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Biodeterioration2
1. 1. Biodeterioration of fruit juices and fruit juice
concentrates
2. Microbial spoilage of wine, beer and other
fermented beverages
3. Microbial deterioration of plant pectin and the
development of soft rot in fruit and vegetables
4. Microbial spoilage of milk
5. Microbial spoilage of raw sugar and sugar
confectionery
3. Pectic Substances
A group of polysaccharides made up primarily of sugar acids. They
are important constituents of plant cell walls and the middle lamella
between adjacent cell walls. Normally they are present in an
insoluble form, but in ripening fruits and in tissues affected by
certain diseases they change into a soluble form, which is
evidenced by softening of the tissues.
They have multifunctional properties:
โข Control cell wall integrity and porosity,
โข Protect plants against phytopathogens,
โข Have gelling, emulsifying, stabilising, thickening and health-beneficial
properties.
4. Types of Pectic Substances
1. Protopectin A water-insoluble polymer that
gives pectic acid on hydrolysis
2. Pectic acid A high-molecular-weight polymer
of galacturonic acid units, with no methoxyl
groups, in which all the units are free
3. Pectinic acid A polygalacturonic acid with
some of its carboxyl groups methylated. It has a
low methoxyl value and forms gels with sugars
and water.
4. Pectins Water-soluble pectinic acids
containing about 6-7% methoxyl that form gels
with sugars and acids
5. Pectins
โข All fruit and vegetables contain plant pectins.
โข Plant pectins are a mixture of polysaccharides from
polymers of anhydrogalacturonic acid residues in
which the carboxyl groups may be methylated.
โข In a typical plant pectin, galacturonic acid residues
are linked by a-1-4 glycosidic bonds in a chain to
which chains of rhamnose and galacturonic acid are
bound and carboxyl groups are esterified to methanol
in a random manner.
7. Schematic Structure of Pectin
Pectin consists of four different types of polysaccharides
Kdo, 3-Deoxy-d-manno-2-octulosonic acid; DHA, 3-deoxy-d-lyxo-2-heptulosaric acid
Harholt J et al. Plant Physiol. 2010;153:384-395
ยฉ2010 by American Society of Plant Biologists
8. The bioterioration of pectins is carried out by
a mixture of biodeteriogen-produced
pectolytic enzymes, of which there are three
main classes.
1. Polygalacturonidases
2. Pectin transeliminases
3. Pectin esterases
9. Polygalacturonidases
โข Polygalacturonide glycanohydrolases
โข Hydrolyze the a-1-4 glycosidic linkages between galacturonic
acid residues
โข Have two subgroups that differ in substrate specificity and
mechanism involved.
1. Exo-polygalaturonidases
Polygalacturonidase Polymethylgalacturonidase
2. Endo-polygalacturonidases
Endo-polygalacturonidase Endo-polymethylgalacturonidase
10. Pectin transeliminases
โข Pectic lyases
โข Cleave the a-1-4 glycosidic bonds between
galacturonic acid residues by the transelimination of a
proton from carbon atom 5 of the
anhydromethylgalacturonate residue together with the
oxygen of the adjacent atom of the a-1-4 glycosidic
bond, to give a methyl galacturonide with a double
bond between carbon atoms 4 and 5.
11. Pectin esterases
โข Hydrolyze methyl ester groups to give free
carboxyl groups and methanol
12. โข Most microorganisms produce AT LEAST ONE
pectolytic enzymes.
โข Almost all fungi and many bacteria contain pectolytic
enzymes that readily degrade pectin layers that bind
the individual cells of fruit and vegetable tissue
together into mixtures of oligosaccharides and
galacturonic acids, a process that destroys the
structural organization of plant tissue, which then
becomes a soft amorphous mass.
โข This degradation of pectin layers in plant tissue is
responsible for the spoilage process known as โsoft
rotโ in fruit and vegetables.
14. Some strains of S. sclerotiorum produce
photodynamic toxins, 8-methoxy psoralen and
4,5,8-trimethyl psoralen, which are responsible
for dermatitis among celery harvesters.
16. โข Microbial spoilage of milk is a well-known
phenomenon, milk being a perfect medium for
microbial growth.
