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1.0037 Arthroconidia: usually square-shaped conidia

originating directly by fragmentation of a hypha

(e.g., Geotrichum candidum,or‘machinery mold’

because of its characteristic enrichment in samples

from improperly sanitized machinery).

0038 Blastoconidia: unbranched chains with dark hila

(basal scars) that arise directly from a distinct

conidiophore; those of Cladosporium are small

and typically apiculate (lemon-shaped).

0039 Poroconidia: dark-walled (dematiaceous) conidia

in chains, each of them developing through a pore

in the apex of the preceding conidium. The

youngest conidia (smaller) are at the tip of the

chain, while the most mature conidia are closer

to the conidiogenous cell. The claviform shape,

large dimensions, and both longitudinal and

transversal septa give a typical appearance to

the conidia of Alternaria (Figure 2).

0040 Phialoconidia: produced in a long chain from an

opening of conidiogenous cell called a phialide

because of the distinctive amphora (flask) shape.

Both Penicillium and Aspergillus produce phialo-

conidia in structures which look different under the

microscope because of (1) the presence in the former

mold of the typical ‘penicillus’ (brush), a conidio-

phore with several terminal branches each bearing

a phialide (Figure 3), while (2) in the latter the con-

idiophore is formed by an unbranched stipe, which

enlarges at its end into a vesicle from which

phialides arise with their chains of very small conidia

(Figure 4). The sickle-shaped, septate phialoconidia of

Fusarium, a powerful mycotoxigenic mold, are called

macroconidia (Figure 5), in contrast to the smaller

and usually nonseptate chains of microconidia.

Contamination and spoilage of food

0041Factors contributing to the wide distribution of molds

in the natural environment are their capacity to utilize

almost all carbonaceous substrates, their ability to

develop in the presence of very low concentrations

of nutrients, and their tolerance to many adverse

conditions. Fungal spores are present in dust and

dead materials where, because of their ability to

utilize cellulose and lignin, they may grow extensively

during those materials’ decomposition.

0042During cultivation, grains, and to a lesser extent

vegetables and fruits, are exposed to fungal spore

contamination from many sources, such as dust,

fig0002 Figure 2 Alternaria tenuis. Chains of large brown clavate

conidia with transverse septa, blowing out from a conidiophore.

Reproduced from Spoilage, Encyclopaedia of Food Science, Food

Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ

(eds) 1993, Academic Press.

fig0003Figure 3 Penicillium. Branched conidiophore bearing conidial

chains, in the form of a brush (‘penicillus’). Reproduced from

Spoilage, Encyclopaedia of Food Science, Food Technology and Nu-

trition, Macrae R, Robinson RK and Sadler MJ (eds) 1993, Aca-

demic Press.

fig0004Figure 4 Conidiophore of Aspergillus enlarged in a round

vesicle covered with a mass of round conidia. Reproduced

from Spoilage, Encyclopaedia of Food Science, Food Technology

and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds)

1993, Academic Press.

SPOILAGE/Molds in Spoilage 5527

insects, and rainfall, so that it is not surprising to find

propagule counts on their surface between 10 and 10

colony-forming units (CFU) per square centimeter,

i.e., between 100 and 10

CFU g

1

. Invasion of in-

ternal tissues is usually prevented by the integrity of

the outer skin. During storage, control of relative

humidity and temperature and the use of modified

gaseous atmospheres (high levels of carbon dioxide)

retard germination and growth rate of molds, thus

preventing spoilage. Moisture content–deterioration

risk relationships for given kinds of grain are shown

in Table 1.

0043 Handling and processing may either give mold

propagules the chance to invade the body of injured

fruits or vegetables or remove them when the

grain pericarp is separated from endosperm during

milling. Mold counts in flour may therefore be re-

duced to 10–10

CFU g

1

0044 ‘Field fungi’ include molds that grow on grains in

the field, where the a

is in the range 0.95–1.00,

while ‘storage fungi’ can grow at the lower a

levels

found in grains during storage (0.70–0.90). Most

species of the genus Aspergillus grow well when

the moisture content is in the range of growing grains

(i.e., > 18%, or a

> 0.85) as well as when the mois-

ture is that typical of stored grains (i.e., < 18% or a

< 0.85). An overview of field and stored grain molds

is shown in Table 2.(See Cereals: Handling of Grain

for Storage; Controlled-atmosphere Storage: Effects

on Fruit and Vegetables.)

0045 Other dried agricultural commodities, such as

spices, may retain their original fungal bioload (i.e.,

counts between 100 and 10

CFU g

1

) if sufficient

attention is given to protection from dust and water

condensation during processing. Unfortunately, most

important producers are located in the Third and

Fourth World areas, where conditions of harvesting

and processing are sometimes primitive, so that mold

counts may reach levels as high as 10

–10

CFU g

1

posing a risk of spoilage in foods with which spices

are mixed.

0046Well-slaughtered and dressed meat is contaminated

with only a few mold spores, usually less than 10 CFU

2

, which increase when skin and hide are not

completely removed. If the surface of meat becomes

dry, because of too long storage in the chiller, yeasts

and molds (e.g., Cladosporium cladosporioides,

agent of the ‘black spot’) may develop and give visible

spoilage. (See Meat: Preservation.)

0047Molds are sometimes isolated from egg shells.

