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Yeasts associated with Danablu - A Taxonomic Study
Abstract
The regular occurrence of yeasts in raw milk, during processing and maturation of Danablu has been demonstrated. High numbers (108 cfu g-1) were found in Danablu after 4 weeks of maturation. Based on traditional methods (morphological, biochemical and physical criteria) and assimilation tests (API ID 32C, BioMerieux, Marcy l’Etoile, France), 166 yeast isolates from four dairies were identified. Candida famata was the most predominant yeast in Danablu, followed by Candida catenulata. Candida famata was found in all samples of raw milk and in increasing numbers during processing and maturation. Similar results were obtained for all dairies. The strains of C. famata isolated were lipolytic and assimilated lactose, glucose, galactose, lactate and citrate important for the micro-ecology of Danablu. C. catenulata had similar pattern of assimilation with the exception that it did not metabolise lactose. Some strains of C. famata produced brownish pigment and significant subspecies variations were observed in the growth characteristics at the combinations of temperature, concentration of NaCl and pH corresponding to the conditions in Danablu.
... In some cheese types, the yeasts contribute to the spoilage and cause changes in the texture (gassiness, softening), flavour (fruity, bitter, or yeasty off-flavours), or colour (pigmentation or discoloration), and, in some other types, they positively affect the maturation process [15,46,47,49,53]. In particular, their positive impact on the cheese ripening process consists in the utilisation of lactic acid causing the pH level to increase and the bacterial growth to be enhanced, especially in the semi-soft cheeses with a surface film, e.g., Limburger, Tilsit, and mould ripened cheeses, such as Camembert and Roquefort [1,14]. ...
... In particular, their positive impact on the cheese ripening process consists in the utilisation of lactic acid causing the pH level to increase and the bacterial growth to be enhanced, especially in the semi-soft cheeses with a surface film, e.g., Limburger, Tilsit, and mould ripened cheeses, such as Camembert and Roquefort [1,14]. In the blue veined cheeses, the yeasts are assumed to enhance the development of Penicillium roqueforti by gas production, leading to curd openness [20,46,47,48]. ...
... One of the yeast species occurring in cheese is Yarrowia lipolytica, but its populations are not usually numerous [26,33,46,47,55]. Our earlier investigations proved that the reason why there were low quantities of Y. lipolytica cells in the Polish blueveined cheese was their vulnerability to killer toxins produced by the species that prevail in that cheese: Candida famata (imperfect form of Debaryomyces hansenii) and C. sphaerica [26]. ...
A Yarrowia lipolytica JII1c yeast strain, isolated from the Polish 'Rokpol' mould cheese, was used as an adjunct culture in the production of a Dutch-type cheese. Its effect on the microbiological and biochemical characteristics of the cheese was evaluated in this research study. Milk used to produce the cheese was inoculated with 105 cfu/mL yeast cells. During the ripening process, the yeast population grew systematically to reach a maximum level of 7.9 log cfu/g in the sixth week of maturation, whereas the number of lactic acid bacteria increased until the fourth week of ripening. Thereafter, the number of microorganisms in the both groups decreased. After 8 weeks of ripening, the pH value of cheese inoculated with yeasts was significantly higher than that of the control cheese sample (produced without those microorganisms) and reached the levels of 6.37 and 5.47, respectively. In the experimental cheeses, it was also found that the utilization rate of lactic and citric acids was higher. Additionally, the concentration levels of water-soluble nitrogen (WSN) and free amino groups (FAG) in the experimental cheeses were about twice as high as in the control cheese sample. A more intensive proteolysis in the experimental cheese was accompanied by a higher accumulation of biogenic amines, especially of tyramine, putrescine, and 2-phenylethylamine; in the experimental cheese, after 8 weeks, their contents amounted to: 167.01, 77.90, and 69.54 mg/100 g, respectively. In contrast, the concentration of histamine was similar in both cheeses (9.47 and 9.81 mg/100 g in the control and experimental cheese samples, respectively). Also, the experimental cheese revealed more pronounced lipolysis resulting in a higher accumulation of free fatty acids, especially of butyric, myristic, palmitic, stearic, and oleic acids. It can be concluded that the Y. lipolytica JII1c grew well in the cheese causing the ripening process of the cheese to significantly accelerate.
... marxianus [42] S. lactis K. lactis var. lactis [42,91] C. catenulata C. catenulata [14,31,32,121,134,141] C. famata D. hansenii [5,14,42,91,134] C. intermedia C. intermedia [14,16,31,32,71,121,128] C. kefyr K. marxianus var. marxianus [14,121] C. krusei Issatchenkia occidentalis [14] C. lipolytica Y. lipolytica [14,42,71,121] C. pseudotropicalis K. marxianus var. ...
