International Journal of Food Science and Technology 2012
1
Original article
New applications for Schizosaccharomyces pombe in the alcoholic
fermentation of red wines
Santiago Benito,* Felipe Palomero, Antonio Morata, Fernando Calderón & José A. Suárez-Lepe
Depto. de Tecnologı́a de Alimentos, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Ciudad Universitaria
S ⁄ N, 28040 Madrid, Spain
(Received 28 March 2012; Accepted in revised form 2 April 2012)
Summary
The fermentation of grape must using non-Saccharomyces yeasts with particular metabolic and biochemical
properties is of growing interest. In the present work, red grape must was fermented using four strains of
Schizosaccharomyces pombe (935, 936, 938 and 2139), Saccharomyces cerevisiae 7VA and Saccharomyces
uvarum S6U, and comparisons were made over the fermentation period in terms of must sugar
(glucose + fructose), malic acid, acetic acid, ammonia, primary amino nitrogen, lactic acid, urea (a
possible fermentation activator or precursor of other metabolites) and pyruvic acid (a molecule affecting
vitisin formation and therefore colour stability) concentration. The colour intensity of the fermenting musts
was also recorded. The Schizosaccharomyces strains consumed less primary amino nitrogen and produced
less urea and more pyruvic acid than other Saccharomyces species. Further, three of the four
Schizosaccharomyces strains completed the breakdown of malic acid by day 4 of fermentation. The main
negative effect of the use of Schizosaccharomyces was strong acetic acid production. The Schizosaccharomyces strains that produced most pyruvic acid (938 and 936) were associated with better ‘wine’ colour than
the remaining yeasts. The studied Schizosaccharomyces could therefore be of oenological interest.
Keywords
Malic acid, pyruvic acid, Saccharomyces spp., Schizosaccharomyces pombe, urea.
Introduction
The yeasts of the genus Schizosaccharomyces have
traditionally been described as wine spoilage organisms
owing to their production of compounds with negative
sensorial impacts, such as acetaldehyde, H2S and volatile
acids (Gallander, 1977; Snow & Gallander, 1979; Unterholzner et al., 1988; Yokotsuka et al., 1993; Pitt &
Hocking, 1999). However, the industrial use of Schizosaccharomyces has been described in the fermentation of
cane sugar in rum-making (Pech et al., 1984; Fahrasmane
et al., 1988), the production of palm wine (Christopher &
Theivendirarajah, 1988; Sanni & Loenner, 1993) and
cocoa fermentation (Ravelomanana et al., 1984; Mazigh,
1994). The genus has also been studied at the laboratory
and semi-industrial scales in the winemaking industry
given the notable capacity of some of its members to
deacidify wines via the ability to metabolise malic acid
with the production of ethanol (Gallander, 1977; Snow &
Gallander, 1979; Sousa et al., 1993, 1995; Yokotsuka
*Correspondent: Fax: +34 91 336 57 46;
e-mail: santiago.benito@upm.es
et al., 1993; Gao & Fleet, 1995; Thornton & Rodrı́guez,
1996; Dharmadhikari & Wilker, 1998). In northerly
viticultural regions, where grape malic acid contents can
be high, the possible use of non-Saccharomyces yeasts,
such as Schizosaccharomyces spp., to reduce malic acid
concentrations is awakening much interest (Gallander,
1977; Magyar & Panik, 1989; Seo et al., 2007; Fleet, 2008;
Kim et al., 2008; Kunicka-Styczynska, 2009). Recently,
the OIV approved ‘deacidification by Schizosaccharomyces, (Resolution OENO ⁄ MICRO ⁄ 97 ⁄ 75 ⁄ phase 7), but
the number of commercial strains available for this is very
limited. Mixed and sequential cultures with Saccharomyces (Kim et al., 2008; Kunicka-Styczynska, 2009) have
been used to mitigate the negative effects of the currently
available Schizosaccharomyces strains’ scant oenological
aptitude (Unterholzner et al., 1988). Understanding how
to isolate and select more appropriate Schizosaccharomyces strains is therefore of great interest.