โข To prevent milk spoilage, heat treatment is a
standard procedure in milk processing, i.e.,
pasteurization (i.e., LTHT, HTST, UHT)
โข Pasteurization is normally sufficient to destroy
all bacteria in milk. However, even after this
process, contamination by nonpathogenic
bacteria may still occur, causing spoilage.
17. Milk microbial spoilage defects
โข Production of lactic acid (souring)
โข Gas production
โขDevelopment of a viscous ropy texture
โขCoagulation of milk proteins
โข Lipolysis of milk fats (rancidity)
โขDevelopment of off-flavors
Predominant causal organisms: lactic-acid-producing
bacteria, which ferment lactose to
lactic acid.
18. Common lactic-acid-producing milk
spoilage organisms
โข Homofermentative species (produce only lactic acid)
Streptococcus lactis, S. cremoris, Lactobacillus casei, L.
acidophilus, L. plantarum, L. helveticus, L. bulgaris
โข Heterofermentative species (produce lactic acid, acetic acid,
ethanol, CO2)
L. brevis, L. buchneri, L. fermenti, L. thermophiles,
Leuconostoc citrovorum, L. mesenteroides, Microbacterium
lacticum, Micrococcus luteus, M. varians, M. freudenreichii
19. Steps in lactose metabolism by lactic-acid-
producing milk spoilage organisms
1. Hydrolysis of lactose to galactose and glucose by lactase
2. Conversion of galactose to glucose via a galactose inverting system,
catalyzed by glucose-4-epimerase.
3. Utilization of glucose to produce more galactose, which serves as an
intermediate for the conversion of galactose-1-phosphate to glucose-1-
phosphate, catalyzed by hexose-1-phosphate uridyl transferase.
4. Conversion of glucose-1-phosphate to glucose-6-phosphate by
phophoglucomutase
5. Metabolism of glucose-6-phosphate to pyruvate via the EMP
pathway.
6. Reduction of pyruvate to lactic acid by lactate dehydrogenase.
25. The principal effect of microbial
deterioration of raw sugar is the LOSS
OF SUCROSE, due to the inversion of
sucrose to fructose and glucose by
invertase-producing yeasts and fungi,
as catalyzed by invertase.
27. The susceptibility of raw sugar to microbial
attack depends on the composition of the
molasses film on sugar crystals, particularly on
the water activity of the sugar.
Most sugars have aw = 0.60-0.75; thus, only
osmophilic yeasts (aw = 0.60) and xerophilic
fungi (aw = 0.65) are the main contaminants of
raw sugars. This is helped by the slightly acidic
pH (5-6) of sugars, which inhibits bacterial
growth.
28. Microorganisms Associated with
Biodeterioration of Raw Cane Sugars
โข Fungi
Aspergillus niger Penicillium expansum
Alternaria brassicae Aspergillus flavus
Monilla nigra Cladosporium herbarum
โข Yeasts
Hansenula anomala Saccharomyces cerevisiae
Candida utilis Pichia fermentans
โข Bacteria
Bacillus subtilis B. megaterium
Clostridia nigrificans C. thermosaccharolyticum
30. โข Microbial deterioration of
carbohydrates
โข Microbial deterioration of
proteins and protein foods
โข Microbial deterioration of
edible oils and fats
31. PROTEINS are large biological molecules
or macromolecules consisting of one or more
long chains of amino acid residues.
The term comes from the Greek word proteios,
meaning โprimaryโ, โin the leadโ or โstanding in
frontโ.
โข Essential to all life
โข They are the major constituents of enzymes,
antibodies, many hormones and body fluids such
as blood, milk, and egg white.
32. Of the many kinds of food spoilage, the
microbial spoilage of proteins and protein
foods is the most complex and perhaps the
least understood, owing to the enormous
complexity of structural proteins in nature
and the wide variety of spoilage
microorganisms associated with protein
spoilage.
33. Structural Proteins of Protein Foods
1. Myoglobin
2. Myofibrillar proteins
3. Collagen
4. Elastins
5. Keratins
34. โข An iron- and oxygen-binding protein found in the muscle
tissue of vertebrates in general and in almost all mammals.
โข Is formed from one polypeptide chain and one heme
molecule.
โข Heme is a pigment responsible for the color of red meat;
thus, the color that meat takes is partly determined by the
degree of oxidation of myoglobin.
