A Review. Received 2 August 1989

Transcription

1 Journal of Applied Bacteriology , I 3 164/08/89 A Review Yeasts in dairy products G.H. FLEET Department of Food Science and Technology, The University of New South Wales, PO Box I, Kensington, New South Wales, 2033, Australia Received 2 August Introduction, Taxonomy of yeasts in dairy products, Milk, Cream, butter, other non-fermented products, Yogurt, Cheese, General occurrence of yeasts, Yeast spoilage of cheese, Contribution of yeasts to cheese maturation, Other fermented dairy products, Further research, References, Introduction Yeasts are commercially significant in foods because they cause spoilage or conduct desirable fermentations (Rose & Harrison 1970; Reed & Peppler 1973). Infections arising from the few, known pathogenic yeasts, such as Candida albicans or Cryptococcus neoforrnans (Hurley et al., 1987) are not transmitted through foods. Consequently, the public health significance of yeasts in foods has been considered by most health authorities to be minimal, if not negligible. However, this attitude may need revision. In summaries of statistics on food-borne disease in Canada, Todd (1983) noted cases where yeasts were suspected of causing food poisoning. The allergic reactions of consumers to foods and their contaminants are of increasing concern to health authorities, and yeasts have been mentioned in this connection (Taylor 1980; Anon. 1984). Although the fermentative and spoilage activities of yeasts are well known in many food and beverage commodities, little consideration has been given to the specific occurrence and significance of yeasts in dairy products. Indeed, recent reviews on the microbiology of milk and dairy products (Robinson 1981; Cousin 1982; Law & Mabbitt 1983; Bishop & White 1986) deal mostly with bacteria and make scant reference to yeasts. Consequently, one is left with the general impression that yeasts are of little economic significance to the modern dairy industry. This review focuses attention on the occurrence and importance of yeasts in dairy products, highlighting their involvement in the spoilage of some products and their beneficial role in the fermentation of others. Ingram (1958), Walker & Ayres (1970), Peppler (1976) and Walker (1977) have reviewed the spoilage of foods by yeasts, in general, and these contributions include significant reference to dairy products. Marth (1978) discusses some specific aspects of yeasts in dairy products. 2. Taxonomy of yeasts in dairy products Throughout the last 75 years, many yeasts have been isolated from dairy products and taxonomically identified. During this same period, there have been many changes to the classification and nomenclature of yeasts, making it a difficult task to trace chronologically the findings with respect to any

2 200 G. H. Fleet particular species. For example, strains described as Saccharomyces lactis and S. fragilis in the early literature, were subsequently described as Kluyueromyces lactis and Kluy.fragilis, respectively, but are now described as the single species, Kluy. marxianus. The current system of yeast classification and identification is that described by Kreger-van-Rij (1984). To minimize the risk of confusion, the names of yeast used in this review are those presented in the original article. The current name may be found by reference to Kreger-van-Rij (1984) or Barnett et al. (1983). As a means of assisting this task, Table 1 lists some of the main species found in dairy products and gives their names as presented by Kreger-van-Rij (1984), along with those mentioned in earlier literature. Deak & Beuchat (1987) give a good discussion of current difficulties associated with the nomenclature and identification of food-borne yeasts. A scan of the literature suggests that a multitude of different yeast species may be isolated from dairy products. However, the studies of Schmidt et al. (1979), Schmidt & Daudin (1983) and Baroiller & Schmidt (1984), which provide a taxonomic focus on yeasts isolated from cheeses, and the compilation of species in Table 1, suggest otherwise. It would appear from these observations that dairy products, as a whole, present a unique ecological niche, selecting for the growth and occurrence of only a few main species. Debaryomyces hansenii and Kluy. marxianus, as well as their asporogenous equivalents, Candida famata and C. kefyr, respectively, convincingly emerge as the most prevalent yeasts in dairy products. Some key properties that select for the growth and predominance of yeasts in dairy products are listed in Table 2, and relate to the chemico-physical properties of the product. Kluyueromyces marxianus, for example, is well known for its production of B-galactosidase and, consequently, it can ferment or assimilate lactose which is the main carbohydrate of milk. Debaryornyces hansenii has good tolerance of salt which is a main ingredient used in the manufacture of some cheeses. Strains of both species have varying abilities to produce proteolytic and lipolytic enzymes and, so metabolize milk protein and fat. Peppier (1977) and Miller (1979) provide good general accounts of the bio- Table 1. Yeast species frequently found in dairy products Identification according to Kreger-van-Rij (1984) Debaryomyces hansenii Candida