Yeasts and molds

1.1  Yeasts and molds in foods

Molds and yeasts form a very large group of microorganisms, with most coming from the air or soil. Molds are extremely versatile, with the majority of the species able to assimilate any source of carbon present in foods. Most species can also use different sources of nitrogen, including nitrate, ammonia ions and organic nitrogen. However, when the use of proteins or amino acids as sources of nitrogen or carbon is required, several species will present limited growth. Yeasts, in general, are more demanding than molds. Most species are unable to assimilate nitrate and complex carbohydrates, some require vitamins, while others, such as Zygosaccharomyces bailii, for example, cannot use sucrose as sole source of carbon. All these factors limit in a certain way the variety of foods susceptible to spoilage caused by yeasts.

 Molds and  yeasts are also quite resistant to adverse conditions, such as acidic pH and low water activity. With regard to the pH, fungi are little affected by variations within the 3.0 to 8.0 range. Several molds grow at pH values below 2.0 and several yeasts even below 1.5. However, as the pH moves away from the optimal range (generally close to pH 5.0), growth rates are considerably reduced and, if any other inhibitory factors are present (water activity, temperature, etc.), its restrictive effect on the growth rate becomes more pronounced.

The optimal growth temperature of most fungi falls in the 25 to 28°C range, and they do not grow well at mesophilic temperatures (35–37°C) and rarely at temperatures of thermotolerant bacteria (45°C). Their growth is not uncommon under cold storage conditions (5°C), however, when kept at temperatures lower than −10°C, foods can be considered microbiologically stable.

Food spoilage  molds, like nearly most other  filamentous fungi, require oxygen for their growth, and for that reason have been strictly aerobic. Nonetheless, several species are effective at using small amounts of oxygen, in such a way that the effect of O₂ is dependent on the absolute amount dissolved in the substrate, and not on the concentration present in the atmosphere. Contrary to molds, many species of yeasts are capable of growing in the complete absence of O₂ and at different concen-trations of CO₂. This makes them the most common spoilage microorganisms of bottled liquid foods, in which the growth of molds is limited by the availability of oxygen. Eventually, some species of the  Mucor,  Rhizopus,  Byssochlamys and  Fusarium genera can grow in these products, causing their spoilage.

The consistency of the food, along with the storage atmosphere, exerts a considerable influence on the types of fungi that will spoil a product. As a general rule, yeasts predominate in liquid foods because they are unicellular microorganisms that disperse more easily in liquids. In addition, liquid substrates offer a greater opportunity for the development of anaerobic conditions, which are ideal for fermentative  yeasts.  Molds, on the contrary, are favored by firm and solid substrates, the surface of which greatly facilitates easy access to oxygen. However, this statement should not be understood as absolute, suggesting that yeasts cannot contribute to the deterioration of solid foods or molds to the spoilage of liquid foods. It simply means that yeasts are more competitive in liquids, causing changes that are perceived more easily or more rapidly.

Infectious  fungi are rarely associated with foods, how-ever, certain  yeasts from foods and related sources may trigger allergic reactions and some  molds may cause infections in immunodepressed individuals. Several molds produce  mycotoxins, which are toxic metabolites formed during their growth. The most important toxigenic mold genera are  Aspergillus,  Penicillium and  Fusarium.

1.2  Methods of  analysis for total  yeast and  mold counts

Yeasts and molds in foods are enumerated using the standard  plate count method and the results are expressed in number of colony-forming units (CFU). The most recommended method of analysis is  spread plate which has the advantage of increasing exposure to oxygen and, at the same time, avoiding stress caused by hot or warm culture medium. Several growth media may be used: Chapter 20 of the Compendium (Beuchat and Cousin, 2001) recommends  Dicloran Rose Bengal Chloramphenicol Agar (DRBC), to examine foods with a water activity greater than 0.95 and  Dicloran Glycerol 18% Agar (DG18), for foods with a water activity smaller than or equal to 0.95. DRBC agar contains chloramphenicol, which inhibits bacterial growth, in addition to dicloran and rose bengal, which restrict spreading of the colonies. DG 18% agar, in addition to chloramphenicol and dicloran, also contains glycerol, which reduces the water activity of the medium. Other methods that have already been officially recognized by the AOAC International are the microbiological test kits described in Table 1.

1.3  Psychrotrophic fungi

Some strains of yeasts and molds are also psychrotrophic, although they have been much less studied than bacterial psychrotrophic strains. Fungi predominate in refrigerated foods with a low water activity, high acidity, or packaging conditions that inhibit bacteria, including fruits, jams and jellies and fermented products (yogurt, cheeses, sausages, etc.). The yeast genera most commonly encountered are  Candida,  Cryptococcus,  Debaromyces, Hansenula,  Kluveromyces,  Pichia,  Saccharomyces,  Rhodotorula,  Torulopsis and  Trichosporon. The most commonly found mold genera include  Alternaria,  Aspergillus,  Botrytis,  Cladosporium,  Colletotrichum,  Fusarium,  Geotrichum, Monascus,  Mucor,  Penicillium,  Rhizopus,  Sporotrichum, Thamnidium and  Trichothecium.