โข Degraded by microbial proteases to amino acids and
oligopeptides
Myoglobin
35. Myofibrillar proteins
โข Consist mainly of actomyosin and form the major part of
muscle proteins.
โข Degraded by trypsin-like microbial proteinases
36. Collagen
โข Found in connective tissues such as tendons and bone cartilage
โข Very resistant to degradation
โข Contain a high proportion of nonpolar amino acids (valine, leucine,
and isoleucine, with proline and hydroxyproline groups) but no
cysteine
โข Degraded by collagenases from Clostridium
Clostridiopeptidase A Hydrolyzes glycylprolyl bonds and has
pH optima of 7.7-8.0. Activated by Ca and inhibited by EDTA.
Clostridiopeptidase B Cleaves peptide bonds adjacent to
lysine and arginine and has pH optima in the range of 7.2-7.4.
Specific for collagen and gelatin.
37. Elastins
โข Found in connective tissues such as tendons and ligaments
โข Contain 90% nonpolar amino acids arranged in a random
structure
โข Highly resistant to hydrolysis, heat, and maceration
โข Degraded by elastinases, which have been isolated from
Flavobacterium elastolyticum, Aeromonas salmonicida, Bacillus
subtilis, Pseudomonas aeruginosa, and P. mallei
โข These elastinases have the following characteristics:
Have pH optima in the range of 7.0-9.0
Hydrolyze peptide bonds adjacent to glycine and proline
Highly specific for elastin
Inhibited by diisopropylfluorophosphate
38. Keratins
โข The major constituents of wool, hair, nails, hooves, and fish scales
โข Contain large amounts of glycine and proline with about 8%
cysteine.
โข Degraded by keratinases, which have been isolated from
Streptomyces fradiae and S. microflavus. They have the following
characteristics:
Have pH optima in the range of pH 8.5-9.0
Degrade keratin
Activated by calcium and magnesium ions
Inhibited by EDTA
39. In general, most bacteria are unable to colonize
pure proteins unless sufficient peptides, amino
acids and vitamins are present to enable them to
produce proteases necessary for protein digestion.
Food such as meat, fish, and cheese contain
abundant quantities of amino acids and other
nutrients; they are therefore readily colonized by
most microorganisms.
40. Principal Changes Associated with
Protein Spoilage
1. Putrefaction
Characterized by the production of foul odors and offensive
textures and flavors, which arise from spoilage metabolites
that result from the catabolic metabolism of low-molecular-weight
peptides and amino acids by spoilage organisms.
2. Degradation of protein constituents
Indicated by protein coagulation and liquefaction, rot
development, and destruction of structural proteins such as
collagen and elastin.
41. Stages in the Protein Spoilage Process
1. Initial contamination and colonization of the protein food by microorganisms.
2. Rapid utilization and metabolism of low-molecular-weight compounds such
as amino acids, dipeptides, lactic acid, and sugars present in meat or fish
juices, which yield spoilage metabolites, e.g., cadaverine, putrescine, organic
acids, CO2, H2S and NH3. At this stage, there is explosive microbial growth.
3. Increased production of microbial proteases by proteolytic spoilage
microorganisms. The proteolytic breakdown of high-molecular-weight proteins
to oligopeptides provides a continued supply of nutrients. The oligopeptides
are then hydrolyzed to free amino acids, which are then metabolized to
additional metabolites. This accumulation of metabolites eventually poisons
the microorganisms themselves, slowing down the putrefactive processes.
42. Amines Produced during Protein Spoilage
Analysis of putrefied protein foods shows that a mixture of amines are
produced by the anaerobic decarboxylation of amino acids. Such
amines include the following:
1. Cadaverine from L-lysine by Bacillus cadaveris, E. coli, and
Clostridium histolycum
2. Putrescine from L-ornithine by Clostridium septicum and C. welchii
3. Aminoburyic acid from glutamic acid by S. faecalis
4. Isobutylamine from L-valine by Proteus vulgaris and Pseudomonas
cocovenans
5. Tyramine from tyrosine by S. faecalis
6. Tryptamine from tryptophan by S. faecalis and C. welchii
NOTE: All reactions are catalyzed by decarboxylases.