famata.kluyoeromyces marxianus Candida kefyr Candida stellata Saccharomycopsis (Yarrowia) lipolytica Candida holmii Saccharomyces exiguus Pichia membranaefaciens Pichia fermentans Rhodotorula glutinis Rhodotorula rubra Some equivalent names in earlier literature D. subglobosus; Torulaspora hanseni Torulopsis candida; T. famata Kluy. fragilis; Kluy. lactis; Kluy. bulgaricus; Saccharomyces lactis; S. fragilis C. pseudotropicalis; Torulopsis kefyr Torula cremoris Torulopsis stellata Candida lipolytica Torulopsis holmii Candida krusei Candida krusei Table 2. Some properties of yeasts that select for their growth and predominance in dairy products 1. Fermentation or assimilation of lactose 2. Production of extracellular proteolytic enzymes 3. Production of extracellular lipolytic enzymes 4. Assimilation of lactic acid 5. Assimilation of citric acid 6. Growth at low temperatures 7. Tolerance of elevated salt concentration

3 Yeasts in dairy products 20 1 chemical and physiological properties of yeasts in relation to their activities in foods. More specific discussions of the properties of yeasts isolated from dairy products are given by Schmidt et al. (1979), Baroiller & Schmidt (1984) and Fleet & Mian (1987). 3. Milk Although milk is the raw material of most dairy products, surprisingly few studies have been conducted on the specific occurrence of yeasts in either raw or pasteurized milks. Often, information on yeasts is reported as an appendage to more detailed bacteriological studies. Generally, the available information shows that yeasts occur in both raw (Foster et al. 1957; Ingram 1958; Randolph et al. 1973; Engel 1986a) and pasteurized (Jones & Langlois 1977; Fleet & Mian 1987; Vadillo et al. 1987) milks, but at low, insignificant populations. Populations less than lo3 cells/ml are mostly reported but, occasionally, counts as high as lo4 cells/ml can occur. Such yeasts rarely grow in milk during refrigerated storage and are quickly overgrown by psychrotrophic bacteria (Cousin 1982; Bishop & White 1986). However, yeast growth might occur in milk where bacterial growth has been inhibited by residual antibiotics. Also, yeasts might develop in milk as secondary flora, after bacterial growth and spoilage. Sweetened condensed milks are particularly prone to gassy fermentation by yeasts because bacterial growth is restricted by the high sugar concentration and low water activity of this product (Walker & Ayres 1970). The taxonomy of yeasts found in milk has attracted little attention. Early studies, reviewed by Walker & Ayres (1970), suggest the frequent occurrence of pigmented yeasts of the genus Rhodotorula. Engel (1986a) reported a prevalence of Candida curvata in samples of raw milk. In a survey of 26 samples of pasteurized milks, Fleet & Mian (1987) described, in descending order of frequency, the isolation of C. famata, Kluy. marxianus, Cryptococcusflavus and C. dij7uens. In addition, they showed that these species, as well as Saccharomyces cerevisiae, could grow to cells/ml when they were inoculated and incubated in UHT-treated milk. While the growth of Candida, Kluyoeromyces and Cryptococcus species could be explained in terms of their ability to utilize either milk lactose, protein or fat, this explanation would not apply to S. cerevisae, which lacks these properties. Further studies are required to determine the substrates that S. cerevisiae utilizes for its growth in milk (Fleet & Mian 1987). Although there are many possible environmental sources of yeast contamination of raw milk, this is not so for pasteurized milks. The frequent occurrence of yeasts in pasteurized milks suggests that they have some degree of tolerance to the pasteurization process (Fleet & Mian 1987; Vadillo et al. 1987), but this possibility requires investigation. 4. Cream, butter, other non-fermented products Recent reviews on the microbiology of cream (Davis 1981) and butter (Murphy 1981) make little mention of yeasts as potential spoilage organisms. Nevertheless, yeast standards for acceptability of these products were stated as less than 10 yeast cells/g. Spoilage of cream by yeasts is well described in the early literature, especially if the cream had been sweetened or soured. Typically, the cream becomes foamy in appearance and yeasty in odour. Yeasts able to ferment residual lactose in the cream, or hydrolyse fat, were responsible (Garrison 1959; Walker & Ayres 1970; Thomas 1970). In a survey of 21 pasteurized cream samples purchased in Sydney, Australia, 48% had yeast counts of cells/ml and 14% exhibited counts of cells/ml (Fleet & Mian 1987). The main species isolated were C. famata, R. glutinis, C. diffluens, Cryp. laurentii and R. rubra. As might be expected, these species showed good lipolytic activity. Cream samples stored at 5 C for 10 days gave a 100-fold increase in their yeast population (Fleet & Mian 1987). Lipolytic species of yeasts, especially those of Rhodotorula, have been reported to grow on the surface of butter (Walker & Ayres 1970; Thomas 1971) but the incidence of this problem is very low. Fleet & Mian (1987) were not able to detect any yeasts in nine of 16 butter samples examined. The remaining samples had counts less than lo3 cells/g. Rhodotorula glutinis and R. rubra were the main species present in these samples.