One of the new applications of Schizosaccharomyces is
ageing over lees, made possible by these yeasts’ strong
autolytic release of cell wall polysaccharides (Palomero
et al., 2009). Further, certain Schizosaccharomyces
mutants may be able to reduce the gluconic acid
doi:10.1111/j.1365-2621.2012.03076.x
2012 The Authors. International Journal of Food Science and Technology 2012 Institute of Food Science and Technology
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New Schizosaccharomyces pombe applications S. Benito et al.
contents of spoiled musts (Peinado et al., 2007, 2009).
The urease activity of Schizosaccharomyces spp. (Casas,
1999; Barnett et al., 2000; Deák, 2008) is also of interest
with respect to food safety; its production could reduce
high wine ethyl carbamate contents by reducing urea
concentrations (a precursor of ethyl carbamate)
(Uthurry et al., 2004).
To further our knowledge of the fermentative activity
of Schizosaccharomyces, the present work examined the
fermentation kinetics of four strains of Schizosaccharomyces pombe, along with the consumption of nitrogenated compounds and the production of acetic and
pyruvic acids. The must colour changes that occurred
over fermentation were also recorded.
Materials and methods
tents of the fermentations were monitored over a period
of 31 days. The colour intensity of the fermenting must
was also recorded. The urea concentration was determined at the end of fermentation.
Determination of glucose + fructose, malic acid, lactic acid,
acetic acid, ammonia, primary amino nitrogen, urea,
pyruvic acid and colour intensity
All analyses were undertaken using a Y15 Autoanalyzer
(Biosystems, Barcelona, Spain). Enzymatic analyses for
G + F, malic acid, lactic acid, acetic acid, ammonia,
PAN and urea, and the colour intensity analysis, were
performed using kits from Biosystems (http://www.bio
systems.es). Pyruvic acid was determined using the
appropriate kit (Megazyme, Bry, Ireland).
Yeast strains
Statistical analysis
The yeasts used in this study were Schizosaccharomyces
pombe strains 935, 936, 938 and 2139 from the type
collection of the Instituto de Fermentaciones Industriales (IFI, CSIC, Madrid, Spain), Saccharomyces cerevisiae 7VA from the collection of the Departamento de
Tecnologı́a de Alimentos de la Escuela Técnica Superior
de Ingenieros Agrónomos (Univeridad Politécnica,
Madrid, Spain) and Saccharomyces uvarum S6U supplied by the Lallemand company (Danstar Ferment,
Montreal, Canada).
Means and standard deviations were calculated, and
anova and least significant differences (LSD) tests were
performed using PC Statgraphics v.5 software (Graphics
Software Systems, Rockville, MD, USA). Significance
was set at P < 0.05 for the anova matrix F-value. The
multiple range test was used to compare the means.
Must preparation
All fermentations were performed using grape red must
stock (224 g L)1 glucose + fructose [G + F]) from the
Ribera del Duero denomination of origin region (grape
variety Vitis vinifera cv. Syrah). The malic acid content
of the stock was adjusted to 2 g L)1 (Panreac, Barcelona, Spain) via the addition of this compound (final pH
3.5).
Fermentations
Microfermentations were performed using 50 mL of
must inoculated with 1 mL of liquid YEDP medium
containing 108 cfu mL)1 (determined using a Thomas
chamber) of one of the above-mentioned yeasts. All
fermentations were performed in 100-mL flasks sealed
with a Müller valve filled with 98% H2SO4 (Panreac);
this allowed the release of CO2 while avoiding microbial
contamination (Vaughnan-Martini & Martini, 1999).
The temperature was maintained at 25 C. The fermentations proceeded without aeration, oxygen injection or
agitation. All fermentations were performed in triplicate.
The G + F, malic acid, acetic acid, ammonia,
primary amino nitrogen (PAN) and pyruvic acid con-
International Journal of Food Science and Technology 2012
Results and discussion
Glucose + fructose fermentation
Figure 1 shows the fermentation kinetics of the different
yeasts examined. Differences can be seen between the
members of Saccharomyces and Schizosaccharomyces.