43. Organic Acids Produced during Protein
Spoilage
1. Pyruvate from alanine by Bacillus subtilis, as catalyzed by
alanine dehydrogenase (NAD-dependent)
2. ๏ข-Methyl-ฮฑ-ketovalerate from isoleucine, as catalyzed
isoleucine oxidase
3. Indole from tryptophan to by E. coli and Proteus vulgaris
4. ฮฑ-Ketoglutarate from glutamate by E. coli, S. cerevisiae, C.
sporogenes, as catalyzed by glutamate dehydrogenase
5. Fumarate from aspartate by E. coli, as catalyzed by
aspartase
6. Pyruvate from serine, as catalyzed by serine dehydratase
44. Organic Acids Produced during Protein
Spoilage
7. ฮฑ-Ketobutyrate from threonine, as catalyzed by threonine
dehydratase
8. Pyruvate from cysteine, as catalyzed by cysteine desulfhydrase
9. Acetate from pyruvate by L. delbrueckii, Proteus vulgaris, and P.
fluorescens
9. Acetate from alanine and glycine by C. sporogenes
10. Isobutyric acid from valine and alanine
11. Isovaleric acid from leucine and alanine
12. Methylbutyric acid from isoleucine and alanine
13. Aminovaleric acid from proline
14. Aminohydroxyvaleric acid from hydroxyproline
45. During the putrefactive process, anaerobic clostridia such
as Clostridium butyricum, C. pasteurianum, C. acetobutylicum
and C. sporogenes also produce large amounts of H2 via the
reduction of hydrogen ions or protons as catalyzed by a specific
hydrogenase requiring reduced ferredoxin as its cofactor.
Ferredoxin
2 H+ H2
Fe3+ Fe3+
Other bacteria produce hydrogen and carbon dioxide from
glucose, which is then degraded to pyruvate via the EMP
pathway, which is then converted to acetate, formate and CO2.
46. โข In the late stage of putrefaction, spoilage microflora
also produce proteinases that degrade various
protein constituents by hydrolyzing peptide bonds
to give low-molecular-weight oligopeptides and
free amino acids.
โข Spoilage microorganisms also utilize muscle
glycogen to produce trimethylamine, ammonia and
dimethylamine, whose chemical determination
forms the basis of several methods for determining
fish and fish product spoilage.
47. The pH optima of collagenase, elastinases, and
keratinases lie in the alkaline range, i.e., 7.2-9.0;
thus, the degradation of collagens, elastins, and
keratins in protein foods is favored during the
advanced stages of putrefaction when the various
putrefactive amines such as cadaverine and
putrescine produced increase the pH of the food
from 5.5 to above 8.0 when proteolysis has
become extensive.
48. During extensive proteolysis, both bacterial
and fungal proteolytic enzymes hydrolyze
casein proteins, elastins, gelatins and
collagens.
49. โข Microbial deterioration of
carbohydrates
โข Microbial deterioration of
proteins and protein foods
โข Microbial deterioration of
edible oils and fats
50. FATSare a wide group of compounds composed of long-chain
organic acids, called fatty acids. A typical fat molecule
consists of glycerol combined with three fatty acids, i.e., it is
a triol (i.e, it has three chemically active -OH groups). Fats
are formed when each of these three -OH groups reacts
with a fatty acid, resulting in triglycerides.
โข Hydrophobic, and generally soluble in organic solvents
but insoluble in water.
โข Shorter-chain fats are usually liquid at room temperature,
whereas longer-chain fats are solid.
NOTE: Fats differ from carbohydrates and proteins in that they are not polymers
of repeating molecular units
51. โOilโ, โfatโ, and โlipidโ are often used
interchangeably. Of these, lipid is the
general term. Oil is the term usually
used to refer to fats that are liquid at
room temperature, while fat to those
that are solid at room temperature.
52. In fat-containing foods, the biodeterioration of
edible oils and fats by bacteria and fungi is the
principal cause of spoilage indicated by the
following:
Rancidity
Acidity
Soapiness
Off-flavors
Discolorations
53. Butter and Margarine
Butter is an emulsion of water in butterfat of the following
composition: 80-83% butterfat
16% water
1% nonfat milk solids
0-3% sodium chloride
Margarine is also a water-in-fat emulsion:
80% fat (a mixture palm, coco and marine oils)
20% water
Both are subject to microbial spoilage characterized by rancidity,
acidity, off-flavors and discolorations.