... marxianus [42] S. lactis K. lactis var. lactis [42,91] C. catenulata C. catenulata [14,31,32,121,134,141] C. famata D. hansenii [5,14,42,91,134] C. intermedia C. intermedia [14,16,31,32,71,121,128] C. kefyr K. marxianus var. marxianus [14,121] C. krusei Issatchenkia occidentalis [14] C. lipolytica Y. lipolytica [14,42,71,121] C. pseudotropicalis K. marxianus var. ...
... D. hansenii and K. marxianus occur mainly in blue-veined cheeses. D. hansenii, K. marxianus and G. candidum are mainly found in acid-curd cheeses and K. marxianus and C. zeylanoides are usually isolated from fresh cheeses [5,31,112,134]. ...
8.1 Introduction In 1680 yeasts were discovered by the Dutch scientist Antonie van Leeuwenhoek. During the second half of the nineteenth century, the French biochemist Louis Pasteur showed that yeasts were responsible for the conversion of sugar to ethanol and carbon dioxide [111]. It was only with the development of a technique to isolate pure cultures on solid media by Robert Koch that it became possible to select yeast strains on the basis of their fermentative characteristics [111, 115]. Yeasts may be defined as unicellular fungi reproducing by bud-ding or fission [80]. Some authors regard yeasts merely as fungi that produce unicellular growth, but that are otherwise not different from filamentous fungi. Consequently, yeasts are ascomycetous or basidiomycetous fungi that reproduce vegetatively by budding or fis-sion and are capable of forming sexual states that are not enclosed in a fruiting body [9]. Fungi for which no sexual stage is known, are traditionally included into the deuteromyc-etes or fungi imperfecti [7]. However, due to recent molecular studies these asexual yeasts are currently placed in their proper phylogenetic position [40, 83–85]. At present, approxi-mately 800 yeast species are recognized, but only a few are commonly used or isolated. Yeasts are the most important microorganisms ever exploited by man, because they have been used during several thousand of years for the production of a wide range of foods such as bread, wine, beer and kefyr, and more recently for the production of ethanol for fuel, bio-chemicals for the pharmaceutical industry and many other substances. Yeasts present on fruits, vegetables, equipments, in homemade starters, and in all kinds of raw biological ma-terial, such as milk, are responsible for the occurrence of spontaneous fermentations. As an example, the raw milk and the environment of a cheese factory, such as the brine, are im-portant sources for yeast contamination of the cheese surface. During ripening of smeared cheeses, these yeasts are indispensable.
... As in many other cheese types, the microbial analysis of blue cheeses has been addressed to the search for and selection of acidifying (LAB) and maturing (P. roqueforti) cultures [10,13,15,16,20,24]. As a result of artisan-like manufacture, where uncontrolled environmental conditions are common, large microbial differences between batches, producers, and seasons have been reported [13,14,27]. ...
... Both P. roqueforti and yeasts possess potent proteolytic and lipolytic systems that help transform the milk components into flavour compounds. Indeed, selected yeast strains have been proposed as adjuncts and maturing cultures for certain blue cheeses [10,15]. Geotrichum candidum (teleomorph state of Galactomyces candidus) is among the dominant yeast species in the surface and interior of the cheeses. ...
Citation: López-Díaz, T.M.; Alegría, Á.; Rodríguez-Calleja, J.M.; Combarros-Fuertes, P.; Fresno, J.M.; Santos, J.A.; Flórez, A.B.; Mayo, B. Blue Cheeses: Microbiology and Its Role in the Sensory Characteristics. Dairy 2023, 4, 410-422. https:// Abstract: Blue cheeses are those whose matrix is veined with a blue, blue-grey, or blue-green colour due to the development of Penicillium roqueforti. There are more than 45 varieties of blue cheese produced worldwide, with some distinct features, although the manufacture process is similar. In addition to P. roqueforti, complex microbial populations interact and succeed throughout the manufacturing and ripening at the cheese's surface (the rind) and interior (matrix). The microbiota of blue cheeses is made up of a vast array of both prokaryotic and eukaryotic microorganisms. Acidification of the curd relies on the action of lactococci and other lactic acid bacteria (LAB) species. The ripened cheeses' final quality and shelf-life properties largely depend on the enzymatic systems of the components of the microbiota, particularly on those of LAB, P. roqueforti, and yeast species. Proteolysis is the most complex and important primary biochemical process involved in blue-veined cheeses during ripening, with P. roqueforti being considered the main proteolytic agent. Lipolysis is also strong, originating, among other compounds, ketones, which are the main aroma compounds in blue-veined cheeses. In addition, several bioactive compounds are produced during ripening. The biochemical activities, mainly of microbial origin, are responsible for the sensory characteristics of these very appreciated cheese varieties worldwide.
