Yeasts and Molds: Spoilage Mold in Dairy Productsq

Nicole H Martin, Abigail Snyder, and Martin Wiedmann, Department of Food Science, Cornell University, Ithaca, NY, United States
© 2020 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by T.Sørhaug, volume 4, pp 780-784, ©2011 Elsevier Ltd.

Introduction 1
Molds Involved in the Spoilage of Dairy Products 1
Cheese 1
Other Dairy Products 2
Control of Mold Growth in Dairy Products 2
Methods for Enumeration and Identification of Molds From Dairy Products 3
References 4
Further Reading 4

Introduction

Spoilage fungi (yeast and molds) relevant to dairy product spoilage can be found in primary production environments (e.g., dairy
farms and equipment) as well as in food processing environments (e.g., cheese caves, manufacturing equipment). Their introduc-
tion into dairy products can result in desirable fermented end-products or undesirable spoilage defects. Importantly, spoilage
caused by yeast and molds is responsible for considerable food loss and waste with significant economic impacts. A report by
Pitt and Hocking (2009) estimates that fungal spoilage of food accounts for 5%–10% of food loss and waste worldwide (Pitt
and Hocking, 2009). Spoilage attributes caused by molds include flavor, odor, and body degradation. Diverse dairy products
including cheese, yogurt, sour cream, and other fermented or concentrated dairy products are susceptible to fungal spoilage.
Whereas fluid milk is more often spoiled by bacteria, products with a higher acid content or lower water activity often select for
fungal spoilage. Dairy-relevant spoilage molds are often capable of growth at low temperature, low water activity (aw), low pH,
and low oxygen tension atmospheres. Indeed, some spoilage molds can metabolize the weak acid antimicrobials commonly relied
upon as commercial food preservatives. The presence of spoilage molds in dairy products, which are formulated, packaged, and
handled under conditions, which support their growth, can lead to quality deterioration that may negatively affect consumer
perceptions as well as have important business, economic, and sustainability consequences for the dairy industry. Interventions
to reduce the incidence of dairy product spoilage by molds should focus on preventing environmental contamination throughout
the production-processing continuum as well as the use of barriers in the finished dairy product (e.g., protective cultures or modified
atmosphere packaging). Often, the application of multiple interventions or hurdles are necessary to ensure product quality.

Molds Involved in the Spoilage of Dairy Products

Cheese
Although fungi can be isolated from many dairy products, mold spoilage is a significant quality issue within cheese due to its ability
to proliferate under low oxygen, water activity, and carbohydrate levels in the presence of chemical preservatives and free fatty acids.
Aided by the relatively long shelf-life of many cheeses, spoilage molds can grow slowly under ambient or refrigerator temperatures
to cause visible and organoleptic defects. The susceptibility of any given cheese product to spoilage by molds depends on many
intrinsic and extrinsic factors, namely; (i) quality of raw materials; (ii) water activity (aw) and pH; (iii) length, degree and conditions
of ripening; (iv) storage conditions (temperature, relative humidity, type and extent of packaging), and; (v) environmental condi-
tions during processing and ripening (e.g., air quality and adherence to good manufacturing practices).
During the ripening of cheese, species from several genera are found on cheeses in cold storage, at temperatures as low as 1–5
C.
Penicillium is the most common spoilage mold found in cheese, with studies reporting50 to >90% of spoilage mold being Penicil-
lium species. Of particular importance are P. commune, P. nalgiovense, and P. roqueforti dominating the spoilage microbiota and other
Penicillium species (P. brevicompactum, P. chrysogenum, P. citrinum, P. cyclopium, P. expansum, P. glabrum, P. granulatum, P. palitans,
P. solitum, P. verrucosum, P. viridicatum) appear to be isolated less often. These Penicillium species have been isolated on cheese
sourced from Australia, New Zealand, South Africa, Argentina, Norway, Turkey, Spain, Italy, Denmark, Greece, France, Germany,

q
Change History: November 2019, N. Martin, A. Snyder and M. Wiedmann updated the Abstract, Introduction, Molds Involved in the Spoilage of Dairy
Products section, Control of Mold Growth in Dairy Products section, and Methods for Enumeration and Identification of Molds from Dairy Products section.
The Toxic Metabolites Produced by Molds in Dairy Products section was removed. This is an update of T. Sørhaug, Yeasts and Molds j Spoilage Molds in Dairy
Products, Editor(s): John W. Fuquay, Encyclopedia of Dairy Sciences (Second Edition), Academic Press, 2011, Pages 780–784, ISBN 9780123744074, https://
doi.org/10.1016/B978-0-12-374407-4.00366-6.