54. Causes of Butter and Margarine Rancidity
1. Autooxidative deterioration
โข Oxygen absorption and oxidation of unsaturated fatty acids (e.g.,
linoleic, linolenic and arachidonic) to hydroperoxides, which are
oxidized to ketones and aldehydes.
โข Generally occurs during prolonged storage at ambient temperature
โข Catalyzed by cupric and ferric ions, UV and high storage
temperatures (>5ยฐC)
2. Lipolysis of natural and synthetic triglycerides in fats
โข Effected by milk and microbial lipases
โข Prevented by pasteurization of milk and heat treatment of butter
3. Lipoxidation
โข Hydroperoxide production by specific microbial lipoxidases.
55. Microorganisms Associated with Butter and
Margarine Rancidity
Butter and margarine rancidity is generally associated with
lipolytic molds and yeasts.
Aspergillus tamari A. chevalieri
Cladosporium suaveolens Cladosporium butyri
Candida lipolytica Ospora lactis
Paecilomyces aureocinnamoneum Pseodomonas fluorescens
Margarinomyces bubaki Penicillium glaucum
Epicoccum purpurescens Micrococci
56. Rancidity and acidity are caused by the production
of free fatty acids, particularly butyric, caproic, caprylic
and capric acids, and their corresponding methyl
ketones. These volatile free fatty acids and methyl
ketones directly arise from the metabolism of liberated
free fatty acids by ๏ข-oxidation to the corresponding ๏ข-keto
acid, which is decarboxylated to methyl ketones or is
cleaved to give acetyl-coA and a lower fatty acid that is
two carbons shorter.
Secondary alcohols are also formed by the
reduction of various methyl ketones.
57. On the other hand, characteristic soapy flavors are
produced by the liberated lauric and myristic acids that
are present as triglycerides in butterfat and coconut
oils.
58. Factors Affecting Microbial
Growth in Food
1. Temperature
2. Water activity
3. Humidity
4. pH
5. Oxygen availability
6. Osmotic pressure
59. Temperature
Storage temperature is considered the most important factor
that affects food spoilage, as it determines the type of
microfolora that will cause spoilage; however, the relative
humidity and availability of oxygen must also be controlled.
Microorganisms have been reported to grow over a wide
temperature range, the lowest being โ34ยฐC and the highest
being 90ยฐC. All microorganisms, however, have an optimum
temperature as well as a range in which they will grow. This
preference for temperature forms the basis of dividing
microorganisms into the following groups:
Psychrohiles Psychrotrophs Mesophiles Thermophiles
60. Types of Organisms by Growth
Temperature
Psychrophiles Grow best between -2 and 7ยฐC
Psychrotrophs Optimum growth from 20 to 30ยฐC,
but can grow at ca. 7ยฐC
Mesophiles Optimum growth at 30โ40ยฐC, but
can grow between 20 and 45ยฐC
Thermophiles Optimum growth between 55 and
65ยฐC, but can grow at temperatures
as low as 45ยฐC.
61. Temperature
Just as molds can grow over a wide range of pHs
and moisture contents, they can also tolerate a
wider temperature range than bacteria. Many
molds can grow in the refrigerator. Yeasts are not
usually found growing in the thermophilic
temperature range, but prefer psychrotrophic and
mesophilic temperatures.
62. Water Activity
Microorganisms cannot grow in a water-free environment,
as enzyme activity is absent and most chemical reactions
are greatly slowed down. Fresh vegetables, fruit, meat, fish
and some other foods naturally have a high moisture
content, averaging about 80%. Drying is one of the oldest
methods of food preservation as it reduces moisture
availability, thereby limiting the number and types of
microorganisms that can grow and reducing the rate at
which they can do so. A measure of this parameter is
called water activity, denoted by aw.
63. Water Activity
Water activity is a measure of water available to
microorganisms. Pure water has a water activity of 1.0
while most fresh foods have a water activity of about 0.99.
In general bacteria require a higher aw than yeasts
and molds. Most spoilage bacteria cannot grow at aw <
0.91, with Clostridium botulinum having a minimum growth
level of 0.94. Staphylococcus aureus, has, however, been
found to grow at aw as low as 0.84. The lowest reported aw
for bacterial growth is 0.75. Most spoilage molds cannot
grow at aw < 0.80. The lowest reported aw for any mold
growth is 0.65, and that for yeasts 0.61.