Saccharomyces cerevisiae 7VA and S. uvarum S6U
finished fermentation on days 4 and 11, respectively,
although S6U left some residual sugar. Schizosaccharomyces pombe 935, 936, 938 and 2139 required 15 days
to complete fermentation, leaving very little residual
sugar. This result agrees with the high fermentative
power of this species reported by other authors (Peynaud & Sudraud, 1962; Suárez-Lepe & Iñigo, 2004). The
slower kinetics of Schizosaccharomyces would likely
allow the easier control of the temperature rises that
occur over fermentation.
Degradation of malic acid
Schizosaccharomyces pombe 936, 938 and 2139 consumed
all the malic acid present, while strain 935 reduced its
presence by 50% (Fig. 2). This result agrees with that
reported by other authors (75–100% depending on the
strain and culture medium) (Snow & Gallander, 1978;
Magyar & Panik, 1989; Gao & Fleet, 1995; Taillandier
et al., 1995; Thornton & Rodrı́guez, 1996; Silva et al.,
2003; De Fátima et al., 2007). Malic acid can be metabolised by species other than Schizosaccharomyces
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New Schizosaccharomyces pombe applications S. Benito et al.
240
7VA
S6U
935
936
938
2139
190
G+F (g L–1)
140
90
40
Figure 1 Consumption of glucose + fructose
a
by the studied yeast strains. Points are
means ± SD for three fermentations. Means
with the same letter are not significantly different (P > 0.05).
0
0
5
10
15
20
25
30
b
35
Time (days)
2.5
7VA
S6U
935
936
938
2139
2.0
a
Malic acid (g L–1)
1.5
b
1.0
c
0.5
d
0.0
Figure 2 Consumption of malic acid by the
studied yeast strains. Points are means ± SD
for three fermentations. Means with the same
letter are not significantly different
(P > 0.05).
0
(Côrte-Real et al., 1989; Côrte-Real & Leâo, 1990;
Rodriguez & Thornton, 1990, 1990; Suárez-Lepe & Iñigo,
2004), although they reduce its presence by only some
25%. The present Saccharomyces species also reduced the
malic acid content, but this only amounted to 20% with
S. cerevisiae 7VA and 30% with S. uvarum S6U.
At the first control point (4 days), Schiz. pombe 936
and 938 had already consumed nearly all the malic acid
present, with their fermentations showing a remaining
G + F concentration of 144.9 and 129.9 g L)1 (Fig. 1),
respectively. The corresponding acetic acid levels were,
however, 0.70 and 0.64 g L)1. The latter results indicate
5
10
15
20
25
30
35
Time (days)
these strains would not be appropriate for use in
conventional fermentations, although they might be of
service in mixed or sequential fermentations. Table 1
shows the final concentrations of lactic acid in the
fermentations with the different yeasts; the absence of
malolactic fermentation shows that no contamination
by lactic acid bacteria occurred.
Production of acetic acid
Differences in acetic acid production were seen
between the yeast species, as well as between
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New Schizosaccharomyces pombe applications S. Benito et al.
Table 1 Lactic acid content at the end of alcoholic fermentation.
Values are expressed as the means ± SD of three determinations
Yeast strain
L-Lactic acid (g L)1)
Saccharomyces cerevisiae (7VA)
S. uvarum (S6U)
Schizosaccharomyces pombe (935)
Schiz. pombe (936)
Schiz. pombe (938)
Schiz. pombe (2139)
0.004
0.006
0.002
0.002
0.001
0.001
±
±
±
±
±
±
0.002
0.001
0.001
0.001
0.001
0.001
Schizosaccharomyces strains. Saccharomyces cerevisiae
7VA and S. uvarum S6U produced mean acetic acid
concentrations of 0.23 and 0.36 g L)1, respectively
(P < 0.05). The Schiz. pombe strains, however, produced concentrations of 0.86–1.01 g L)1 (Fig. 3), rendering them unsuitable for use on their own in
winemaking. These results agree with those of other
authors who report Schiz. pombe to be associated with
olfactory defects (Unterholzner et al., 1988; Pitt &
Hocking, 1999; Tristezza et al., 2010). The studied
Schizosaccharomyces strains are the only representatives of this species in the IFI collection, which in
contrast has nearly 800 S. cerevisiae strains, highlighting the formers’ relative scarceness. All the present
Schizosaccharomyces strains were accidentally isolated
and certainly with no oenological criteria in mind. An
exhaustive study of the species might discover strains
with more moderate acetic acid production. Some
authors recommend biological malic deacidification be
performed with Schizosaccharomyces ssp. before the
addition of a selected Saccharomyces strain to avoid
this negative effect (Yang, 1973; Munyon & Nagel,
1977).
