosaccharomyces, 73<br />
bailii, 78, 81–83, 245<br />
bisporus, 79, 82<br />
rouxii, 79