Their presence is mainly as a result of dust and mois-

ture in the hatchery and the cold store, but their

growth is prevented by the low E

value of the egg.

0048Molds entering raw milk from soil, bedding, feed

residues, manure, and so forth, during milking are so

greatly outnumbered by bacteria, and so effectively

inhibited by natural antimicrobial substances such as

lactoperoxidase and lactoferritin, that their number

fig0005 Figure 5 Sickle-shaped macroconidia of Fusarium with three

transverse septa. Reproduced from spoilage, Encyclopaedia of

Food Science, Food Technology and Nutrition, Macrae R, Robinson

RK and Sadler MJ (eds) 1993, Academic Press.

tbl0001Table 1 Risk relationships for moisture content against

deterioration

Grain Minimum moisture level to

prevent spoilage (20^30



Barley (Hordeum sativum) 15–14.5

Corn (Zea mays) 14.5–13.5

Cotton (Gossypium barbadense) 8.5–8

Rice (Oryza sativa) 13.5–13

Soybean (Glycine soja) 12–11.5

Wheat (Triticum vulgare) 14.5–13.5

tbl0002Table 2 Overview of molds occurring in field and stored grain

Mold genus/species Minimum moisture

level for growth

Minimumwater

activitylevel for growth

Field molds

Fusarium spp. 20–22% 0.87–0.89

Fusarium moniliforme 0.87

Fusarium graminearum 0.89

Alternaria alternata 18–20% 0.85–0.87

Cladosporium spp. 18–20% 0.85–0.87

Storage molds

Penicillium spp. 16–18% 0.80–0.85

Penicillium viridicatum 0.81

Penicillium griseofulvum 0.83

Penicillium citrinum 0.82

Others

Aspergillus spp. 14–20% 0.72–0.87

Aspergillus flavus 0.85

Aspergillus ochraceus 0.80

Aspergillus glaucus 0.74

5528 SPOILAGE/Molds in Spoilage

never exceeds a few tens of spores per liter. (See Milk:

Processing of Liquid Milk.)

0049 Molds pose a real problem to the food industry

mainly as secondary invaders of heat-treated (not

aseptically packed) products. These treatments are

usually sufficient to inactivate all but the most heat-

resistant microorganisms. Airborne mold spores may

contaminate products after heating (e.g., baking)

and, when chemical and physical properties of the

substrate are unsuitable to bacteria, may invade

the food and become the main agent of spoilage.

The main sources of airborne contamination are

(1) crops and trees in close proximity of the factory;

(2) areas where dusty raw materials, such as flour,

cardboard packages, and woody materials such as

pallets are stored; and (3) aerosol-generating oper-

ations, such as in waste treatment plants. Molds

carried by air movements and/or by the clothes and

shoes of people coming from these areas may pene-

trate processing rooms, where they fall directly on to

the product before packaging, and are also deposited

over the floor and wall surface. In this case the spores

may later recirculate and contaminate the product.

0050 When large volumes of air are needed, e.g., for

dehydration or forced cooling of foods, the number

of airborne organisms needs to be reduced by suitable

filtration. High-efficiency nonabsolute filters, which

remove 90–99% of the 1-mm or larger particles, are

the minimum requirement for drying and cooling

operations. Ultrahigh-efficiency (removing at least

99–99.9% of all particles of 0.1–0.2 mm or larger)

or absolute filters (efficiency > 99%), even in the

form of hoods or ceilings, are suggested to insure

trouble-free production in aseptic or semiaseptic

(such as yogurt) packaging lines.

0051 To prevent condensation and subsequent mold

growth on surfaces, good ventilation is required,

which removes the excess moisture released during

processing and storage of certain products, e.g., during

cooking, salting, aging, and other common operations.

0052 Possible fallout of airborne mold spores on to the

surface of exposed products during high-risk oper-

ations such as cooling after baking or during packaging

may be prevented by a careful separation between

these environments and sources of molds such as stor-

age of raw commodities and packaging materials.

0053 The microbiological condition of packaging mater-

ials may directly contribute to the fungal contamin-

ation of packed foods, owing to the presence of spores

on the material. The high temperatures used in the

fabrication of plastic articles cause most of them to be

virtually sterile (usually less than 1 mold spore per

100 cm

). The maintenance of hygiene during hand-

ling and storage is therefore important. Materials and

containers should be stored in clean and dust-free

warehouses, well sealed in their original bags, until

the moment of use. At that time, packaging materials

should be taken out of their wrapper before entering

the processing room, in order to limit the entry of dust

and associated fungal spores.

0054Unless a food has been previously heated and subse-

quently aseptically placed in a hermetically sealed con-

tainer, the presence of mold spores arising from raw

materials or processing environments has to be con-

sidered as ‘normal’. Fortunately, the growth of mold

colonies on incubated Petri dish does not necessarily

imply their development in the food substrate, because

food is usually protected through suitable preservation

techniques that prevent or delay the mold spores’ ger-

mination and growth. Cooling and lowering the E

value of cheese and sausages and drying and storing

dry wheat and flour are examples of appropriate

means for avoiding overt spoilage by molds.