The traditional method for enumerating psychrotrophiles in foods is the standard plate count, used to determine the number of colony-forming units (CFU) of aerobic microorganisms per gram or milliliter. The most commonly used growth media are  Dicloran Rose Bengal Chloramphenicol agar (DRBC) (for foods in general) and  Dicloran Glycerol 18% Agar (DG18) (for foods with a water activity smaller than 0.95). The incubation temperature is 7±1°C for 10 days or 17±1°C for 16 hours, followed by three more days at 7±1°C.

1.4  Heat-resistant molds

 Fungi, in general, have low resistance to heat and are relatively easily destroyed by mild heat treatments. There are, however, exceptions, since certain filamentous fungi produce spores that are capable of surviving heat treat-ments. Known heat-resistant mold species include  Byssochlamys fulva,  Byssochlamys nivea,  Neosartoria fisheri,  Talaromyces flavus,  Talaromyces bacillisporus and

Table.1   Analytical kits adopted as  AOAC Official Methods for the yeasts and molds count in foods (Horwitz and Latimer, 2010, AOAC International, 2010).

Eupenicillium brefeldianum, all of which produce spores exhibiting levels of heat-resistance comparable to those of bacterial spores. The spores of  B. fulva have a D90ºC value of 1–12 min (Bayne and Michener, 1979) and a z value between 6ºC and 7ºC (King et al., 1969). The heat resistance level of B. nivea is slightly lower, with a D88ºC of 0.75 to 0.8 min and a z value between 6ºC and 7ºC (Casella et al., 1990), while that of  N. fischeri is similar to that of B. fulva (Splittstoesser and Splittstoesser, 1977).

The heat-resistant molds are commonly associated with the deterioration of fruits and heat-processed fruit-based products. Their survival of heat treatment may result in growth with the formation of mycelia and, in the case of  Byssochlamys, in complete change of texture, caused by the production of pectinases.

 Heat-resistant molds are widely distributed through-out the soil but the number of spores in fruits is generally low, not exceeding 1–10/100 g or ml of processed products. Even so, in the step immediately prior to heat treatment, the presence of only five spores/g of product is already considered a serious problem. In products aseptically processed at high temperatures for short periods of time (UHT or HTST), without the addition of preservative agents, even lower counts are unacceptable.

For that reason, the success in detecting the presence of heat-resistant mold spores depends on the collecting of samples that are significantly greater in size than the samples normally taken for the purpose of microbiological examination of foods. These samples can be frozen until analysis, since the spores are not affected by freezing.

Detection is based on the heat-treatment of the samples, intended to eliminate vegetative cells of molds, yeasts and bacteria, followed by plating on a culture medium adequate for the growth of molds, such as  Potato Dextrose Agar (PDA) with antibiotics or  Malt Extract Agar (MEA) with antibiotics.

1.5   Preservative-resistant yeasts (PRY)

Some yeast species known as  PRY (preservative-resistant yeasts) are capable of growing in the presence of preservatives, such as sulphur dioxide and sorbic, benzoic, propionic, and acetic acids. The most important among these species is  Zygosaccharomyces bailii, but  Zygosaccharomyces bisporus,  Schizosacharomyces pombe,  Cândida krusei, and  Pichia membranaefaciens are also capable of growing in the presence of preservative compounds (Pitt and Hocking, 2009).

1.5.1   Zigosaccharomyces bailii (Lindner) Guilliermond 1912

Data collected by Pitt and Hocking (2009) highlight Zigosaccharomyces bailii as the most well-known and feared yeast species in the food industry processing acid foods. First of all, this species ferments glucose vigorously with CO₂ production, and is not inhibited by pressures in the order of 80 psig (560 kPa). Secondly, it is resistant to the majority of preservatives used to pre-vent fungal growth, and grows in the presence of 400 to 800 mg/kg or more of benzoic or sorbic acid. Thirdly, it presents mechanisms to adapt itself to preservatives and is able to acquire or develop resistance, after first exposure, to increasingly greater amounts. Fourthly, it is xerophilic (which means that it grows at water activity values in the order of 0.80 at 25ºC and 0.86 at 30ºC) and mesophilic in nature (it grows within the 5 to 40°C temperature range). Fortunately, it has low heat-resistance (D_50ºC  = 0.1 to 0.3 min for vegetative cells and D_60ºC = 8 to 14 min for ascospores), in addition to not using saccharose, which, when added in place of glucose, may prevent deterioration. The minimum pH for growth is 2.2–2.5.