4 202 G. H. Fleet Other products in which low levels of non-specific yeasts have been found include icecream (de Graft-Johnson 1974; Khayyat et al. 1977; Fleet & Mian 1987), milk powder (Jarvis & Shapton 1986), dried infant foods (Collins-Thompson et al. 1980; Singh et al. 1980) and frozen cream pies (Todd et al. 1983). 5. Yogurt Yogurt is a fermented milk product that, traditionally, was prepared by allowing milk to sour at 4W5"C. Modern yogurt production is a well-controlled process that utilizes ingredients of milk, milk powder, sugar, fruit, flavours, colouring, emulsifiers and stabilizers, and pure cultures of lactic acid bacteria to conduct the fermentation. Although fruit and flavoured yogurts are now very popular, plain yogurts, that contain no non-milk ingredients, are still prepared. Details of the production (Humphreys & Plunkett 1969; Robinson & Tamine 1986), composition (Robinson & Tamine 1975; Kroger 1976) and microbiology (Davis 1975; Bottazzi 1983) of yogurts are well reviewed. Yeasts are not involved in the fermentation for producing yogurt, but they are a major cause of spoilage of the final product. When they are produced under conditions of good manufacturing practice, yogurt should contain less than 10 yeast cells/g (but preferably less than 1 cell/g) and, if refrigerated at 5 C or less, they should not undergo spoilage by yeasts (Davis 1970, 1975). In these cases, a shelf-life of 4 weeks is expected and is limited by factors other than yeasts. Yogurt which is contaminated with an initial load of 100 or more yeast cells/g will probably spoil as the yeast cells multiply. Spoilage becomes evident when the yeast population reaches cells/g, and is first seen as a swelling of the yogurt package due to gas production by yeast fermentation. Eventually, the package ruptures and the yogurt acquires a yeasty, fermentative flavour and odour, and gassy appearance (Suriyarachchi & Fleet 1981; Green & Ibe 1987). Occasionally, yeast colonies are seen on the under surface of the package lid. There are two main mechanisms by which yogurts become contaminated with yeasts. First, they may originate from contaminated ingredients such as fruits, nuts and honey which, in most operations, are added to the fermented yogurt base just before packaging. Second, yeasts may develop on the surfaces of production equipment, such as mixing vessels and filling machines, that have been poorly cleaned and sanitized (Davis 1975; Suriyarachchi & Fleet 1981). Usually, yogurt base is free of yeasts because the ingredients may already have been mixed and heated to approximately 90 C for 30min. Growth of yeasts during fermentation of the mix is most unlikely because high concentrations of starter cultures of lactic acid bacteria are used, and because the fermentation temperature of 4WYC is restrictive to the growth of most yeast species. Contamination of the starter culture with Kluy. marxianus could pose a potential risk, however, as this yeast can grow at 4WYC and ferment lactose (Kreger-van-Rij 1984). It is not uncommon to find yeast populations of lo3 cells/g or more in retail samples of either plain or fruit yogurts. In many cases, counts of cells/g have been recorded (van Uden & Carmo Sousa 1957; Green & Ibe 1987; Fleet & Mian 1987). Surveys of retail yogurts in the UK (Davis 1974, 1975) and Canada (Arnott et al. 1974) found that 2&30% of samples had yeast counts exceeding lo3 cells/g. Similar surveys in Portugal (van Uden & Carmo Sousa 1957), Australia (Suriyarachchi & Fleet 1981 ; Fleet & Mian 1987) and Nigeria (Green & Ibe 1987) revealed a much higher incidence of contamination, with some 60% of samples having counts exceeding lo4 cells/g. Lesser degrees of contamination were reported for retail yogurts in the USA (Kroger 1976; Hankin & Shields 1980) and the Netherlands (Hup & Stadhouders 1972). Yogurts purchased in Spain (Garcia & Fernandez 1984) and Saudi Arabia (Salji et al. 1987) also exhibited contamination with yeasts, but quantitative statistics were not reported. Identification of the yeast species found in yogurts has been undertaken in a few studies. Lactose fermenting strains of Torulopsis spp. (Soulides 1956), C. pseudotropicalis (van Uden & Carmo Sousa 1957) and Kluy. bulgaricus (Dubois et al. 1980) have been implicated in the spoilage of plain yogurts. Torulopsis candida, T. versatilis, C. pelliculosa, C. intermedia and Hansenula anomala were the species most frequently isolated from retail yogurts in the United Kingdom (Tillbury et al. 1974). Candida lusitaniae, C. krusei and Kluy. fragilis were the main species isolated from 100 samples of yogurts