0055None the less, molds are involved in the spoilage of

both fresh, perishable foods and stored or processed

foods. They are the main cause of market disease of

fresh fruits and vegetables. Some of the spoilage

organisms are true plant pathogens in that they have

the ability to invade healthy, viable plant tissue. An

example is Phytophthora infestans, the causal organ-

ism of late blight of the potato. Other fungi are sapro-

phytes in that their development is restricted to dead

or damaged plant tissue. Species of penicillia and

aspergilli fall into this category. Molds produce

enzymes which digest the main fruit and vegetable

skin components, i.e., pectinases and esterases for

the pectic substance and glucanases and glycosidases

(cellulases) for the soluble cellulose. Growth of fungi

on fruits and vegetables therefore results in tissue

disintegration, i.e., rotting.

0056The ubiquity of molds in the environments where

foods are handled, stored, and processed is such that

these organisms will always be found in, and on,

items which have not been heat-treated in a sealed

container or aseptically packed after heating. Not-

withstanding this, molds are involved in the spoilage

of foods only under special circumstances. They dom-

inate in spoiled food whose intrinsic properties are

suited for their physiology, i.e., moist acidic foods

rich in carbohydrates (fruits), those having a tendency

to dry out during storage (vegetables), or with added

salt (cheese, sausages), and dry foods rich in carbohy-

drates, such as cereals and flour. Development of

grain spoilage molds is usually limited to high-mois-

ture, temperature-abused stored cereals. The water

content of flour is usually just below the critical level

for mold growth (i.e., under 16–17%), hence flour

carries fungal propagules to cereal products rather

than undergoing spoilage. When growth is present

flour gives a very pungent musty smell.

SPOILAGE/Molds in Spoilage 5529

0057 Infection of the surface of bakery products, par-

ticularly bread and cakes, may arise from dispersal

of fungal propagules by air when products are cooled

after baking. Enough water and a long storage time

are required for fungal growth, which appears in the

form of colored spots. French-style high-moisture

breads, although wrapped and treated with preserva-

tives, spoil very rapidly (i.e., within 2–7 days). Cakes

have a longer mold-free life, depending on the

individual a

, processing technology, sanitation, and

selection of ingredients.

0058 Herbs and spices often contain large numbers of

fungal spores and, even if the proportion of spices in

recipes is usually low, under some conditions they

contribute to an overt spoilage of the final product.

The most important factors in determining the qual-

ity of dried herbs and spices are the initial bioload of

the source crop and subsequent storage conditions.

The desired properties of many spices are lost if

storage is too extended or under poor conditions.

(See Herbs: Herbs and Their Uses.)

0059 Under special circumstances, molds may spoil even

protein-rich foods, e.g., fresh meat whose surface

dries following prolonged cool storage, and long-

term ripened cheese whose rind becomes too dry to

support the growth of competing bacteria. Penetra-

tion of mold mycelium through cracks in the rind and

spreading of fungal enzymes across the rind may

result in localized off-colored and off-flavored areas

in the body of the cheese. Growth of spoilage molds

is a problem on all cheeses but it is particularly rapid

on soft cheese where cutting for prepacking is

involved – even more so where untreated ingredients,

pepper, nuts, herbs, and mushrooms are used.

0060 Conversion of an oil-in-water emulsion, i.e., cream,

to a water-in-oil emulsion, i.e., butter, makes butter a

substrate with such a low a

that surface molding

is seldom a major problem. When present it is mani-

fested by discolored areas, appearing more frequently

on the surface of items manufactured from unpasteur-

ized cream (farm-related contamination) or under un-

sanitary processing conditions (process-related

contamination). (See Butter: The Product and its

Manufacture.)

0061 Both fresh and fermented sausages represent an

ecological niche where inherent (pH, a

) and envir-

onmental (temperature and moisture in the cellar)

factors promote mold growth. Traditionally some

salami-type sausages in Europe are mold-ripened.

The organisms involved, mainly belonging to the

genus Penicillium, are chosen on the basis of their

ability to develop pleasant flavors during growth

with no adverse side-reactions such as the production

of mycotoxins. (See Meat: Sausages and Comminuted

Products.)

See also: Butter: The Product and its Manufacture;

Cereals: Handling of Grain for Storage; Controlled-

atmosphere Storage: Effects on Fruit and Vegetables;

Heat Treatment: Chemical and Microbiological Changes;

Herbs: Herbs and Their Uses; Listeria: Properties and

Occurrence; Meat: Preservation; Sausages and

Comminuted Products; Milk: Processing of Liquid Milk;

Mycotoxins: Occurrence and Determination;

Pasteurization: Principles; pH – Principles and

Measurement; Preservation of Food; Water Activity:

Effect on Food Stability

Further Reading

Doyle MP, Beuchat LR and Montville TJ (eds) (1997) Food

Microbiology. Fundamentals and Frontiers. Washing-

ton, DC: ASM Press.

Gams W, van der Aa HA, van der Plaats-Niterink AJ,

Samson RA and Staplers JA (1987) CBS Course of My-

cology. Baarn: Centraalbureau voor Schimmelcultures.

Moss MO, Jarvis B and Skinner FA (eds) (1989) Filament-

ous Fungi in Foods and Feeds. Society for Applied Bac-

teriology Symposium Series no. 18. Oxford: Blackwell

Scientific.

Ottaviani F and Disegna L (1987) Moulds and yeasts in

dairy products and environment. In: Cominazzini C (ed.)

Microbial Recontamination in Dairy Processing, pp.

22–54. Thiene: Food and Dairy Institute of Thiene.