... According to the review by Tudor and Board (1993), S. cerevsiae has been reported to be associated with cheese, whereas C. kefyr has been shown to occur frequently in dairy products (Fleet, 1990). Both K. marximanus and S. cerevisiae have been isolated from blue-veined cheeses (De Boer and Kuik, 1987; van den Tempel and Jakobsen, 1998), samples of yogurt (Suriyarachchi and Fleet, 1981; Fleet and Mian, 1987), and considered as normal to Kefir (Iwasawa, Ueda, Miyata, Hirota, and Ahiko, 1982; Engel, Krusch, and Teuber, 1986; Marshall, 1986). S. cerevisiae was the most dominant yeast despite its inability to ferment and assimilate lactose. ...
... The possible probiotic properties of S. cerevisiae were mentioned by Gedek (1991). In other works, S. cerevisiae has been isolated from raw milk but in low numbers (van den Tempel and Jakobsen, 1998). C.kefyr was the second most dominant yeast, probably because it is able to ferment lactose. ...
Nunu is a spontaneously fermented yoghurt-like milk product consumed as a staple food commodity in parts of the Saharan West Africa. Its production and consumption derives much food security and economic benefits to the rural people in the region. However, the process characteristics result in products which are not appealing to many people, have very short shelf-life and could have food safety concerns. In a framework of research to improve the product quality and increase consumption, a study was conducted to determine the process characteristics and elucidate the predominant microflora associate with the production of the product. A survey was done on the technology of nunu production in three major towns of the Upper East region, during which fermenting nunu were taken from 15 processors for laboratory analysis. pH, and titratable acidity were determined, as well as isolation and identification of the predominant microorganisms. Processing of nunu in northern Ghana takes place at ambient conditions in calabashes or plastic containers, and spontaneously. No pre-fermentation heating of the milk is done. The duration of fermentation is 24 to 48 hours. Several microorganisms were isolated from the fermenting samples, including LAB classified as lactobacillus, Leuconostoc, Lactococcus, Enterococcus, and Streptococcus; yeasts classified as Saccharomyces cerevisiae, Saccharomyces pastorianus, Candida kefyr, Yarrowia lipolytica, Candida stellata, Kluyveromyce maxianus, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii. The enterobacteriaceae, which were associated with the early stages of fermentation but eliminated as fermentation progressed were identified as Enterobacter, Klebsiella, Escherichia, Proteus vulgaris, and Shigella. The fermentation of nunu is spontaneous.
... Nevertheless, and as previously described, different microorganisms may respond differently to heat treatments, depending on a variety of factors [19]. In this sense, a few authors have reported the survival of some yeasts [146], that some pathogens may remain viable at the lower end of the thermisation temperature range, where the lethal effect is more reduced [30,117], and that thermisation may not be enough to significantly reduce the population of vegetative cells of the more heat-resistant bacterial species (Enterococcus, for example) [117,145]. Besides the possibility of some bacteria remaining viable in thermised milk, other shortcomings associated with this thermal treatment are the possible germination of spores present in milk during subsequent cold storage (for example, thermisation at 65 • C for 10 s may be sufficient to stimulate the germination of B. cereus spores [115]) and the possible selection for heat-resistant microorganisms such as M. tuberculosis and C. burnetii, by enabling their survival while reducing competitive flora [2,116]. ...
The microbial quality of raw milk artisanal cheeses is not always guaranteed due to the possible presence of pathogens in raw milk that can survive during manufacture and maturation. In this work, an overview of the existing information concerning lactic acid bacteria and plant extracts as antimicrobial agents is provided, as well as thermisation as a strategy to avoid pasteurisation and its negative impact on the sensory characteristics of artisanal cheeses. The mechanisms of antimicrobial action, advantages, limitations and, when applicable, relevant commercial applications are discussed. Plant extracts and lactic acid bacteria appear to be effective approaches to reduce microbial contamination in artisanal raw milk cheeses as a result of their constituents (for example, phenolic compounds in plant extracts), production of antimicrobial substances (such as organic acids and bacteriocins, in the case of lactic acid bacteria), or other mechanisms and their combinations. Thermisation was also confirmed as an effective heat inactivation strategy, causing the impairment of cellular structures and functions. This review also provides insight into the potential constraints of each of the approaches, hence pointing towards the direction of future research.
... Early studies, reviewed by Walker and Ayres (1970), suggest the frequent occurrence of pigmented yeasts of the genus Rhodotorula in raw milk and a recent study confirmed this finding (Pereira-Dias et al., 2000). Van den Tempel and Jakobsen (1998) found T. cutaneum in cow's milk for Danablu cheese manufacture and Corbo et al. (2001) isolated this species from milk of different animal origin. Moreover, T. cutaneum is a synonym of T. beigelii (Barnett et al., 1990) which was found by Tornadijo et al. (1998) in goat's milk. ...
The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64±1 log10 cfu ml−1 in milk to 0.65±1 log10 cfu g−1 in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein. G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.