Ammonia consumption
Ammonia is the primary nitrogen source for Saccharomyces (Bell & Henschke, 2005). This would also appear
to be true for Schizosaccharomyces as these strains all
consumed ammonia before PAN. A residual 4 mg L)1
of ammonia was recorded for all the studied yeasts,
although their consumption kinetics were different
(Fig. 4). Saccharomyces cerevisiae 7VA, S. uvarum
S6U and Schiz. pombe 938 had consumed nearly all
the ammonia available by the 4-day check point, while
Schiz. pombe 2139, 936 and 935 did not consume it all
until day 11.
Consumption of primary amino nitrogen
Saccharomyces cerevisiae 7VA and S. uvarum S6U
showed minimum PAN concentrations of 23 and
33 mg L)1, respectively, on day 4 (Fig. 5). However,
the kinetics of the Schiz. pombe strains were slower;
minimum concentrations of 54–60 mg L)1 were not seen
until day 15. The PAN needs of this species therefore
seem to be smaller, probably due to its lower growth
rate. An eventual increase in the PAN concentration was
seen in all fermentations, perhaps owing to autolysis at
the end of this process (Dizy & Polo, 1996; FornaironBonnefond et al., 2002; Alexandre & Guilloux-Benatier,
2006; Moreno-Arribas & Polo, 2009).
Pyruvic acid production
Saccharomyces cerevisiae 7 VA and S. uvarum S6U
showed maximum pyruvic acid production at 4 days,
reaching 0.061 and 0.045 g L)1, respectively (Fig. 6).
1.4
7VA
S6U
935
936
938
2139
1.2
a
ab
ab
b
1.0
Acetic acid (g L–1)
4
0.8
0.6
0.4
c
d
0.2
0.0
0
5
10
15
20
Time (days)
International Journal of Food Science and Technology 2012
25
30
35
Figure 3 Acetic acid production by the studied yeast strains. Points are means ± SD for
three fermentations. Means with the same
letter are not significantly different
(P > 0.05).
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110
7VA
S6U
935
936
938
2139
90
Amonia (mg L–1)
70
50
a
30
b
bc
10
a
d
Figure 4 Ammonia consumption by the
studied yeast strains. Points are means ± SD
for three fermentations. Means with the same
letter are not significantly different
(P > 0.05).
–10
0
5
10
15
20
25
30
35
Time (days)
200.0
7VA
S6U
935
936
938
2139
180.0
Primary amino nitrogen (mg L–1)
160.0
140.0
120.0
100.0
80.0
a
ab
b
60.0
c
d
40.0
Figure 5 Primary amino nitrogen consump-
tion by the studied yeast strains. Points are
means ± SD for three fermentations. Means
with the same letter are not significantly different (P > 0.05).
20.0
0.0
0
The Schizosaccharomyces strains produced more, however, within the same timeframe and with significant
(P < 0.05) differences between most of the member
strains. Strain 938 reached a maximum of 0.386 g L)1
while 2139, 936 and 935 reached maxima of 0.292, 0.207
and 0.199 g L)1, respectively. Pyruvic acid-based selection studies on S. cerevisiae returned maximum values
of 60–132 mg L)1 after 4 days of fermentation (Morata,
2004) – values below those for the present Schiz. pombe
strains. The same author earlier reported a strong
correlation between the amount of pyruvic acid released
into the medium and the formation of vitisin A (Morata
5
10
15
20
25
30
35
Time (days)
et al., 2003). Strains with high pyruvate production
might therefore be of interest in terms of pigment
production and stability.
Changes in colour intensity
A gradual decline in colour intensity was seen in all
fermentations, but especially with S. uvarum S6U
(Fig. 7). Final values of 8.26, 8.09, 7.78, 7.26 and 7.43
OD were returned for Schiz. pombe 938, 936, 935,
S. cerevisiae 7VA, S. uvarum S6U and Schiz. pombe
2139, respectively (see graph for significant differences).