Pedersen LH, Skouboe P, Boysen M, Soule J and Rossen L

(1997) Detection of Penicillium species in complex

food samples using the polymerase chain reaction.

International Journal of Food Microbiology 35:

169–177.

Pitt JI and Hocking AD (1985) Fungi and Food Spoilage.

North Ryde: Academic Press.

Samson RA, Hocking AD, Pitt JI and King AD (eds) (1992)

Modern Methods in Food Mycology, Developments in

Food Science 31. Amsterdam: Elsevier Science.

Samson RA, Hoekstra ES, Frisvard JC and Filtenborg O

(1995) Introduction to Food-borne Fungi. Barn:

Centraalbureau voor Schimmelcultures.

Yeasts in Spoilage

V Loureiro and M Malfeito-Ferreira, Universidade

cnica de Lisboa, Tapada da Ajuda, Portugal

Background

0001Yeasts are microorganisms generally perceived as fa-

vorable components in food systems, since their roles

are well known in the production of foods and

fermented beverages – bread, beer, wine, cider, sake,

5530 SPOILAGE/Yeasts in Spoilage

distilled spirits, etc. More recently, this image has

been strengthened by the fact that yeasts can be

obtained in capsules, as sources of B-complex vita-

mins, and are medically prescribed for several condi-

tions. However, yeasts are also responsible for

undesirable effects such as food spoilage. This article

summarizes the main aspects of the occurrence,

effects, and monitoring of yeasts in the spoilage of

foods and beverages.

Competition among Bacteria, Molds, and

Yeasts

0002 The contaminant microflora of food is constituted, in

most cases, of mixed populations of bacteria, molds,

and yeasts. Microbial alteration becomes a competi-

tive process among the different groups of micro-

organisms, prevailing over those which show a

better adaptation to the environmental conditions in

each product.

0003 In food commodities, under optimal growth condi-

tions, the populations of most bacteria, owing to their

faster growth, overtake those of molds and yeasts.

While bacteria have typical generation times of less

than 1 h and, in some cases, around 10–15 min, mold

and yeast populations may take several hours (or days)

to duplicate. Thus, molds or yeasts can compete with

bacteria only when environmental conditions severely

affect bacterial activity. Low pH and low water activ-

ity (a

) favor the growth of molds and yeasts, as they

are more acid-resistant and xerotolerant than bac-

teria. The presence of chemical preservatives, mainly

those of an acidic nature, is also less inhibitory to-

wards molds and yeasts.

0004Yeast activity can lead to subsequent bacterial ac-

tivity, as in acid-fermented foods; in this case, organic

acids can be consumed by film-forming yeasts,

leading to deacidification and promotion of bacterial

activity. The competition between molds and yeasts

depends, to a large extent, on oxygen availability.

While the former are obligate aerobes, the latter

may be facultative, growing either under aerobic or

anaerobic conditions. For this reason, yeasts are

found much more frequently on liquids, where good

aerobic conditions exist only at the air–liquid inter-

face, whereas filamentous fungi predominate in solid

foods.

Concept of Spoilage Yeasts

0005Of all yeasts isolated from nature, about 500 species

are presently recognized by taxonomists. Of these,

about a quarter may be readily isolated from foods,

but only a few play a significant role in food alter-

ation. Those which do not affect foods are generally

called adventitious or innocent yeasts; those respon-

sible for undesirable changes in foods and beverages

are called spoilage yeasts. For food technologists, the

concept of spoilage yeast generally has a stricter

sense. It takes into account only whether a particular

species is able to cause deterioration in foods and

beverages which have been processed and packaged

according to the standards of good manufacturing

practice (Table 1). It should be emphasized that the

concept of spoilage yeast is conditioned by the type of

product and by the moment when the yeasts exert

their activity. For instance, in the production

of wines, the yeast Saccharomyces cerevisiae is

tbl0001 Table 1 Principal yeast species causing spoilage of foods and beverages processed according to the standards of good

manufacturing practices

Type of food Foodproduct Yeast species

Fresh fruits and vegetables Strawberries, figs, tomatoes Hanseniaspora/Kloeckera spp.

Refrigerated and frozen foods Icecreams, frozen peas Rhodotorula spp.

Pasteurized foods Yogurts, fruit juices, ketchup Kluyveromyces marxianus, Saccharomyces

cerevisiae, Zygosaccharomyces bailii

Preserved foods Mayonnaise, salad dressings, sauces, soft

drinks, chutneys

Brettanomyces/Dekkera spp., Issatchenkia

orientalis, Pichia membranifaciens,

S. cerevisiae, Saccharomycodes ludwigii,

Schizosaccharomyces pombe, Z. bailii

Fermented foods Pickles, olive brines, sauerkraut, cheeses,

sausages

Debaryomyces hansenii, I. orientalis,

P. membranifaciens, Ya r r owia lipo l y t i c a , Z. bailii

Alcoholic beverages Wine, beer, cider Brettanomyces/Dekkera spp., Pichia anomala,

P. membranifaciens, S. cerevisiae, S. cerevisiae

var. diastaticus, Sac. ludwigii, Sc. pombe,

Torulaspora delbrueckii, Z. bailii

Concentrated products Dry fruits, jams, jellies, honey, fruit

concentrates, filled chocolates, marzipan

Candida versatilis, D. hansenii, P. anomala,

T. delbrueckii, S. cerevisiae, Sc. pombe, Z. bailii,

Z. rouxii, Z. bisporus

SPOILAGE/Yeasts in Spoilage 5531

recommended for its good fermentation characteris-

tics. However, later in the production process, this

species is considered a dangerous spoiler in sweet

bottled wines, because it is able to ferment residual

sugar. (See Wines: Production of Table Wines; Pro-

duction of Sparkling Wines.)