The products susceptible to fermentative deterioration or explosive fermentation by  Z. bailii include tomato sauces, mustard, olives, mayonnaise and other salad dressings, beverages and soft drinks (carbonated or not), fruit juices (concentrated or not), fruit syrups and other toppings, cider, wines and balsamic vinegar. Prevention should be based on the complete exclusion of cells of Z. bailii from the product, since experience has shown that the distribution of only five adapted cells per package is sufficient to result in the deterioration of a high percentage of production. The most efficient alter-native is the product pasteurization in the final package, maintaining the product in the center of the package at temperatures of 65–68ºC for several seconds. If the product is pasteurized before filling, then a very stringent and rigorous sanitation and cleaning program of the entire processing line and environment will have to be put in place and maintained to prevent entrance and harboring of contamination sources. Membrane filtration before filling the product into final packages may also be an effective alternative, in case pasteurization is not feasible. Whenever possible, it is recommended to replace glucose by saccharose, and, in synthetic products, to avoid the addition of natural fruit juices and other ingredients that can be used by the microorganism as a source of carbon and nitrogen.

1.5.2   Zygosaccharomyces bisporus (Naganishi) Lodder and Kreger 1952

According to Pitt and Hocking (2009),  Z. bisporus presents physiological characteristics that are similar to those of Z. bailii, however, this species is even more xerophilic (capable of growing at a water activity of 0.70 in glucose/glycerol syrup). The ascospores of this species survive at 60ºC for 10 min, but not for 20 min. There are few reports on this species in the literature and its presence is less common in spoiled products, although it has the same spoilage potential as Z. bailii.

1.5.3   Schizosaccharomyces pombe Lindner 1893

S. pombe presents two characteristics that distinguish it from the majority of spoilage yeasts in foods (Pitt and Hocking, 2009). In the first place, vegetative reproduction does not rely on budding, but takes place by lateral fission and, secondly, this species grows better and more rapidly at 37°C than at 25°C, which makes it an important potential spoilage microorganism in tropical countries. It is osmophilic (grows at a water activity of 0.81 in glucose-based media), it is resistant to preservatives (resists 120 mg/kg free SO₂ and 600 mg/l benzoic acid) and its heat-resistance depends on the water activity and the solute present, being greater in the presence of saccharose (aW 0,95/D_65ºC  = 1.48 min) than in the presence of glucose (a_W 0,95/D_65ºC  = 0.41 min), fructose (a_W 0,95/D_65ºC = 0.27 min) or glycerol (aW 0,95/D_65ºC = 0.21 min). It is relatively uncommon as a spoilage microorganism, but has been isolated from sugar syrups and cranberry liquor preserved with SO₂.

1.5.4   Candida krusei (Castellani) Berkhout 1923

Pitt and Hocking (2009) highlight, as main characteristics of C. krusei: the capacity to grow at an extremely low pH (1.3 to 1.9, depending on the acid present), within a wide temperature range (8–47°C), and excel-lent development at 37°C. It is resistant to common food preservatives, and grows in the presence of 335 ppm of sorbic acid, 360 ppm of benzoic acid, and 30 ppm of free SO₂. For cells adapted to benzoic acid the minimum inhibitory concentration (MIC) found was 13.5 g/l of acetic acid, 8 g/l of propionic acid, 440 mg/l of benzoic acid and 1 g/l of methylparaben. The species has a relatively high heat resistance level (survives for 80 min at 56ºC), although it is inactivated after 2 min at 65ºC. The type of deterioration most commonly caused in foods is the formation of surface films and the species has been isolated from cocoa seeds, figs, tomato sauces, citrus products, concentrated orange juice and other fruit-based products, beverages, soft drinks, olives, cheeses, yogurt and other fermented milk products.

1.5.5   Pichia membranaefaciens Hansen 1904

Pitt and Hocking (2009) highlight the following characteristics of P. membranaefaciens: produces hat-shaped ascospores, grows in the 5–37°C temperature range and is halophilic, which means that it is capable of growing in the presence of up to 15.2% NaCl (aW 0.90), depending on the pH. Resistant to preservatives, it has been reported to grow at concentrations of sodium benzoate that, depending on the pH, varied from 250 mg/kg (pH 3.0) to 3000 mg/kg (pH 4.5). At pH 5.0, its growth has already been observed in the presence of 250 mg/kg of sorbate, but not at pH 3.0. The mini-mum pH for growth varies from 1.9 to 2.2, depending on the acid present. The species is very sensitive to heat, resisting for 30 min at 56ºC or for 10 min at 55°C, but not for 20 min at 55 or 10 minutes at 60°C. It has been isolated from olive brine, in which it causes “stuck fermentation” (reducing the amount of carbohydrate without the consequent production of lactic acid), and from several other products preserved in ace-tic acid (onions, cucumbers, pickles and sauerkraut), from tomato sauces, from mayonnaise and other salad dressings, in which it typically forms films, from concentrated orange juice and other citric products, from grape must and from processing lines of beverages and carbonated soft drinks. The detection of yeasts resistant to preservatives is based on the inoculation in culture media containing acetic acid. Pitt and Hocking (2009) recommend  Malt Extract Agar supplemented with 0.5% of acetic acid (MAA) or  Tryptone Glucose Yeast Extract Agar or Broth supplemented with 0.5% of acetic Acid (TGYA-TGYB). Inoculation in MAA or TGYA is accomplished by spread plating and incubation of the inoculated plates at 30ºC for two to three days. TGYB broth is used for previous enrichment of the samples, when populations are very low, thereby increasing the probability of recovering the strains in  MAA or  TGYA.