... Information on the microbial diversity of raw milk can be used to judge its sanitary quality and the conditions of production [5,14]. Yeasts can have a negative action as spoilage microorganisms in dairy products, such as cheese, besides being able of causing several infections in man and animals [8,10,12,16]. Milk quality can also be affected by the enzymes produced by contaminating microorganisms [15]. The most important of these are proteases and lipases [4]. ...
Raw milk is generally considered an ideal environment for the growth of many microorganisms because of its nutrient richness. Although there have been numerous reports on the occurrence of yeasts in dairy products in the world, few have attempted to determine the diversity of these microorganisms in samples from raw milk, mainly in Brazil. Thirty six samples were collected from 25 dairy farms located at different districts of Rio Grande do Sul, during a period of 5 months. A total of 80 isolates were obtained and identified according to standard methods. Of these, 63 strains corresponded to true yeasts (51 with ascomycetic and 12 with basidiomycetic affinity), and 17 isolates were recognized as yeast-like strains. Yeast distribution was uneven among the producers analyzed. The most frequent yeasts isolated in this study belong to the genera Kluyveromyces, Rhodotorula, Candida, Geotrichum, and Trichosporon. The high frequency of isolation of Geotrichum and Trichosporon from raw milk is of great concern. Almost 79% of our isolates were lipolytic, while only 6% were proteolytic. Although these microorganisms are not expected to survive the pasteurization/sterilization treatments applied during milk processing, their enzymatic activities may alter milk constituents and affect its quality.
... RBCA was selected for the subsequent experiments because it consolidated a series of characteristics: gave higher counts, suppressed the spreading of P. roqueforti making the development, counting and observation of the other fungi possible, and a high diversity of yeast species was recovered. Yeast counts were within the range 10 5 –10 8 cfu/g and within the values reported for blue cheeses in the literature (Gobbetti et al., 1997; Roostita and Fleet, 1996b; Van den Tempel and Jakobsen, 1998; Viljoen et al., 2003). The outer crust counts (8.32 ± 0.02 log cfu/g) were approximately 10-fold higher than those in the blue veins (7.60 ± 0.02 log cfu/g) and about 1000-fold more than those in the white core (5.07 ± 0.02 log cfu/g). ...
Blue cheeses are very complex food matrices presenting significant spatial differentiation between sections and the Stilton variety also has a hard brown crust making its matrix even more complex. The mycobiota communities in the three sections (blue veins, white core and outer crust) of a Stilton blue cheese were studied by employing culture-independent (TRFLP, DGGE) and culture-dependent analyses. Yeasts isolated from the cheese were studied for aroma production in a dairy model system with and without the starter Lactococcus lactis and filamentous fungus Penicillium roqueforti using SPME GC–MS. Significant qualitative and quantitative differences were observed in the yeast communities between the cheese sections with all the techniques. Yarrowia lipolytica presented strong synergistic activity with P. roqueforti enhancing the production of ketone aroma compounds, characteristic of blue cheeses. Culture techniques allowed the observation of the presence and uneven distribution of two different morphological groups of Debaryomyces hansenii in the different sections and of Trichosporon ovoides but failed to isolate Candida catenulata which dominated some parts of the cheese in the culture-independent analysis. This suggests that this species may be an important early coloniser but fails to survive into the final cheese. The study indicated that the yeast flora in the cheese sections differ including isolates that could affect their aroma profiles.
... Yeasts are essential microorganisms in the production of various foods and drinks (Senses-Ergul et al. 2006). Due to their diverse metabolic potential, yeasts can play an important role in the fermentation and ripening of many cheese varieties and make a positive contribution to the development of taste and aroma (Fleet 1990;Jakobsen and Narvhus 1996;van den Tempel and Jakobsen 1998;Westall and Filtenborg 1998;Cosentino et al. 2001;Fadda et al. 2004). Unfortunately, yeasts can also be involved in the spoilage of food products caused by either failure in fermentation process or post-process contamination (Senses-Ergul et al. 2006). ...
In this research, 43 yeast strains were isolated from different types of cheese in Turkey and these isolates were identified
with both phenotypic tests and genotypic tests with 5.8S ITS-PCR. Also, the sequence of PCR fragments obtained from ITS-PCR
was analyzed by DNA sequencing. In the results of the phenotypic tests, 9 of 43 isolates were identified as Yarrowia lipolytica. After the phenotypic identification, all the isolates were identified with ITS-PCR and 13 were identified as Y. lipolytica. Yarrowia lipolytica secretes several hydrolytic enzymes including alkaline and acid proteases, lipases, a ribonuclease, and phosphotases. In
this paper, the effect of carbon and nitrogen sources and pH on production of AEP and RNase were examined for our identified
strains of Y. lipolytica. The best production of AEP were found in skim milk medium with pH 7.0 by TEM YL 5 and Y. lipolytica CBS 6124 type strain. An extra carbon source (glucose) reduced AEP production at all the pH studied. Glutamine which is a
nitrogen source did not affect AEP production. In contrast to AEP production, glucose increased RNase production at all the
pH studied.