Importance of Yeasts in Food Spoilage

0006 The presence of yeasts in food is generally believed to

be harmless to public health, although occasional

allergies have recently been attributed to them. The

absence of pathogenic foodborne yeasts diminished

interest compared with bacteria and molds, and so,

for a long time, their importance as food contaminant

microorganisms has been underestimated. More

recently, however, yeasts have been established as

important factors in food spoilage, which involves

the visible or detectable deterioration of physical

and sensorial properties of foods. These undesirable

activities are responsible for serious economical

losses, which remain mostly unreported due to the

confidentiality assumed by the companies involved

(e.g., manufacturers, suppliers of raw materials and

packaging, retailers).

0007 Among the factors that have contributed to this

increasing importance are the use of modern tech-

nologies in food processing, the great variety of new

formulations of foods and beverages, and the ten-

dency to reduce the use of preservatives, particularly

those effective against yeasts (e.g., sulfur dioxide and

benzoic acid). Modern technologies tend to utilize

less severe processing conditions so as to preserve as

much as possible their flavors, tastes, and natural

colors. Thus, for instance, in the thermal processing

of products of vegetable origin, it is becoming more

commonplace to use cold filling, after a gentle pas-

teurization treatment, which significantly increases

the risk of yeast contamination and, consequently,

the risk of yeast spoiling. New food and beverage

formulations, using juices and concentrated fruits,

sugar syrups, sliced fruits, etc., such as the different

types of yogurts, have also contributed significantly

to the importance of yeasts as spoilage agents. In the

same way, low-calorie foods, where preservation no

longer depends on the effect of high sugar concen-

trations, providing low water activities, are good

habitats for yeast activity.

Effects and Extent of Yeast Spoilage

0008The most common spoiling effects occur in sweet acid

drinks and are characterized by abundant gas produc-

tion, which may deform or explode the packages,

haziness, sediment or film formation, off-flavors,

and off-tastings. Deterioration of food and drinks by

yeasts may present other effects, more or less evident,

according to the type of food (Table 2). However,

in certain cases, yeast spoilage is not so easily

defined, mainly in fermented foods and drinks (e.g.,

wines, artisanal beers, black olives, soy sauce,

cheeses), where the metabolites produced contribute

to the flavor and aroma. In fact, the distinction

between detrimental and beneficial activities is not

always clear-cut. For instance, the 4-ethylphenol

production by Brettanomyces/Dekkera yeasts in red

wines is only regarded as spoilage when this second-

ary metabolite is present at levels above about

700 mgl

1

. At levels below 400 mgl

1

it contributes

favorably to the complexity of wine aroma.

0009The level of contamination in foods is not a clear

indicator of product deterioration. For instance, in

many cheeses, cured meats, and cloudy beers, yeast

counts as high as 10

–10

cells g

1

do not cause any

detectable depreciation; on the contrary, they are sup-

posed to improve aromatic complexity. The reverse

tbl0002 Table 2 Principal spoilage effects of food products resulting from yeast activity

Foodproduct Spoilage effect

Color

spots/discoloration

Gas

production

Haze/

cloudiness

Sediments Films Off-flavors/

off-tastings

Texture changes

Fresh fruits and vegetables X X X X

Brined vegetables X X X X X X X

Fruit juices, wine, beer X X X X X

Mayonnaise, salad dressings X X X X X

Confectionery X X X X X

Syrups, honey, fruit concentrates X X X X

Butter, cream X X

Cheeses, yogurts X X X

Sliced bread, unbaked bread dough X X

Sausages, meat products X X X

Gas production may lead to distortion or explosion of the container or product.

5532 SPOILAGE/Yeasts in Spoilage

situation also occurs, when counts below 10

cells g

1

induce spoilage. This is the case of the formation of

visible cell clusters, in bottled white wines, or of the

development of surface color spots due to yeast col-

onies, in certain solid foods (e.g., skinless sausages,

sliced bread, etc.).

Factors Affecting Yeast Activity

0010 The microbiological stability of foods and beverages

can be achieved by complete removal of microorgan-

isms before packaging of the final product, as in sterile

filtration of beverages without suspended solids. It can

also be achieved by total destruction of contaminant

microorganisms, as in heat sterilization, pascalization,

or g-irradiation, or by inhibiting metabolic activity by

freezing. However, in most cases, food microbio-

logical stability is dependent upon the inhibition (or

reduction) of microbial activity by the creation of one

or more adverse environmental factors, such as low

water activity (dried, concentrated, and salted foods),

low pH (acid foods), low temperatures (refrigerated

foods), and the presence of inhibitors (preserved

foods). In the case of sterile filtration, heat, pascaliza-

tion, or g-irradiation, microbial contamination is pos-

sible only when there is process failure or when there is

contamination after treatment. When thermal pro-

cesses, pascalization, and g-radiation are used, heat-,

high-pressure-, or radioresistant yeasts predominate

as contaminant organisms. For food preservation

based on microbial inhibition through the action of

adverse environmental conditions, the spoilage micro-

flora is restricted to organisms resistant to the factor(s)

used in the preservation process, e.g., low water activ-

ity, organic acids, and so on. (See Freezing: Structural

and Flavor (Flavour) Changes; Irradiation of Foods:

Basic Principles; Sterilization of Foods.)