5 Yeasts in dairy products 203 produced in Lagos, Nigeria (Green & Ibe 1987). In a survey of 169 samples of yogurts retailed in Sydney, the species most frequently isolated in descending order were: C. famata, Kluy. marxianus, S. cerevisiae, C. stellata and C. difjluens (Suriyarachchi & Fleet 1981; Fleet & Mian 1987). Each of the species was able to establish good growth (populations exceeding lo7 cells/g) when they were inoculated into freshly prepared plain and fruit yogurts and incubated at either 20"C, 15 C or 5' C. At 5"C, best growth was exhibited by C. famata and C. dfluens which reached cells/g by 9 days (Fleet & Mian 1987). These authors concluded that the growth and predominance of particular species in yogurts is related to ability of the species to: (1) grow at the low temperatures (< 10 C) of yogurt storage; (2) produce lipolytic and proteolytic enzymes to hydrolyse milk fat and protein; (3) ferment lactose or sucrose which are the main carbohydrates of either plain or flavoured yogurts respectively; and (4) assimilate lactic and citric acids which are the main organic acids in yogurt. The most prevalent yeasts in yogurts, C. famata and Kluy. marxianus, were positive for many of these properties (Fleet & Mian 1987). Control over the occurrence and growth of yeasts in yogurts is straightforward, and embraces the general principles of good manufacturing practice. The critical points to monitor are: (1) proper mixing and heating of ingredients before fermentation; (2) absence of yeasts in the starter culture of lactic acid bacteria; (3) absence of yeasts (not detectable in 1.0 g) in fruit and other ingredients added to the fermented yogurt base; (4) regular, effective cleaning and sanitation of processing equipment; and (5) rapid cooling of the final product to 5 C and maintenance of this temperature throughout retailing (Davis 1975; Suriyarachchi & Fleet 1981). In some countries, the use of sorbate or benzoate preservatives may be permitted, but it is noteworthy that two of the main yeasts, C.famata and Kluy. marxianus, found in yogurts are tolerant of these substances at concentrations of 500 mg/l (Fleet & Mian 1987). 6. Cheese 6.1 GENERAL OCCURRENCE OF YEASTS The occurrence of yeasts in cheese is not unexpected because of the low ph, low moisture content, elevated salt concentration and refrigerated storage of these products. It is not widely recognized, however, that yeasts can be a major component of cheese microflora. The significance of this presence needs stronger emphasis, and depends on the particular style of cheese. In some cheeses, yeasts contribute to spoilage, but in others they make a positive contribution to flavour development during the stage of maturation. The chapters in the book by Fox (1987) provide good overviews of the chemistry, physics and microbiology of the different types of cheeses. The literature on the isolation of yeasts and moulds from foods in general, contains scattered references to the occurrence of yeasts in many samples of soft, semi-soft and hard cheeses. In some samples, yeast counts as high as cells/g have been reported (Koburger 1971; Hup & Stadhouders 1972; Anagnostakis & Hankin 1975; Koburger & Farhat 1975; Henson et al. 1982; Brodsky et al. 1982; Williams 1986; Jarvis & Shapton 1986; Banks & Board 1987). More specific surveys have confirmed this frequent occurrence. In a survey of imported European and North American cheeses, Nakase & Komagata (1977) found that 5 of 12 samples had yeast counts of cells/g. Debaryomyces hansenii and Candida lipolytica were the species most frequently isolated (Nakase et al. 1977). Retail samples of Burgos and Villalon cheeses produced in Spain had yeast counts of cells/g (Chavarri et al. 1985). Eleven of 50 samples of the Greek cheese, Kopanisti had yeast populations of cells/g. The predominant species, Pichia memhranaefaciens and P. fermentans were recovered from 80% and 24%, respectively, of the samples (Tzanetakis et al. 1987). Fleet & Mian (1987) reported that 48% of 23 samples of Australian cheddar cheese had yeast counts in the range cells/g. Thirty seven Yn of 19 cottage cheeses contained yeasts at cells/g. The most frequently isolated species from both cheese types were C. famata (38% of samples), Kluy. marxianus (19%) and C. difjluens (14%). These authors demonstrated the ability of yeasts to grow in cheddar and cottage cheeses during storage at 5 C for 10 days. In a comprehensive survey of 256 samples of blue-veined cheeses, representing mostly Danablue, Roquefort and Gorgonzola varieties, de Boer & Kuik (1987)

6 204 G. H. Fleet recorded a very high incidence of yeasts. Eighty seven o/o of Gorgonzola samples and 77% of Roquefort samples contained yeast populations exceeding lo6 cells/g, with some samples having cells/g. Debaryomyces hansenii was the most frequently occurring species, but isolates of Kluy. marxianus, S. cereuisiae and Yarrowia lipolytica were also obtained. a similar, large survey of Brie and Camembert cheeses purchased in Holland revealed that 60% of samples had yeast populations greater than lo6 cells/g. Yarrowia lipolytica, D. hansenii, Kluy. marxianus and several Candida species were the predominating yeasts (Nooitgedagt & Hartog 1988). 