... Although D. hansenii is a widely used starter culture in dairy and meat products, few studies on the combined effect of pH, NaCl and temperature have been carried out (Sørensen & Jakobsen, 1997;Van den Tempel & Jakobsen, 1998, 2000. Further, few studies have been undertaken concerning the growth of coryneforms under environmental conditions similar to surface ripening; such as the effect of salt concentration (El-Erian, 1972;Eliskases-Lechner & Ginizinger, 1995a) and the effect of pH and salt (Stadhouders & Langeveld, 1966) on their growth. ...
Growth of five strains each of Debaryomyces hansenii, Brevibacterium linens and Corynerbacterium spp. in a laboratory substrate was investigated at combinations of pH, NaCl concentration and temperature similar to those in surface ripening of cheeses. For D. hansenii the effect of pH on growth was found to be insignificant, but a combination of NaCl and temperature had a significant effect. For strains of B. linens and the Corynebacterium spp., the combined effects of pH, NaCl and temperature were found to be significant. All strains of B. linens, C. ammoniagenes and C. flavescens were stimulated by 4.0% (w/v) NaCl. Quadratic polynomial models were developed that described the growth of D. hansenii as a function of NaCl and temperature and growth of B. linens and Corynebacterium spp. as a function of pH, NaCl and temperature. Model validation in the laboratory substrate showed a good agreement between predicted and observed values.
... This finding is in agreement with results obtained for surface-ripened cheeses of the Tilsiter type (Lechner and Ginzinger, 1995). Also, studies on the surface of blue-veined cheeses demonstrated D. hansenii as the dominant yeast species (Roostita and Fleet, 1996, Van den Tempel and Jakobsen, 1998). However, in another study including different types of surface-ripened cheeses (Limburger, Romadour, Tilsiter, Mü nster, Weinkäse, and Harzer), the dominant yeast flora was found to consist of D. hansenii as well as one or more of the following species; Galactomyces geotrichum, Pichia membranifaciens, and Kluyveromyces marxianus (Valdés-Stauber et al., 1997). ...
Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].
... doi:10.1016/j.ijfoodmicro.2003.09.013 secondary starter culture have been added. Yeasts are not traditionally added as part of the starter culture or as adjunct starter cultures, but are often reported to develop as natural contaminants to high numbers (>10 6 CFU/g) in these cheeses Roostita and Fleet, 1996;Van den Tempel and Jakobsen, 1998). The complicated interactions between these contaminating yeasts and the starter cultures have been investigated (Hansen and Jakobsen, 1996). ...
Five countries representative of laboratories 1-5 evaluated 11 different selective media, designed to suppress mould and bacterial growth and support yeasts growth, for the recovery of yeast populations from blue veined cheeses. In addition, qualitative results were also incorporated. The yeast enumeration values were subjected to statistical analysis using analysis of variance (ANOVA) and the Tukey-Kramer multiple comparison test. With the exception of Laboratory 3, none of the other laboratories was successful in recovering yeasts on all the media. Six of the media proved inadequate for the enumeration of yeasts in the mould invested environment and were therefore omitted from statistical analysis. No significant differences in quantitative data obtained on Rose-Bengal Chloramphenicol Agar (RBCA), Dichloran Rose-Bengal Chloramphenicol Agar (DRBC), Dichloran 18% Glycerol Agar (DG18), and Malt extract agar supplemented with NaCl and oxytetracycline (MES) were detected by four of the collaborating laboratories whereas one laboratory found RBCA to be superior for yeast enumeration. DG18 and Malt Extract Agar with Biphenyl (MEB), however, were ranked superior based on qualitative results compared to the other media, attributed to distinctive individual yeast colonies and mould inhibition. RBCA, DRBC, DG18, and MES on the other hand, all proved to be adequate in supporting yeast colony development for quantitative analysis in samples obtained from blue veined cheeses.
... Early studies, reviewed by Walker and Ayres (1970), suggest the frequent occurrence of pigmented yeasts of the genus Rhodotorula in raw milk and a recent study confirmed this finding (Pereira-Dias et al., 2000). Van den Tempel and Jakobsen (1998) found T. cutaneum in cow's milk for Danablu cheese manufacture and Corbo et al. (2001) isolated this species from milk of different animal origin. Moreover, T. cutaneum is a synonym of T. beigelii (Barnett et al., 1990) which was found by Tornadijo et al. (1998) in goat's milk. ...
The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64+/-1 log(10) cfu ml(-1) in milk to 0.65+/-1 log(10) cfu g(-1) in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein. G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.