Water Activity (

)

0011 Water activity is, probably, the environmental factor

which most strongly affects living organisms, given

the narrow range of values allowing growth. Like

other organisms, yeasts are also markedly affected by

water availability, although, together with molds, they

are the least sensitive. Thus, it is not surprising that

molds and yeasts are the dominant flora of low-a

foods. (See Water Activity: Effect on Food Stability.)

0012 For identical a

values, different solutes have dif-

ferent effects on yeast growth, salts (e.g., NaCl) being

generally less well tolerated than sugars (e.g., su-

crose). The most resistant yeasts to low a

, in the

presence of either sugar or salt, belong to the genus

Zygosaccharomyces, Z. rouxii being the most resist-

ant species (with an a

of approximately 0.65 in the

presence of sugar). Other species resistant to high

sugar and salt levels are Debaryomyces hansenii,

Candida etchelsii, Candida parapsilosis, Torulas-

pora delbrueckii, Pichia membranifaciens,andPichia

anomala.

0013Yeasts and molds are generally more resistant than

bacteria to low pH values and constitute the typical

flora of acid food. Acetic acid bacteria, and particu-

larly lactic acid bacteria, can compete with yeasts

in acid foods, although they are more sensitive to

low pH, low a

, and some preservatives, such as

sulfur dioxide. (See Lactic Acid Bacteria; pH –

Principles and Measurement.)

0014Yeasts usually grow in the pH range 2.0–8.0, with

an optimum at 4.0–4.5. The minimum pH for yeast

growth is not easy to define, owing to the strong

influence of the particular acid, other inhibitors, and

environmental factors. The nature of acids present in

foods significantly affects yeast activity, since their

inhibitory effect is dependent on the concentration

of undissociated form of the acid. Among other

species resistant to low pH, Zygosaccharomyces bailii

is considered the most resistant to organic acids.

This feature, associated with resistance to low a

makes this species one of the most feared yeasts

by food technologists, particularly in the production

of fruit juices, fruit concentrates, marzipan, and

acid-preserved foods like mayonnaises and salad

dressings.

Temperature

0015The influence of temperature on yeast activity should

be considered in two ways: as a lethal agent, during

thermal processing, and as an environmental factor,

during storage. Concerning the first, the thermal

resistance of yeasts is considered to be low, as yeasts

are destroyed at pasteurization temperatures. The

higher resistance of some species is related to

the formation of ascospores, which are slightly

more heat-resistant than vegetative cells. Ascospores

are not, however, thermally resistant forms in the

same sense as bacterial endospores. (See Pasteuriza-

tion: Principles.)

0016The yeasts more frequently referred to as contam-

inants of pasteurized foods belong to the species Klu-

yveromyces marxianus, S. cerevisiae, and Z. bailli.

Thermal resistance varies with the nature of the

media, the protective effect of high sugar concentra-

tions being well known. In concentrated foods, there-

fore, the pasteurization conditions need to be more

severe to obtain absolute safety. The following treat-

ments are recommended: 20 s at 85



C for concen-

trated orange juices of 50



Brix, 30 s at 90



C for

chocolate syrups of 75



Brix.

SPOILAGE/Yeasts in Spoilage 5533

0017 As an environmental factor, temperature does not

favor spoilage yeasts in relation to other groups of

microorganisms. Food yeasts are essentially meso-

philes, capable of growing in the temperature range

0–40



C, with an optimum temperature of about

25–27



C. Some species capable of growing at low

temperatures – psychrotrophic yeasts – are frequently

isolated from refrigerated products, suggesting that,

in this case, temperature plays an important selective

role. These yeasts are nearly always pigmented, and

consequently, colored spots may develop on foods.

Storage temperature has a strong effect on spoilage

yeast activity, often accelerating the food-alteration

process. For this reason, some foods that are very

susceptible to dangerous spoilage yeasts (e.g., Z. bai-

lii), such as fruit concentrates, are generally stored

under refrigeration.

Oxygen Availability

0018 The presence of oxygen always enhances yeast activ-

ity, because it allows oxidative metabolism, which is

energetically more efficient, as well as enabling the

utilization of a wider range of energy-rich substrates

(such as ethanol and organic acids). For this reason, in

all foods susceptible to yeast activity, it is necessary to

exclude oxygen so as to prevent film-yeast growth

and reduce the yeast metabolism.

Nutrients

0019 In general, the range of chemical compounds found in

foods is adequate to supply the nutrient demands for

the majority of spoilage yeasts. Essentially all food

yeasts can utilize glucose and fructose as carbon and

energy sources; less frequent is the utilization of or-

ganic acids (e.g., acetic and lactic acid) and alcohols

(e.g., ethanol and glycerol); the utilization of starch

and lipids is rare.

0020 Organic nitrogen is not necessary for yeast

metabolism, all yeasts being able to utilize ammonia

as the sole nitrogen source. They can also utilize

some amino acids owing to their ability to deaminate

or transminate acids. Few yeasts have proteolytic

activity. Consequently, foods with a low carbon-

to-nitrogen ratio tend to be spoiled by bacteria,

while those with a high carbon-to-nitrogen ratio are

preferentially affected by yeasts. (See Amino Acids:

Metabolism.)