Reference Module in Food Sciences https://doi.org/10.1016/B978-0-08-100596-5.23017-5 1

2 Yeasts and Molds: Spoilage Mold in Dairy Products

Belgium, Japan, the Netherlands, the Czech Republic, Switzerland, Malta, Costa Rica, Azores, the United Kingdom, and the United
States. Penicillium species can grow at aw levels that approach 0.80, whereas other spoilage molds (e.g., Cladosporium species) only
grow down to aw of only 0.86. Some Penicillium species, particularly P. roqueforti, can grow in low oxygen (at 1%); however, carbon
dioxide at levels of 40% or more can prevent growth. It is important to note that the development of molecular tools for classifi-
cation of fungi has led to the revision of species and genus names, with names reported in older publications often being incon-
sistent with current designations. As fungal taxonomy and nomenclature continues to develop rapidly, these designations will
continue to be updated in the near future, which should be considered by the dairy industry when consulting with literature.
In addition, species of Aspergillus, Cladosporium, Galactomyces (formerly Geotrichum), Phoma, Scopulariopsis, and the Mucoralean
species Mucor, Rhizopus, and Syncephalastrum have also been isolated from cheese. In bloomy and washed-rind cheeses, such as
Camembert, Galactomyces geotrichum and G. candidum are used as desirable fermentative cultures. However, their extensive proteolytic
and lipolytic activities can result in off-flavors and aromas in dairy foods when they are introduced through post-pasteurization
contamination from the processing environment (Cai and Snyder, 2019). Some molds can produce bitter peptides in surface ripened
or blue-veined cheeses, such as some strains of P. camemberti, P. roqueforti, and G. candidum. Other fungi growing on cheese surfaces
result in visual defects. For example, darkly pigmented hyphal growth from Aspergillus and Penicillium that colonize cheese surfaces
cause consumer dissatisfaction. Similarly, cheese makers refer to the growth of Mucor or Rhizopus on the surface of cheese rinds the
“cat hair defect” (Snyder and Worobo, 2019). In vacuum-packaged cheeses, several fungal species produce a defect termed “thread
mold” because these molds grow in the wrinkles of packaging under relatively low oxygen tension.
Shredded cheese is particularly susceptible to spoilage by yeasts and molds due to the increased surface area and oxygen avail-
ability. Many commercial producers utilize the bacterially synthesized antimicrobial peptide natamycin to inhibit mycelial devel-
opment. Modified atmosphere packaging in CO2/N2 (e.g., 73%/27%) has been explored and may be necessary to control mold
growth.

Other Dairy Products

Fungal spoilage by molds of yogurt, butter, sweetened condensed milk, cream, and other dairy products has also been reported. Simi-
larly to cheese, the primary mold found in yogurt spoilage incidents is Penicillium (Buehler et al., 2017; Garnier et al., 2017).
However, a number of other genera have been reported in yogurt including species of Absidia, Alternaria, Aspergillus, Monilia, and
Rhizopus. Although the food industry refers to mold and yeast as distinct classes of fungi, they are morphological designations
and many fungi can grow in either a yeast or a hyphal phase. Yogurt spoilage can occur as the result of either mold or yeast growth.
In some cases, contamination of yogurt products with dimorphic mold (e.g., Mucor), or those that can grow as yeast or mold depend-
ing on environmental conditions, have also been shown to cause yogurt spoilage (Snyder et al., 2016). Heat-resistant fungi, which
produce ascospores, do not normally spoil dairy products; however, there are reports of Paecilomyces byssochlamys (formerly Byssochl-
amys nivea), Penicillium brefeldianum, Aspergillus fischerianus (formerly Neosartorya fischeri), and Talaromyces avellaneus causing spoilage
in products, such as ultra-heat-treated (UHT) custard and cream cheese. Perhaps more notably, these so-called heat resistant molds
are more commonly associated with thermally processed fruit products and may be introduced into finished products via ingredients,
such as fruit preparations. For example, strawberry yogurt has been shown to harbor Talaromyces contamination.
Mold spoilage genera found in butter include lipase-producing species of Aspergillus, Cladosporium, Galactomyces, and Penicillium.
In particular, Penicillium and Cladosporium species have been observed; these molds are lipolytic and also produce off-flavors
including 2-methylisoborneol and geosmin, thus contributing to an undesirable earthy flavor. In sweetened condensed milk,
mold growth is limited to xerophilic or xerotolerant fungi (Swanson, 2011), such as Wallemia; however, species of Aspergillus
and Penicillium can grow on the surface if there is poor sanitation in the processing plant that allows entry of mold spores and a large
enough headspace in the can to provide oxygen for growth. Sometimes, cream that is stored for long periods at temperatures close
to 0
C can have Penicillium species growing on the top. In addition, Fusarium oxysporum has been isolated from UHT flavored milks
in Australia, possibly due to the production of thick walled chlamydospores and the ability to tolerate low oxygen tensions.