1.6  Osmophilic yeasts

In general, yeasts present minimal water activity (a_W) levels for growth around 0.88 and most molds around 0.80. The molds capable of growing at aW levels below the normal limit of 0.80 are called xerophilic (or xerophiles), while the yeasts that can grow below the aW limit of 0.88 are commonly known as osmophiles or osmophilic (those that are capable of growth in high concentrations of sugar) or halophiles (those that are capable of growth in high concentrations of salt). According to Pitt and Hocking (2009), the majority of osmophilic yeasts belong to the   Zygosaccharomyces genus, including  Z. rouxi,  Z. bailii and  Z. bisporus.

1.6.1   Zygosaccharomyces rouxii (Boutroux) Yarrow

Pitt and Hocking (2009) highlight the following characteristics of  Z. rouxii: It is a species extremely resist-ant to low water activities, and is considered to be the 2nd most xerophilic microorganism known in nature (the first being another fungus, the mold  Xeromyces bisporus). The minimum water activity levels reported are 0.62 in fructose syrup and 0.65 in glucose/glycerol syrup, while it may also grow and produce ascospores in dry plums with a_W 0.70. The optimal growth temperature varies from 24ºC (in 10% glucose weight/weight) to about 33ºC (in 60% glucose weight/weight, water activity of 0.87). Depending on the glucose concentration, the minimum growth temperature varies from 4ºC (in 10% glucose) to 7ºC (in 60% glucose) and the maximum varies from 37ºC (in 10% glucose) to 42ºC (in 60% glucose). In the presence of 46% glucose, the pH growth range extends from 1.8 to 8.0.

The heat resistance of Z. rouxii varies with the substrate, being greater in the presence of saccharose (a_W 0.95/D_65ºC = 1.9 min) than in the presence of glucose, fructose, or glycerol (a_W 0.95/D_65ºC  = 0.2–0.6 min). Water activity exerts a great influence on the heat survival capacity, which increases with the reduction of the level of free water:

The combination between the growth capacity at extremely low water activities and the ability to vigorously ferment hexoses makes Z. rouxii the second most frequent yeast associated with the deterioration of processed foods (the first is Z. bailii). The products most at risk include sugarcane juice, malt extract, concentrated fruit juices, syrups and caramel-based, soft-centered bonbons, dried fruits and sugar syrups. Preservation of these products may not be solely based on reduc-ing water activity since the products cannot be concentrated to values below their minimum growth limit. In this way, exactly like with Z. bailii, prevention requires complete exclusion of the cells from the product, with pasteurization prior to concentration being considered the most efficient alternative, in addition to refrigeration at a temperature around 0°C. Preservatives are effective in controlling their growth, but the use of these substances is rarely permitted in concentrated products. Putting in place and maintaining a rigorous cleaning and sanitation program of the processing lines is of essential importance, since Z. rouxii may grow continuously on equipment and processing devices and its presence in the product will only be detected after a considerable period of time.

The detection of osmophilic yeasts is based on the inoculation in culture media with high concentrations of glucose. Inoculation can be done either by surface plating or membrane filtration, with incubation of the inoculated plates at 30ºC for five to seven days.

References

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Beuchat, L.R. & Cousin, M.A. (2001) Yeasts and molds. In: Downes, F.P. & Ito, K. (eds).  Compendium of Methods for the Microbio-logical Examination of Foods. 4th edition. Washington, American Public Health Association. Chapter 20, pp. 209–215.

Horwitz, W. & Latimer, G.W. (eds) (2010)  Official Methods of Analysis of  AOAC International. 18th edition, revision 3. Gaithersburg, Maryland, AOAC International.

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King, A.D., Michener, H.D. & Ito, K.A. (1969) Control of Byssochlamys and related heat-resistant fungi in grape products. Applied Microbiology, 18, 166–173.

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Pitt, J.I. & Hocking, A.D. (eds) (2009) Fungi and Food Spoilage. 3rd edition. London, Springer.