0021 Some yeasts grow very slowly, or do not grow at

all, in certain foods as a result of difficulties in syn-

thesizing one or more vitamins. Z. bailii, for example,

requires group B vitamins, and Brettanomyces/

Dekkera require biotin and thiamin (vitamin B

). In

addition, the inability of Z. bailii to utilize sucrose as

an energy source is of some significance. Extreme care

must be taken when sucrose syrups are substituted by

fructose syrups.

Preservatives and Antimicrobials

0022Most preservatives used in the food industry are weak

organic acids – sorbic, benzoic, acetic, sulfurous, and

propionic acids. Their activity increases with reduced

pH, ceasing to be effective at values much higher than

their pK

, i.e., for pH values where the undissociated

form is insignificant. The acid resistance of yeasts is

the major factor in their predominance over bacteria

in the alteration of preserved food. Certain species,

such as Z. bailii, may grow on foods with levels of

sorbic acid (c. 800 mg/kg) much higher than those

permitted by law. Other spoilage yeast species

known for their high resistance to preservatives are

Saccharomycodes ludwigii, Schizosaccharomyces

pombe, Issatchenkia orientalis, P. membranifaciens,

and S. cerevisiae. The contact of such yeasts with

preservatives contributes to an adaptive process

which can significantly increase their resistance and

subsequent growth. (See Preservation of Food.)

0023The presence of antimicrobials in foods also

strongly affects the contaminating flora. Among

other compounds, ethanol exerts a strong selective

effect, particularly on molds and most bacteria.

Lactic and acetic bacteria are the most resistant to

ethanol. The former may resist up to 20% (v/v) etha-

nol and the latter to about 14% (v/v). Thus, bacteria

frequently compete with yeasts in the alteration

process of alcoholic beverages. The yeasts’ behavior

in the presence of ethanol is quite variable. There

are several very sensitive species such as Hansenias-

pora uvarum and Rhodotorula glutinis and several

quite resistant species, tolerating up to 18–20%

(v/v) ethanol (e.g., some strains of S. cerevisiae and

Z. bailii).

Prevention and Monitoring of Yeast

Spoilage

0024When a food commodity is spoiled by yeasts, the

yeasts are present as contaminants and environmental

conditions make them stronger and quicker than

other microorganisms. The prevention of spoilage,

with fewer chemicals and milder preservation pro-

cesses, requires a sound understanding of the prob-

lem, and so the food microbiologist should have

available tools to evaluate the whole microbiological

ecosystem. In a code of good manufacturing and

distribution practices, several conditions should be

met to avoid or to control the activity of yeast

spoilers: (1) good-quality raw materials, i.e., with

low yeast counts; (2) adequate and efficient hygiene;

5534 SPOILAGE/Yeasts in Spoilage

(3) adequate and efficient processing operations; and

(4) careful monitoring of spoilage yeasts.

Good-quality Raw Materials

0025 The microbiological quality of raw materials is essen-

tial in the prevention and control of spoilage yeasts

because higher initial contaminations increase the

risk of failure of technological control processes. As

a rule, natural raw materials (e.g., fruits and sugar

cane) have an adventitious microbial flora which is

not active in foods, after processing, being easily con-

trolled through good manufacturing practices. How-

ever, when raw materials have already been processed

(e.g., fruit concentrates, fruit juices, and sugar

syrups), as in many foods of complex formulation

(e.g., dessert yogurts, reconstituted fruit juices, and

filled chocolates), the type of contaminant yeast is

much more dangerous. This situation results from

the enrichment of the contaminating flora in yeasts

highly resistant to the environmental stress present

during the processing of natural raw materials.

Processing Operations

0026 One of the most efficient measures for controlling

spoilage yeast activity is thermal processing of pack-

aged foods and beverages. Unfortunately, there are

few products where this is possible without any ad-

verse effects on the quality of the food commodity.

Among other measures that might be taken to prevent

yeast spoilage, the following should be noted:

0027 preservatives should be added immediately before

packaging so as to avoid contact with yeasts, which

might lead to adaptation and increased preserva-

tive resistance;

0028 oxygen should be excluded through air evacuation

or purging with inert gases before packaging to

inhibit yeast metabolism;

0029 cross-contaminations should be avoided in plants

processing more than one kind of product (e.g.,

jams and fruit juices);

0030 food products should be stored at low tempera-

tures, particularly for foods made with ingredient

mixtures with a high susceptibility to yeast activity.

Hygiene

0031 It is generally accepted that the main source of con-

tamination by yeasts is food residues remaining in

processing equipment after inadequate cleaning and

disinfection. It is essential, therefore, to ensure high

standards of hygiene, for which several conditions

should be met:

0032 suitable plant layout and easily accessible equip-

ment;

0033awareness of the most likely sites for spoilage

yeasts (e.g., filler heads, diaphragm valves, pressure

gauges, dead ends of sterilizing filters, pumps, stir-

rer bearings, and glands);

0034cleaning and disinfection programs should include

dismantling of pieces of equipment that are

difficult to sterilize, and adequate contact time

and concentrations of sterilants must be used. (See

Sanitization.)