Control of Mold Growth in Dairy Products

Controlling the entry and outgrowth of spoilage molds in dairy foods is achieved through several approaches that extend beyond
the intrinsic characteristics of the dairy product itself and include; (i) maintaining good manufacturing practices in processing
facilities, which includes proper cleaning and sanitation, control of air quality and in some cases, environmental monitoring;
(ii) managing ingredients, which may introduce fungal contaminants; and (iii) controlling the outgrowth of spoilage molds
through strategies, such as protective cultures, preservatives and packaging strategies. The importance of maintaining good
manufacturing practices in order to reduce the entry of environmental mold contaminants cannot be understated, as studies
have pointed to contamination during processing as a key transmission pathway of fungal microorganisms into dairy foods
(Buehler et al., 2019). Areas that may be improperly cleaned and allow mold buildup include conveyor belts, pumps, and valves.
The choice of sanitizers can be critical because chlorine seems to be more effective against molds than either peracetic acid per
oxidesor iodophors (Dijksterhuis et al., 2018). Molds can grow in moist environments found in dairy plants and establish them-
selves on ceilings, floors, walls, and in floor drains if these areas are not properly cleaned and sanitized.
Mold spores can also enter the processing plant in raw ingredients, on packaging materials, and on people. Cheese processing
enzymes that were synthesized by fungi have resulted in the spoilage of cheese products (Snyder and Worobo, 2019). Cardboard
 Yeasts and Molds: Spoilage Mold in Dairy Products 3

packing materials, slip sheets, wooden pallets, and paper packaging materials often harbor fungal spores. Separation of functions,
protecting filler spaces, and good air-filtration systems are essential in dairy processing plants to reduce the level of mold
spores. Seasonal and regional differences in fungal spore load in circulating air has been reported. Recommendations of fewer
than 50–100 molds and yeasts per cubic meter have been suggested for cheese processing plants. One way to reduce molds in
the air is to use high-efficiency particle air (HEPA) filters because they are designed to remove 90%–99% of particles 0.3 mm in
size. Maintenance and regular filter replacement are critical for good air quality. The use of positive air pressure in critical areas,
such as rooms for cheese ripening and packaging, can significantly decrease the level of mold contamination. The air intake and
exhaust systems in the plant need to be separated from each other to prevent re-contaminated air from coming back into the pro-
cessing plant. In an effort to improve air quality, “clean room technology” has been introduced into some cheese processing plants,
especially in the ripening rooms. The design of the rooms is carefully monitored to control air filtration and circulation, movement
of people who are dressed in cleanroom attire, and microbial contamination by using footbaths and air locks before entering the
area. Generally, clean rooms have air zones that limit particles > 0.5 mm per 0.3 m3 of air, resulting in <0.1bacteria per 0.3 m3 of
air. The humidifiers in these areas also operate by using sterilized and even demineralized water. In addition to humidity, control of
the temperature of the ripening and storage rooms can help to slow down the growth of molds in cheeses. Some manufacturers
choose to routinely monitor their environments for the presence of fungal contaminants, especially when the product is sensitive
to fungal growth (e.g., no preservatives are used).
Once introduced into the product, either through ingredients or from environmental cross-contamination, outgrowth of
spoilage mold in dairy is inhibited by a number of strategies including the use of protective cultures, packaging strategies, and
the use of preservatives. Many commercially available protective cultures have been developed to control fungal spoilage in fer-
mented dairy products, such as cheese and yogurt (Elsser-Gravesen and Elsser-Gravesen, 2014). These cultures are typically
strains of Lactobacillus or Propriono bacterium, which produce weak organic acids (e.g., benzoic acid not lactic, acetic and propionic
acids?), other inhibitory compounds (e.g., antimicrobial peptides), and act through competitive exclusion (Liptakova et al., 2006).
The use of protective cultures may allow processors to maintain a “clean label” with no added preservatives. Alternatively, direct
addition of the inhibitory compounds themselves has been very effective at reducing or preventing the growth of spoilage mold
in dairy products. Chemical preservatives, such as sorbic, benzoic and propionic acid as well as their salts (e.g., potassium sorbate)
are known for their antifungal activity (Garnier et al., 2017). Notably, however, certain species of common dairy spoilage molds
(e.g., Penicillium roqueforti) are able to decarboxylate the preservative sorbic acid to 1,3-pentadiene, which causes the kerosene
off-flavor in chemically preserved dairy products (Pinches and Apps, 2007). Other Penicillium species reduce sorbic acid to 4-
hexanoic acid and 4-hexanol, thereby reducing the antimicrobial activity as well as potentially causing off-odors and flavors.
New packaging technologies have considerably reduced mold spoilage of cheeses. Packaging material can be coated with anti-
mycotic agents, such as sorbates, propionates, or natamycin, or, alternatively, antimycotic agents can be incorporated directly into
the packaging material. Historically, excluding oxygen by the use of vacuum and modified atmosphere packaging has been
commonly used to limit growth of molds on dairy products sold commercially. The use of more than 50% carbon dioxide and
less than 0.5% oxygen prevents some spoilage fungi from growing on cheeses in modified atmosphere packages. However, package
leakage and pinhole defects can be problems with these types of packages, allowing molds to grow and cause spoilage.
Further, while there is little evidence to support a widespread public health threat from mycotoxins and other toxic metabolites
produced by molds in dairy products, controlling the entry and growth of molds in dairy products is of importance to reduce the risk
of consumer exposure to these toxins. The rate at which toxins are biosynthesized, their concentration, and their diffusion into the
cheese have moderated the current understanding of the hazard posed by fungal toxin production. Indeed, the presence of myco-
toxins in the raw milk from the consumption of contaminated grains by the dairy cow may represent the greater risk. Aspergillus
versicolor produces sterigmatocystin and both the mold and its mycotoxin have been isolated from cheese in temperate climates.
However, studies on the migration of mycotoxins into cheese rarely show greater than a 2 cm movement into the cheese; therefore,
cutting more than 2 cm under the moldy portions of the cheese should remove the mycotoxin as well as the mold. Significant addi-
tional research into mycotoxin formation and the risk it poses to human health would help food processors and regulators more
definitively assess and manage this potential hazard. However, the use of low ripening and storage temperatures (<10
C) and
vacuum or modified atmosphere packaging, generally, reduce the potential for mycotoxin production in cheese.