6.2 YEAST SPOILAGE OF CHEESE Ingram (1958) and Walker & Ayres (1970) have reviewed early studies that describe the spoilage of Cheddar and Swiss cheeses by yeasts. The main defects attributable to yeast activity were fruity, bitter or yeasty off-flavours and gassy, open texture. Such problems are now less frequently reported and, presumably, this is due to better understanding and control by the cheesemaker. Nevertheless, they still occur. Ikemiya & Yasumi (1973) reported good growth of D. hansenii on the surface of processed cheese. A fermented, yeasty flavour defect in Australian Cheddar cheese was recently reported by Horwood et al. (1987) and attributed to the activity of an unidentified species of Candida. Kluyueromyces marxianus, originating from the whey, has been implicated in the gassy spoilage of Parmesan cheese (Romano et al. 1989). Assessment of cheese spoilage by yeasts is complicated by subjective judgements on whether yeast activity during maturation is detrimental or beneficial to product quality. Over-ripening during maturation could be interpreted as spoilage. Thus, continued lactose fermentation by yeasts at this stage could lead to increased acidity, gassiness and fruity flavours, and continued hydrolysis of protein and fat could contribute to bitter and rancid flavours as well as a softening of product texture. There is little doubt that yeasts can spoil cottage cheese or similar types of unripened soft cheeses such as quarg (Roth et al. 1971; Hankin et al. 1975; Guiraud & Galzy 1976; Brocklehurst & Lund 1985; Engel 1986b; Engel et al. 1987). Yeast populations of cells/g frequently develop during refrigerated storage of the final product, leading to flavour and odour defects, gassiness and the appearance of surface colonies. Torulopsis sphaerica, C. lipolytica, Sporobolomyces roseus, Cryp. laurentii, Kluy. marxianus, C. ualida and P. membranaefaciens have, on different occasions, been implicated in this spoilage. The mechanism of yeast contamination and methods for controlling this problem are similar to those described for yogurt. It is very likely that the soft, brined cheeses, such as Feta and Domiati, are prone to yeast spoilage but the microbiology of these products has received little attention (Haddadin 1986; Abd El-Salam 1987). Ghoniem (1968) briefly reported the occurrence of yeasts in Domiati cheese and suggested their role in the production of gassy and flavour defects. 6.3 CONTRIBUTION OF YEASTS TO CHEESE MATURATION The growth of yeasts during the maturation (ripening/curing) stages of some cheese varieties is not widely appreciated, although it has been known for some time and is acknowledged in recent reviews on the microbiology of cheesemaking (Marth 1978; Law 1982; Vedamuthu & Washam 1983). It is considered that these yeasts contribute either directly or indirectly to the development of cheese flavour and texture but, in most cases, the chemical and biochemical mechanisms involved are not clearly understood. Generally, these yeasts are vaguely described as part of the secondary, associated or adventitious flora of cheesemaking (Law 1982; Vedamuthu & Washam 1983). In almost all cases, they originate as contaminants of the cheesemaking process and are not added as a starter culture. A most likely source of the yeasts is the milk, as evident from the data given in Section 3, but other sources of contamination include process equipment, starter cultures of lactic acid bacteria, rennet (Martinez et al. 1986), salt and any added fungal cultures Bacterial surface ripened cheeses Early studies in the United States provided convincing evidence that yeasts are involved in the curing of Limburger (Kelly 1937; Kelly & Marquardt 1939; Purko et al. 1951), Brick (Iya & Frazier 1949;

7 Yeasts in dairy products 20 5 Lubert & Frazier 1955) and Trappist-style (Szumski & Cone 1962; Ades & Cone 1969) cheeses. These are semi-soft cheeses, consisting of 40-60% moisture, that are cured at C for several days to weeks. During this stage, a smear of microbial growth develops over the surface of the cheese curd and plays a key role in the production of characteristic flavour. The predominant micro-organisms of the flora are Breuibacterium linens, micrococci, corynebacteria and yeasts (see Olson 1969 and Reps 1987 for reviews of the production and microbiology of these cheeses). The yeasts grow to cells/g during the first few days of curing, then decline to a stable population of about cells/g (Kelly 1937; Langhus et al. 1945; Szumski & Cone 1962). Although different yeast species occur within the total population, they have not been well characterized or identified (Iya & Frazier 1949; Purko et al., 1951; Lubert & Frazier 1955; Szumski & Cone 1962). According to Marth (1978), the species occur within the genera Debaryomyces, Rhodotorula, Torulopsis and Candida. These yeasts make several contributions to the curing process. Some species metabolize lactic acid of the cheese curd, thereby causing the ph to increase from an initial value of about 5.0 to approximately 5.7. This decrease in acidity encourages the growth of bacteria whose proteolytic and lipolytic activities are essential for cheese ripening. Furthermore, through autolysis or excretion, the yeasts liberate vitamins that promote bacterial growth (Kelly & Marquardt 1939; Purko et al ; Lubert & Frazier 1955), and proteolytic and lipolytic enzymes that contribute directly to the ripening process (Szumski & Cone 1962; Ades & Cone 1969) Blue-veined cheese Blue-veined cheeses are semi-soft cheeses that are ripened by the growth of mould, principally Penicillium roqueforti. After