Bu çalışmada, Ortadoğu’da uzun yıllardan beri tüketilen ve son yıllarda Türkiye’de de üretilmeye başlanan Shanklish peynirlerinden mayaların izolasyonu, Start Codon Targeted (SCoT) markör yöntemi kullanılarak identifikasyonu ve enzimatik aktivitelerinin belirlenmesi amaçlanmıştır. Olgunlaştırılmış peynirlerden 24 adet maya izole edilmiş, SCoT markör yöntemiyle DNA parmak izleri elde edilerek gruplandırılmış ve her gruptan temsili izolatlar sekanslanarak identifikasyon sonuçları elde edilmiştir. Bu sonuçlara göre, 19 adet Kluyveromyces lactis, 2 adet Pichia kudriavzevii, 1 adet Pichia fermentans, 1 adet Pichia membranifaciens ve 1 adet Clavispora lusitaniae suşu tanımlanmış ve API-ZYM enzim test kiti yardımıyla enzimatik karakterizasyonları belirlenmiştir. Bu suşlar arasından K. lactis ANO17 suşu yüksek esteraz lipaz, lösin arilamidaz, valin arilamidaz, sistin arilamidaz, asit fostataz, Naftol-as-bi-fosfohidroliz, α-glukosidaz ve β-glukosidaz aktivitesi gösterirken orta seviyede esteraz, β-galaktosidaz ve düşük seviyede alkalin fostataz aktivitesi göstermiş ve bu suş enzimatik aktivite yönünden en umut verici suş olarak tespit edilmiştir. Çalışma sonuçlarına göre, K. lactis ANO17 suşunun olası starter/destek kültür kombinasyonlarında laktik asit bakterileriyle birlikte kullanımının teknolojik yönden üstün peynir elde edilmesinde faydalı olacağı düşünülmektedir.
Cheese ripening involves highly complex biochemical events. Coagulant enzymes as well as the utilized starters play an important role in these events. Two types of starters are used: primary and secondary. The main role of the primary culture, which consists of lactic acid bacteria, is to carry out lactic production during fermentation. They contribute to proteolysis and limited flavor formation with the enzymes they possess. Secondary or adjunct cultures are used to develop the texture and to accelerate the ripening. During the selection of this type of culture, enzyme profiles (i.e., proteolytic and lipolytic activities and their autolyse levels) in cheese are the primary factors to be taken into consideration. Apart from these, the other factors are their positive effects on health, availability, and economy. Adjunct cultures include yeast, molds, and bacteria. Some of the heterofermentative lactobacilli species, in particular weakened strains, are used as adjunct cultures in order to accelerate the ripening and shorten the ripening time in fat-reduced and low-fat cheeses. This chapter explores adjunct cultures in cheese technology.
Yeasts are associated with the secondary flora of many cheese types. Some yeast species such as Debaryomyces hansenii, Yarrowia lipolytica, Geotrichum candidum, Kluyveromyces lactis, Kluyveromyces marxianus, Candida zeylanoides, Candida utilis, Candida kefyr, and Saccharomyces cerevisiae can be frequently isolated from different traditional cheeses. By their metabolic activities, they may either cause spoilage or contribute to the cheese ripening process by causing desirable biochemical changes. Yeasts may contribute to cheese ripening by the metabolism of lactic acid, which in turn increases the pH and favors bacterial growth, as well as by their proteolytic and lipolytic activities, and metabolism of lactose, glucose, galactose, and organic acids. There are also reports about the interaction of yeasts with other microorganisms during cheese ripening. Because of their positive attributes, the yeast strains isolated from different traditional cheeses are proposed to be used as adjunct starters in cheese production. Yeasts originated from traditional cheeses may also be used as starter culture candidates in the production of other foods or they may be evaluated in biotechnological processes based on their biochemical traits.
Samples of milk, curd, cheese whey, and cheese were collected in 10 farms located at the region of Serra do Salitre, Minas Gerais state. These samples were studied in relation to their lactic acid bacteria and yeast populations. The diversity of lactic acid bacteria species was lower than the diversity of yeasts in these samples. The isolated lactic acid bacteria were Lactococcus lactis, Enterococcus spp., Enterococcus faecalis, and Streptococcus agalactiae; and the yeasts were Debaryomyces hansenii and Kluyveromyces lactis. Only the species Enterococcus spp., Enterococcus faecalis, and Leuconostoc mesenteroides showed an increase in their populations during the production of the artisanal cheese. Lactic acid bacteria and yeasts found in this study could be responsible by the sensorial characteristics of the artisanal cheese produced in the region of Serra do Salitre.