Monitoring of Spoilage Yeasts

0035Microbiological control Generally, food microbio-

logical control does not provide satisfactory informa-

tion for an effective prevention against spoilage

yeasts. The main reasons for this are:

0036there is a lack of specificity of culture media used

for yeast identification, which does not allow a

distinction between adventitious and spoilage

species (often, the same media are used for yeast

and mold counts);

0037sampling procedures are usually statistically inad-

equate owing to time and cost restrictions;

0038sampling size is often insufficient to detect low

contamination levels;

0039there is a lack of reference values needed for

adequate interpretation of yeast counts.

Accordingly, it should be noted that for yeasts such as

those from the genus Zygosaccharomyces, which are

potentially dangerous even in very low numbers, the

microbiological control information is often rather

inadequate.

0040Microbiological control allows determination of

the critical points of processing lines and estimation

of their relative importance as origins of outbreak of

spoilage yeasts. These critical points are dependent on

the quality of the equipment. For example, in wine-

bottling lines with poorly designed filling machines,

the main critical points of yeast contamination are as

shown in Figure 1.

0041Besides yeast detection and counting, the microbio-

logical control may be further aimed to the typing or

identification of contaminant yeasts. Classical identi-

fication is based on physiological, biochemical, or

sexual characteristics and cannot be routinely utilized

in the food industry. As a consequence, various mini-

aturized and simplified identification methods have

been developed. However, they are based on the same

approach of the classical method of yeast identifica-

tion and are time-consuming, even when procedures

are automated and computerized, and often lead to

false or equivocal identifications. To overcome these

difficulties, alternative faster typing methods have

been developed, based, among others, on analysis of

SPOILAGE/Yeasts in Spoilage 5535

total proteins, total long-chain fatty acids, and isoen-

zymes. In addition, recent progress in molecular biol-

ogy has led to the development of highly specific

techniques (e.g., restriction-fragment length poly-

morphism of mitochondrial DNA, chromosomal

DNA electrophoresis, restriction-enzyme analysis of

polymerase chain reaction-amplified ribosomal

DNA, and random amplified polymorphic DNA

assay).

0042 Zymological indicators The microbiological quality

of foods is currently evaluated through microbio-

logical indicators. When yeasts are the most danger-

ous microorganisms for the stability of foodstuffs, the

microbiological indicator used is, generally, the ‘yeast

and molds,’ resulting from the utilization of a general-

purpose plating medium to enumerate microfungi.

However, this indicator does not distinguish between

dangerous and adventitious yeasts, and so other more

meaningful indicators may be used. For instance,

yeasts tolerant to environmental stress are detected/

enumerated by general-purpose media modified

according to the stress factor involved. Thus, ‘acid-

resistant yeasts’ is a zymological (zymo ¼ yeast) indi-

cator used to characterize dangerous yeasts in acid

food industries, ‘xerotolerant (osmophilic) yeasts’ is

used for grouping yeast particularly dangerous in

industries that process low-available-water foods.

Occasionally, it may be of interest to detect the pres-

ence of yeasts with specific hydrolytic capacities,

namely ‘lipolytic yeasts,’‘pectinolytic yeasts,’ and

‘proteolytic yeasts,’ using the appropriate culture

media. These indicators may also be directed to detect

individual spoilage yeast species provided the select-

ive or differential media to quantify them in a rapid

way. Examples of this approach include media for

Debaryomyces hansenii (e.g., intermediate moisture

foods and cheeses), Dekkera/Brettanomyces spp.

(e.g., wines, beers, and ciders), Kluyveromyces mar-

xianus and K. lactis (e.g., dairy industry), Yarro-

wia lipolytica (e.g., butter and cheeses) and Z. bailii

and Z. bisporus (e.g., wines, beverages, and fruit

concentrates).

0043An alternative approach to the zymological indi-

cators, determined by microbiological methods, is to

examine food samples for chemical (or sensorial)

evidence of past microbial activity. The use of me-

tabolites as indicators of spoilage is often more con-

venient and faster than using microbiological

methods of examination. Examples are given by

determination of ethanol and acetoin (e.g., fresh

fruits), carbon dioxide (e.g., detection of fermenta-

tive yeasts in foods), 4-ethyl-phenol (chemical

marker for Dekkera/Brettanomyces in wines) and

1,3-pentadiene (chemical marker for Z. rouxii and

D. hansenii in marzipan based products preserved

with sorbate). The two last chemical indicators may

be regarded as ‘spoilage predictors,’ because at low

concentrations, they may reflect future product

deterioration.

Membrane

filter

Filling

machine

Filling

tank

Bottle

washer

Palleted

bottles

Bottled

wineCork

machine

fig0001 Figure 1 Main sampling points in spoilage-yeast monitoring of a cold-sterile wine bottling line. Empty bottles (1) are frequently

infected by fungi, some bacteria (generally spore-forming), and adventitious yeasts; after washing/rinsing (2), an increase in the

microbial load is frequently seen, and spoilage yeast infections may occur (about 5% of all outbreaks). The filling machine is usually

the main cause of contamination (c. 50% of cases), particularly owing to deficient disinfection of the filling heads (3) and self-levelling

system (4). The cork machine is responsible for about 30% of outbreaks, mainly owing to contamination of cork jaws (5) and also the

corks (6); membrane filters occasionally (c. 10% of cases) may be a serious infection point when a membrane rupture occurs.

5536 SPOILAGE/Yeasts in Spoilage