shaping and salting, the cheese curd is pierced with needle-like rods to create a network of pockets that allow entry of air and the escape of carbon dioxide. This encourages veins of mould growth throughout the internal and external portions of the cheese. Some well-known cheeses in this group include Roquefort, Gorgonzola, Danish Blue and Stilton. The Cabrales cheese of Spain and Kopanisti cheese of Greece could also be considered in this category. Aspects of microbiology, biochemistry and technology of these cheeses are reviewed by Coghill (1979), Shaw (1986) and Gripon (1987). Several early studies suggested the growth of yeasts during the ripening of cheeses (Morris et al ; Hartley & Jezeski 1954; Maxa & JiEinskjr 1956; ProkS et al. 1959). Comprehensive investigations of French Roquefort cheese have provided details of the yeast association (Galzin et al. 1970; Devoyod & Sponem 1970). Yeasts were present at less than lo3 cells/g throughout acidification of the milk by lactic acid bacteria. On storage of the curd for curing, yeasts rapidly multiplied, reaching cells/g within 1-2 days. Subsequent addition of salt to the curd caused the population to decrease by about 90% but, on further storage, growth recommenced and populations as high as cells/g developed. This pattern of growth occurred for yeasts at internal and surface locations in the curd, although populations in the center of the curd were about fold less than those on the surface. The species of yeast present varied with the stage of production, and location in the curd. According to Galzin et al. (1970), species of Saccharomyces, Hansenula and Torulopsis developed in the curd before salting. After salting, oxidative species of Torulopsis developed in the surface while fermentative species of Torulopsis were found at interior locations. Devoyod & Sponem (1970) reported a predominance of lactose fermenting strains of S. lactis, S. fragilis and T. sphaerica in the inner parts of the curd before salting. Candida lipolytica was also found. Salt-tolerant species of Torulopsis (T. candida, T. famata) grew within the curd after salting and species of H. anomala, P. membranaefaciens and Debaryomyces subglobosa developed on the surface. A similar profile of yeast evolution was reported during the maturation of Cabrales cheese, but higher yeast populations were found in the center of the curd than on the surface. Pichia membranaefaciens and P. fermentans dominated within the curd while D. hansenii and T. candida were the main species on the surface of the cheese (Nunez et al. 1981). Pichia membranaefaciens and P. fermentans were the predominant yeasts in Kopanisti cheese (Tzanetakis et al., 1987). Yeasts are considered to have several functions during the maturation of blue-veined cheeses. Lactose fermenting species that develop within the curd open up its texture, facilitating the growth

8 206 G. H. Fleet and penetration of P. roqueforti. Many of the yeast species utilize lactic acid, thereby increasing the ph of the curd. This stimulates the growth of bacteria that also form part of the ripening microflora (Devoyod et al. 1968, 1971). Other metabolic end products of yeast growth, and the activities of proteases and lipases produced by some species are believed to contribute to the development of cheese flavour and texture. According to Proks et a/. (1959) some yeast species may influence the production of flavour constituents, such as methyl ketone, by P. roqueforti Camembert cheese Camembert is another variety of semi-soft cheese that is ripened by mould. A complex microflora develops over the surface of the curd during the maturation stage, giving the cheese its characteristic white-grey appearance. The mould, Penicilliurn camemberti (or P. caseicolurnn) is a predominant component of the microflora, and its development is deliberately encouraged by inoculation. Other contributors to the ripening flora include Geotrichum spp., several bacterial species and yeasts (Lenoir 1984; Karahadian et al. 1985; Gripon 1987). This diversity of micro-organisms is clearly evident in scanning electron micrographs of the cheese surface (Rousseau 1984; Richard 1984). Guittonneau et al. (1939) foreshadowed the role of yeasts in the ripening of high quality Camembert. In a study of Camembert cheeses produced in five different French factories, Schmidt & Lenoir (1978, 1980) reported strong growth of yeasts during the maturation stage. The yeasts developed at both the inner and surface locations of the curd, reaching populations of cells/g and cells/g, respectively, during the first 7-15 days. Some 500 yeast strains were isolated and identified. Kluyverornyces lactis and Kluy. fragilis, along with their asporogenous forms, T. sphaerica and C. pseudotropicalis were the species isolated most frequently, and were more prevalent at surface locations. Deharyornyces hansenii and its asporgenous form, T. candida, occurred most frequently at inner locations of the curd. Other species that were isolated less frequently included Zygosaccharornyces rouxii, S. cereoisiae and T. oersatilis. The taxonomic status of these strains was given detailed discussion in later studies (Schmidt & Daudin 1983; Baroiller & Schmidt 1984). The roles that yeasts play in the muturation process is not clear, but are probably related to their capacity to produce lipases, esterases and peptidases, as well as ferment lactose and utilize lactic acid (Schmidt et al. 1979; Noomen 1983; Lenoir 1984) Other cheese oarieties Other cheeses in which yeasts grow during maturation include Gruyere (Zerfiridis et a/. 