In this paper, some technological properties of 8 strains of Yarrovia lipolytica yeast and 13 strains of Debaryomyces hansenii yeasts were analyzed. The analysis focused on the level of their proteolytic and lipolytic activities, their limited ability to generate biogenic amines and pigment, and, also, on the mutual interactions between the strains of those two species. The yeast strains of Y. lipolytica (PII6a, PII6c and Rile) demonstrated the best ability to synthetise both the extra- and the intracellular hydrolases. As regards the D. hansenii yeast strains, only a minor intracellular peptidase activity thereof was found, and, in the majority of cases, an extracellular lipolytic activity. Only the Y. lipolytica yeast strains showed an ability to produce pigment, except for the PII6a, PII6b, and PII6c strains. None of the strains of the two species analyzed degraded histidine to histamine, however, the Y. lipolytica yeast showed an ability to produce more biogenic amines. All the D. hansenii yeast strains produced killer toxins. Only three strains of Y lipolytica (JII1a, JII1b, JII1c) demonstrated resistance to those killer toxins.
In this study, technological and probiotic characteristics of 35 yeast strains isolated from Erzincan tulum cheese, locally produced in the Eastern Anatolia region of Turkey, were investigated. Four strains belonging to G. candidum, C. kefyr, C. lipolytica and C. lambica were found to have proteolytic activities while 80% of the investigated strains had lipolytic activities. Most (69%) of the tested yeast strains had the ability to assimilate galactose. All of the strains could grow at 10°C and 83% of them had the ability to grow at 4°C, while only 34% of them were resistant to 45°C. Most of the strains had also ability to grow at pH 2.5 except one C. colliculosa strain. All of the isolates had the ability to grow in the medium with 5% NaCl. In the media with 10% and 15% NaCl concentration, 89% and 31% of the strains were able to grow, respectively. Tolerance to high bile salt concentrations was a rare property among the strains, of which C. rugosa gave the best result for growing in Ox-Bile medium. Inhibition effects of the tested yeasts on some pathogenic bacteria were generally weak, but a strong inhibition effect of a C. krusei strain was detected on S. aureus.
The growth dynamics of an oxidative yeast Candida maltosa Komagata, Nakase et Katsuya in model liquid media with glucose and yeast extract was determined in relation to different lactic acid concentrations ranging from 0 to 1.6 % (w/v). The strain C. maltosa was isolated from the surface of a spoiled fruit yoghurt probably for the first time in Slovakia. The maximum specific growth rate in the broth without lactic acid at 25 °C was 0.36 h−1 and the minimum lag−phase duration was 2.9 h. The decrease in the natural logarithm of the specific growth rate (ln μ) as well as the increase in the natural logarithm of the lag−phase (ln λ) correlated linearly (Rμ2 = 0.9526; Rλ2 = 0.9577) with the increasing lactic acid concentration (0–1.59 %). Based on these correlations, predictions of the time for C. maltosa to reach a density of 1.106 CFU.ml−1 are presented.
Economic and sensorial losses of dairy products due to spoilage by yeast have been increasing in Turkey because of poor hygienic conditions during processing and to shorten the anticipated shelf life of products and/also reduced use of preservatives that do not strictly control the growth of these organisms. This study reports the results of a survey of yeast species in samples of cow's, ewe's and goat's milk products collected in some regional bazaar of Izmir, Western Turkey. Yeasts were isolated from white pickled cheese, tulum cheese and kashkaval cheese, yoghurt and strained yoghurt respectively. The most frequently occurring yeast species on dairy products were Candida sp., Saccharomyces sp., Kluyveromyces sp. and Trichosporon sp. respectively. Even though yeasts are considered as environmental contaminants, the occurrence of some of them in dairy products at high levels could represent a potential risk for human health especially for immuno compromised patients.
In this paper, some technological properties of 8 strains of Yarrovia lipolytica yeast and 13 strains of Debaryomyces hansenii yeasts were analyzed. The analysis focused on the level of their proteolytic and lipolytic activities, their limited ability to generate biogenic amines and pigment, and, also, on the mutual interactions between the strains of those two species. The yeast strains of Y lipolytica (PII6a, PII6c and JII1c) demonstrated the best ability to synthetise both the extra- and the intracellular hydrolases. As regards the D. hansenii yeast strains, only a minor intracellular peptidase activity thereof was found, and, in the majority of cases, an extracellular lipolytic activity. Only the Y lipolytica yeast strains showed an ability to produce pigment, except for the PII6a, PII6b, and PII6c strains. None of the strains of the two species analyzed degraded histidine to histamine, however, the Y lipolytica yeast showed an ability to produce more biogenic amines. All the D. hansenii yeast strains produced killer toxins. Only three strains of Y lipolytica (JII1a, JII1b, JII1c) demonstrated resistance to those killer toxins.
Blue or blue-veined cheeses are characterized by growth of the mold Penicillium roqueforti, giving them their typical appearance and flavor. Blue cheese is produced in many countries all over the world, where their own types of blue cheeses have been developed, each with different characteristics and involving different manufacturing methods.This chapter aims to review the knowledge on different aspects of blue cheese ripening, emphasizing changes in the microenvironment, for example, pH and salt gradients within the cheese matrix; the microorganisms that contribute to ripening and their interactions, that is, lactic acid bacteria, mold, and yeasts; and the various biochemical changes, that is, lipolysis, proteolysis, and aroma formation and the effect on the texture and consistency of the ripened cheese. Potential mycotoxin production is also covered. Finally, thoughts on the selection of appropriate starter and mold cultures, as well as new, possible adjunct cultures, will be discussed.