Methods for Enumeration and Identification of Molds From Dairy Products

For general mold enumeration in dairy foods, dichloran rose Bengal chloramphenicol (DRBC) agar is an effective choice because
dichloran (2,6-dichloro-4-nitroaniline) and rose bengal prevent the spreading of molds and thereby allow easier counting of colo-
nies and the chloramphenicol inhibits bacterial growth. Spread plates, as opposed to pour plates, are prepared because some fungi
may not be recovered under low oxygen environments. The plates are incubated upright at 25
C for 4–5 days for most molds. Addi-
tional incubation time may be needed for slower-growing molds. The use of acidified media, such as potato dextrose agar, is
discouraged for detection and enumeration of fungi from dairy products because acid-sensitive molds can be prevented from
growing on the media. Other culture-independent approaches include holding product samples under temperature abuse and look-
ing for product defects, such as gas production. Alternatively, cytometric tools have been developed my multiple biotechnology
companies for rapid quantification of yeast and mold levels in products.
4 Yeasts and Molds: Spoilage Mold in Dairy Products

Identification of foodborne fungi increasingly relies upon molecular subtyping, although microscopy and morphological
examination have historically played essential roles. PCR amplification and sequencing of internal transcribed spacer (ITS),
a universal barcoding gene for fungi, has proved a rapid and useful tool and is an industry standard in identification of fungi
(Buehler et al., 2019). The continued development of these rapid and novel methods will allow the dairy industry to better control
and reduce contamination by spoilage mold.