1983; Piton 1988), le Cuartirolo of Argentina (Tesone & Quevedo 1978), Vacherin (Sozzi & Shepherd 1972), Cantal (Millet et a/. 1974), Saint-Nectaire (Vergeade et a/. 1976) and Romano (Deiana et al. 1984). However, more detailed study of the yeast associations involved is required. In the case of Saint- Nectaire, populations of cells/g develop consisting predominantly of lactic acid utilizing strains of D. hansenii and T. candida on the surface and, to a lesser extent, lactose fermenting strains of Kluy. lactis and T. sphaerica within the cheese (Vergeade et al. 1976). Fatichenti et a/. (1983) and Deiana et al. (1984) have proposed the addition of D. hansenii as a starter culture in the production of Romano cheese because it accelerated ripening through faster proteolysis and lipolysis of the curd, metabolized acetic and lactic acids, and had an inhibitory effect on the growth of spoilage species of Clostridiurn. The possibility of using D. hansenii in cheese ripening was earlier mentioned by Yamauchi et a/. (1976) who noted that proteolysis by this species encouraged the survival and growth of lactic acid bacteria during mixed culture in skim milk. The involvement of yeasts in the maturation of Cheddar cheese is not clear. Studies on the microflora of these cheeses have not reported the occurrence of yeasts. (Fryer 1969; Law & Sharpe 1977; Cromie et al. 1987). However, these reports may reflect a lack of specific examination for yeasts rather than a true ecological absence. Fleet & Mian (1987) found that almost 50% of Australian Cheddar cheese samples contained yeast cells/g. The possible use of yeast proteases in the maturation of Cheddar cheese has been reported (Grieve 1982; Grieve et al. 1983; El-Soda 1986).

9 Yeasts in dairy products 7. Other fermented dairy products Other products that derive part of their character from yeast activities include kefir, koumiss and laban. These are produced by the fermentation of milk with a mixture of lactic acid bacteria, yeasts and other bacteria, such as acetic acid bacteria. The final products are acidic, slightly alcoholic, liquid to semi-liquid and effervescent, and are consumed as a beverage (Oberman 1985; Marshall 1986). In the case of kefir, particulate granules, consisting of microbial polysaccharide, denatured milk constituents and entrapped micro-organisms, are produced. Yeasts are located both within and outside these granules at populations of cells/g (Bottazzi & Bianchi 1980; Marshall 1986; Engel et al. 1986). The yeast flora of kefir varies with its source of production, but mixtures of lactose and non-lactose fermenting species, identified as Kluy. marxianus, C. kefyr, C. pseudotropicalis, S. cerevisiae, S. exiguus and T. holmii, have been reported (Iwasawa et al. 1982; Engel et al. 1986; Marshall 1986). Kluyueromyces marxianus and S. cereuisiae appear to be involved in the fermentation of koumiss and laban (Oberman 1985; Marshall 1986). Generally, the taxonomy and biochemistry of yeasts involved in the fermentation of kefir, koumiss and laban require more detailed investigation. A novel dairy food that is produced, in part, by the action of yeasts is gua-nai. This is a sweet-set gel which is formed by clotting pasteurized milk with an enzyme system, that is obtained from the fermentation of steamed glutinous rice, with a mixture of the fungi Amylomyces rouxii, Rhizopus oryzae and Aspergillus oryzae, and the yeast, Endomycopsis burtonii (Onyencho et al. 1987). The possibility of producing an acidophilus milk with a mixed culture of Lactobacillus acidophilus and the lactose fermenting yeasts Kluy. marxianus and C. pseudotropicalis has been described by Subramanian & Shankar (1985). Acidophilus milks, generally, should have the potential for spoilage by yeasts, but this problem is not evident from the literature Further research Knowledge about the general occurrence and growth of yeasts in dairy products remains incomplete. More comprehensive ecological surveys are needed to determine the diversity of yeast presence in dairy products, and to establish the occurrence of any specific yeast-product associations. Such studies must have a strong taxonomic focus to accurately describe the species present, and a strong quantitative emphasis to provide data about the potential for yeast growth during production and retailing of the product. The biochemical mechanisms by which yeasts affect the sensory quality of dairy products are poorly understood. Further research is required to determine the biochemical and physiological properties of the main yeast species found in dairy products. Such information will provide a better understanding of the factors that affect the growth of these species in dairy