64. Minimum Water Activities for growth of
Different Microorganisms
Normal bacteria 0.91
Normal yeasts 0.88
Normal molds 0.80
Xerophilic molds 0.65
Osmophilic yeasts 0.60
65. Humidity
The humidity of the environment is important as it
affects the aw of the food as well as the moisture
content of the food surface. Food can pick up
moisture from the atmosphere. Under conditions of
high relative humidity storage (e.g., in a
refrigerator), surface spoilage can take place,
unless food is adequately protected by packaging.
66. pH
Most microorganisms grow best at neutral pH and only a
few are able to grow at a pH lower than 4.0. Bacteria are
more fastidious about their pH requirements than yeasts
and molds. The fact that pH can limit microbial growth is a
basic principle of food preservation and has been exploited
for thousands of years. Fermentation and pickling extend
the shelf-life of food products by lowering the pH. The fact
that no known spore-forming pathogenic bacteria can grow
at pH < 4.6 is the basis for the food sterilization principle for
low-acid and acid foods.
67. Oxygen Availability
Controlling the availability of free oxygen is one means of
controlling microbial activity in food. Although oxygen is essential for
carrying out metabolic activities that support all forms of life, some
microorganisms use free atmospheric oxygen, while others metabolize
oxygen (reduced form) bound to other compounds such as
carbohydrates.
Microorganisms can be broadly classified into two groups:
aerobic and anaerobic. Aerobes grow in the presence of atmospheric
oxygen, while anaerobes, in the absence of atmospheric oxygen. In
between these two extremes are facultative anaerobes, which adapt
and grow with or without atmospheric oxygen, and microaerophilic
organisms, which grow in the presence of reduced amounts of
atmospheric oxygen.
68. Oxygen Availability
At the surface and within protein foods, oxygen availability
and oxygen tension govern the numbers and type of food-colonizing
spoilage microorganisms. The exposed surface of
fresh meat and fish have a high oxygen tension and therefore
support a large number of aerobic microorganism, such as
Pseudomonas spp., Achromobacter spp., bacilli, micrococci,
yeasts and fungi.
69. Osmotic Pressure
Osmotic pressure is inversely related to water activity.
As the osmotic pressure of any system increases, water
activity decreases. Thus, high osmotic pressures are normally
incompatible with living organisms due to the osmotic effects
that tend to dehydrate living cells.
70.
71. Food spoilage associated with
protein degradation
Type of food Spoilage
Milk Cogulation of caseins, off-flavors, racidity, putrefaction,
cadaverine
Meats Surface slimes, liquefaction, degradation of collagen, elastin,
keratin, putrefaction, cadaverine, putrescine
Fish Fishy odor, TMA, DMA, surface slimes, H2S, cadaverine,
putrescine, indole
Hams, bacon,
chicken, turkey
Greening, putrefaction, liquefaction, bone taint, rancidity
Eggs White, rot, black rot, mixed rot, fungalinfections
Cheese moldy
73. Microbial lipases
โข Cleave triglycerides at either
โข 1,3 position
โข 2-position
74. ๏ข-oxidation
โข Yield keto-acids, methyl ketones, secondary alcohols, shorter fatty
acids such as butyric, propionic acid, acetic acid
75. Acyl CoA dehydrogenase
Enoyl CoA hydratase
๏ข-hydroxylCoA dehydrogenase
Thiolase
Pathway for ๏ข-oxidation
Of a Fatty Acid
Editor's Notes
Schematic structure of pectin. Pectin consists of four different types of polysaccharides, and their structures are shown. Kdo, 3-Deoxy-d-manno-2-octulosonic acid; DHA, 3-deoxy-d-lyxo-2-heptulosaric acid. HG and RGI are much more abundant than the other components (see text). 3-C-carboxy-5-deoxy-l-xylose aceric acid
Some strains of S. Sclerotiorum produc photodynamic toxins, 8-methoxy psoralen and 4,5,8-trimethyl psoralen, which are responsible for dermatitis among celery harvesters.
Pyruvate to lactic acid --REDOX
Xerophilic fungi are yeasts and moulds that are capable of growth at or below a water activity (aw) of 0.85. E.g. Aspergillus,ย Penicilliumย andย Eurotium. Osmophilic organismsย are microorganisms adapted to environments with high osmotic pressures, such as high sugar concentrations.