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New Schizosaccharomyces pombe applications S. Benito et al.
0.45
7VA
S6U
935
936
938
2139
a
0.40
0.35
b
Piruvic acid (g L–1)
0.30
0.25
c
0.20
a
0.15
b
c
d
0.10
d
0.05
e
e
0.00
0
5
10
15
20
25
30
35
Time (days)
Figure 6 Production of pyruvic acid by the
studied yeas strains. Points are means ± SD
for three fermentations. Means with the same
letter are not significantly different
(P > 0.05).
14.0
7VA
S6U
935
936
938
2139
13.0
12.0
11.0
10.0
ICM
6
9.0
a
b
bc
c
8.0
7.0
6.0
5.0
0
5
10
15
20
25
30
Time (days)
Residual urea
The urea concentration (mg L)1) of the ‘wine’ was
determined at the end of fermentation. Saccharomyces
cerevisiae 7VA and S. uvarum S6U returned values of
3.16 and 2.62 mg L)1, respectively (P < 0.05), while the
Schizosaccharomyces strains all returned values of
<0.5 mg L)1 (no significant difference between these
strains, but P < 0.05 compared with the Saccharomyces
species) (Table 2). Although none of these values are
very high, the lower values recorded for the Schiz. pombe strains might be a consequence of their urease activity
(Casas, 1999; Barnett et al., 2000; Deák, 2008). Urease
activity may be of interest with respect to wine safety as
it removes urea, the precursor of ethyl carbamate
International Journal of Food Science and Technology 2012
35
Figure 7 Change in must colour intensity over
fermentation with the studied yeast strains.
Points are means ± SD for three fermentations. Means with the same letter are not
significantly different (P > 0.05).
Table 2 Residual urea content after alcoholic fermentation. Values are
expressed as the means ± SD of three determinations. Means with the
same letter are not significantly different (P > 0.05)
Yeast strain
Urea (mg L)1)
Saccharomyces cerevisiae (7VA)
S. uvarum (S6U)
Schizosaccharomyces pombe (935)
Schiz. pombe (936)
Schiz. pombe (938)
Schiz. pombe (2139)
3.16
2.62
0.38
0.36
0.44
0.32
±
±
±
±
±
±
0.28a
0.32a
0.18b
0.24b
0.31b
0.21b
(Uthurry et al., 2004, 2006). These strains may also be
of interest as they reduce the possibility of lactic acid
bacteria growing by removing malic acid (another of
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New Schizosaccharomyces pombe applications S. Benito et al.
their nutrient sources), thus reducing the risk of biogenic
amine formation (Lonvaud-Funel, 2001; Alcaide-Hidalgo et al., 2007; De Fátima et al., 2007). Schizosaccharomyces strains could therefore make fermentations safer
for human health by reducing the final urea content and
avoiding malolactic fermentation.
Conclusions
The metabolic properties of Schiz. pombe, that is, the
breakdown of malic acid, production of pyruvic acid and
the breakdown of ethyl carbamate precursors, are of great
interest in modern winemaking. However, its major
drawback is its strong acetic acid production at least for
the unselected strains commonly used in wine research.
The selection of Schizosaccharomyces strains with low
production of acetic acid could bring a new oenological
tool for unbalanced musts. This may help remove the
spoilage stigma attached to this species. Other fermentation modalities such as mixed and sequential fermentation between Saccharomyces and Schizosaccharomyces
to minimise the levels of acetic acid could be used.
In addition, Non-Saccharomyces yeasts with high
pyruvic acid production, as the Schizossaccharomyces
strains studied in this research, can improve the formation of stable pigments. The relevance of these compounds to increase chromatic parameters has largely
been described in scientific bibliography.
Finally, the selection of Schizosaccharomyces strains
with high urease activity can be developed as a new tool
to assure wine safety.
Acknowledgments
This work was supported by the Ministerio de Ciencia e
Innovación (MCeI) (Project AGL2008-05603-C0201 ⁄ AGR). The authors are very grateful for the help
received from Biosystems S.A., and in particular to
Pablo Rodrı́guez Plaza for the donation of the enzyme
kits used in this work.
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2012 The Authors
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