products, and of the mechanisms by which their metabolic activities affect product quality. Thus, little information is known about the flavour and aroma volatiles produced by Kluy. marxianus when it ferments lactose. Similarly, little is known about the nature of the proteolytic and lipolytic enzymes produced by Kluy. marxianus or D. hansenii, and how these enzymes act on milk proteins and fat. Finally, the deliberate use of selected yeast species in the maturation of cheese or in the production of other fermented dairy products requires serious exploration, and could give rise to a new starter culture technology for the dairy industry. ABD EL-SALAM, M.H Domiati and Feta Cheeses. In Cheese: Chemistry, Physics and Microbiology, Vol. 2, Major Cheese Groups ed. Fox, P.F. pp London : Elsevier Applied Science. ADm, G.L. & Corn, J.F Proteolytic activity of Breuibacterium linens during ripening of Trappisttype cheese. Journal of Dairy Science, 52, ANAGNOSTAKIS, S.L. & HANKIN, L use of selective media to detect enzyme production by micro- 9. References organisms in food products. Journal of Milk & Food Technology 38,57&572. ANON Adverse reactions to foods. American Academy of Allergy and Immunology Committee on Adverse Reactions to Foods. National Institute of Allergy and Infectious Diseases. U.S. Department of Health and Human Services, Washington: National Institute of Health Publication Number

10 ARNOTT, D.R.. DUITSCHAEUER, C.L. & BULLOCK, D.H Microbiological evaluation of yogurt produced commercially in Ontario. Journal of Milk & Food Technology 37, BANKS, J.G. & BOARD, R.G Some factors influencing the recovery of yeasts and moulds from chilled foods. International Journal of Food Microbiology 4, BARNETT, J.A., PAYNE, R.W. & YARROW, D Yeasts: Characteristics and Identification. Cambridge: Cambridge University Press. BAROILLER, C. & SCHMIDT, J.L Mise au point d une grille simplifib d identification des principales especes de levures presentes dans les homages. Le hit 64, BISHOP, J.R. & WHITE, C.H Assessment of dairy product quality and potential shelf life-a review. Journal of Food Protection 49, DE BOER, E. & KUIK, D A survey of the microbiological quality of blue-veined cheeses. Netherlands Milk & Dairy Journal 41, BOTTAZZI, V Other fermented dairy products. In Biotechnology, Vol. 5, Food and Feed Production with Microorganisms ed. Reed, G. pp Weinheim: Verlag Chemie. BOTTAZZI, V. & BIANCHI, F A note on scanning electron microscopy of microorganisms associated with the Kefir granule. Journal of Applied Bacteriology 48, BROCKLEHURST, T.F. & LUND, B.M Microbiological changes in cottage cheese varieties during storage at + 7 C. Food Microbiology 2, BRODSKY, M.H., ENTIS, P., ENTIS, M.P., SHARPE, A.N. & JARVIS, G.A Determination of aerobic plate and yeast and mould counts in foods using an automated hydrophobic grid-membrane filter technique. Journal of Food Protection 45, CHAVARRI, F.J., NurjEz, J.A., BAUTISTA, L. & Numz, M Factors affecting the microbiological quality of Burgos and Villalon cheeses at the retail level. Journal of Food Protection, 48, COGHIL, D The ripening of blue vein cheese: a review. 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In Dairy Microbiology, Vol. 2, The Microbiology of Milk Products ed. Robinson, R.K. pp London: Applied Science Publishers. DEAK, T. & BEUCHAT, L.R Identification of foodborne yeasts. Journal of Food Protection 50, DEIANA, P., FATICHENTI, F., FARRIS, G.A., MOCQUOT, G., LOW, R., TODWO, R. & CECCHI, L Metabolization of lactic and acetic acids in Pecorino Romano cheese made with a combined starter of lactic acid bacteria and yeast. Le Lait 64, 38G394. DEVOYOD, J.J. & DFSMAZEAUD, M Les associations microbiennes dans le fromage de Roquefort Action des enterocoques et des levures fermentant le lactose vis-a-vis des lactobacilles. Le Lait 51, DEVOYOD, J.J. & SPONEM, D La flore microbienne due fromage de Roquefort. VI. Les levures. Le Lait 50, DEVOYOD, J.J., BRET, G. & AUCLAIR, J.E La flore microbienne du fromage de roquefort. I. Son evolution au course de la fabrication et de I aflinage du fromage. 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11 Yeasts in dairy products 209 GARCIA, A.M. & FERNANDEZ, G.S Contaminating mycoflora in yogurt: general aspects and special reference to the genus Penicillium. Journal of Food Protection 47, GARRISON, E.R Lipolytic yeasts in cream from dairy farms. Journal of Dairy Science 42, GHONIEM, N.A Incidence of yeasts other than Candida species in Damietta cheese. Milchwissenschaji 23, DE GRAFT-JOHNSON, C The microbiological grading of ice-creams manufactured in Ghana. Ghana Journal of Agricultural Science 7, GREEN, M.D. & IBE, S.N Yeasts as primary contaminants in yogurts produced commercially in Lagos, Nigeria. Journal of Food Protection 50, GRIEVE, P.A Utilisation de la protease de levain 26, HURLEY, R., LOUVOIS, J. DE & MULHALL, A Yeasts as human and animal pathogens. In The Yeasts, Vol. 1, BiOlOgY of Yeasts ed. Rose, A.H. & Harrison, J.S. pp London : Academic Press. IKEMIYA, M. & YASUMI, K A strain of Debaryomyces hansenii isolated from processed cheese. 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