References

Buehler, A., Evanowski, R., Martin, N., Boor, K., Wiedmann, M., 2017. Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products. J. Dairy
Sci. 100, 8814–8825.
Buehler, A.J., Evanowski, R.L., Wiedmann, M., Martin, N.H., 2019. Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt
facilitiesda case study. J. Dairy Sci. 102, 3646–3653.
Cai, S., Snyder, A.B., 2019. Machinery mold (Galactomyces geotrichum) survival following thermal and hydrostatic pressure processing. J. Food Protect. 82, 1034–1038.
Dijksterhuis, J., Meijer, M., van Doorn, T., Samson, R., Rico-Munoz, E., 2018. Inactivation of stress-resistant ascospores of Eurotiales by industrial sanitizers. Int. J. Food Microbiol.
27–33. https://doi.org/10.1016/j.ijfoodmicro.2018.06.018.
Elsser-Gravesen, D., Elsser-Gravesen, A., 2014. Biopreservatives. In: Zorn, H., Czermak, P. (Eds.), Biotechnology of Food and Feed Additives. Springer Berlin Heidelberg, Berlin,
Heidelberg, pp. 29–49.
Garnier, L., Valence, F., Mounier, J., 2017. Diversity and control of spoilage fungi in dairy products: an update. Microorganisms 5, 42.
Liptakova, D., Valík, L., Bajusova, B., 2006. Effect of protective culture on the growth of Candida maltosa YP1 in yoghurt. J. Food Nutr. Res. 45, 147–151.
Pinches, S.E., Apps, P., 2007. Production in food of 1,3-pentadiene and styrene by Trichoderma species. Int. J. Food Microbiol. 116, 182–185.
Pitt, J.I., Hocking, A.D., 2009. Fungi and Food Spoilage, vol. 519. Springer.
Snyder, A.B., Worobo, R.W., 2019. Association of fungal genera from spoiled processed foods with physicochemical food properties and processing conditions. Food Microbiol. 83,
211–218.
Snyder, A.B., Churey, J.J., Worobo, R.W., 2016. Characterization and control of Mucor circinelloides spoilage in yogurt. Int. J. Food Microbiol. 228, 14–21.
Swanson, K.M., 2011. Milk and dairy products. In: Microorganisms in Foods 8: Use of Data for Assessing Process Control and Product Acceptance. Springer US, Boston, MA,
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Further Reading

Beuchat, L.R., Cousin, M.A., 2001. Yeasts and molds. In: Downes, F.P., Ito, K. (Eds.), Compendium of Methods for the Microbiological Examination of Foods, fourth ed. American
Public Health Association, Washington, DC, pp. 209–215.
Bullerman, L.B., 1981. Public health significance of moulds and mycotoxins in fermented dairy products. J. Dairy Sci. 64, 2439–2452.
Filtenborg, O., Frisvad, J.C., Thrane, U., 1996. Moulds in food spoilage. Int. J. Food Microbiol. 33, 85–102.
Fox, P.F., McSweeney, P.L.H., 1998. Dairy Chemistry and Biochemistry. Blackie Academic and Professional, London.
Fox, P.F., McSweeney, P.L.H., Cogan, T.M., Guinee, T.P. (Eds.), 2004. Cheese: Chemistry, Physics and Microbiology Major Cheese Groups, third ed., vol. 2. Elsevier Academic
Press, London.
Frisvad, J.C., Thrane, U., 2004. Mycotoxin production by common filamentous fungi. In: Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Filtenborg, O. (Eds.), Introduction to Food- and
Airborne Fungi, seventh edn. Centraalbureau voor Schimmelcultures, Baarn, The Netherlands, pp. 321–331.
Hocking, A.D., 1997. Understanding and controlling mould spoilage in cheese. Aust. J. Dairy Technol. 52, 123–124.
Kure, C.F., Borch, E., Karlson, I., Homleid, J.P., Langsrud, S., 2008. Use of the selective agar medium CREAD for monitoring the level of airborne spoilage moulds in cheese
production. Int. J. Food Microbiol. 122, 29–34.
Ledenbach, L.H., Marshall, R.T., 2009. Microbiological spoilage of dairyproducts. In: Sperber, W.H., Doyle, M.P. (Eds.), Compendium of the Microbiological Spoilage of Foods and
Beverages. In: Doyle, M.P. (Ed.), Food Microbiology and Food Safety Series. Springer, NewYork, pp. 41–67.
Lund, F., Filtenborg, O., Frisvad, J.C., 1995. Associated mycoflora of cheese. Food Microbiol. 12, 173–180.
Robinson, R.K. (Ed.), 2005. Dairy Microbiology Handbook: The Microbiology of Milk and Milk Products, third edn. JohnWiley & Sons, Inc, New York.
Samson, R.A., CBS-KNAW Fungal Biodiversity Centre, 2010. Food and Indoor Fungi. CBS-KNAW Fungal Biodiversity Centre.
Scott, P.M., 1989. Mycotoxigenic fungal contaminants of cheese and other dairy products. In: van Egmond, H.P. (Ed.), Mycotoxins in Dairy Products. Elsevier Applied Science,
London, pp. 193–259.
Walstra, P., Geurts, T.J., Noomen, A., Jellema, A., van Boekel, M.A.J.S., 1999. Dairy technology. In: Principles of Milk Properties and Processes. Marcel Dekker, Inc, New York.