This chapter explains Blue cheeses that are characterized by the growth of the mould Penicillium roqueforti, giving them their typical appearance and flavor. Mesophilic and thermophilic lactic acid bacteria (LAB) are used as the primary starter culture for the production of different varieties of Blue cheese. The benefits obtainable by selecting the right combination of cultures are discussed by the results, showing that the stimulation of the growth and sporulation of P. roqueforti was stronger at higher levels of NaCl. Compounds resulting from different pathways of amino acid catabolism have been found in Blue cheese. Research on texture development and its relationship with the proteolytic activity of P. roqueforti together with texture analysis of Blue cheese is very limited. It would be a valuable tool for further characterization of the cheese itself and the effect thereon of the microbiota, especially the strain of P. roqueforti.
Newly weaned 4-week-old piglets were fed a semi-synthetic diet supplemented with 40g fructo-oligosaccharides (FOS) or 40g transgalacto-oligosaccharides (TOS) per kg replacing 20g maize starch and 20g cellulose per kg in the control diet. Faecal samples were collected on days 0, 3, 7, 14 and 28 after weaning and analysed for dry matter, pH, organic acid concentration and distribution, microbial populations and identity of dominant bacterial species able to ferment FOS.The oligosaccharide addition to diets had no effect on growth performance of the piglets. Dietary oligosaccharides and time after weaning did not affect faecal dry matter content. There was no effect of diet on faecal pH and total organic acid concentration, but the pH decreased (P
Em 10 fazendas da região da Serra do Salitre, MG, foram coletadas amostras de leite, soro fermentado (pingo), coalhada e queijo frescal para avaliar a microbiota de bactérias láticas e leveduras presentes. Uma diversidade menor de bactérias láticas foi observada durante a produção do queijo quando comparada à de leveduras. As espécies de bactérias láticas mais freqüentes foram Lactococcus lactis, Enterococcus spp., Enterococcus faecalis e Streptococcus agalactiae e de leveduras foram Debaryomyces hansenii e Kluyveromyces lactis. Apenas as populações de Enterococcus spp., Enterococcus faecalis e Leuconostoc mesenteroides apresentaram aumento significativo durante a produção do queijo. As espécies de bactérias láticas e leveduras encontradas nos diferentes substratos estudados podem ser responsáveis pelas características de aroma e sabor do queijo artesanal da Serra do Salitre.Samples of milk, curd, cheese whey, and cheese were collected in 10 farms located at the region of Serra do Salitre, Minas Gerais state. These samples were studied in relation to their lactic acid bacteria and yeast populations. The diversity of lactic acid bacteria species was lower than the diversity of yeasts in these samples. The isolated lactic acid bacteria were Lactococcus lactis, Enterococcus spp., Enterococcus faecalis, and Streptococcus agalactiae; and the yeasts were Debaryomyces hansenii and Kluyveromyces lactis. Only the species Enterococcus spp., Enterococcus faecalis, and Leuconostoc mesenteroides showed an increase in their populations during the production of the artisanal cheese. Lactic acid bacteria and yeasts found in this study could be responsible by the sensorial characteristics of the artisanal cheese produced in the region of Serra do Salitre.
Abstract Yarrowia lipolytica has been developed as a production host for a large variety of biotechnological applications. Efficacy and safety studies have demonstrated the safe use of Yarrowia-derived products containing significant proportions of Yarrowia biomass (as for DuPont's eicosapentaenoic acid-rich oil) or with the yeast itself as the final product (as for British Petroleum's single-cell protein product). The natural occurrence of the species in food, particularly cheese, other dairy products and meat, is a further argument supporting its safety. The species causes rare opportunistic infections in severely immunocompromised or otherwise seriously ill people with other underlying diseases or conditions. The infections can be treated effectively by the use of regular antifungal drugs, and in some cases even disappeared spontaneously. Based on our assessment, we conclude that Y. lipolytica is a "safe-to-use" organism.
http://informahealthcare.com/doi/pdf/10.3109/1040841X.2013.770386
Flavor production among 12 strains of Debaryomyces hansenii when grown on a simple cheese model mimicking a cheese surface was investigated by dynamic headspace sampling followed by gas chromatography-mass spectrometry. The present study confirmed that D. hansenii possess the ability to produce important cheese flavor compounds, primarily branched-chain aldehydes and alcohols, and thus important for the final cheese flavor. Quantification of representative aldehydes (2-Methylpropanal, 3-Methylbutanal) and alcohols (2-Methyl-1-propanol, 3-Methyl-1-butanol, and 3-Methyl-3-buten-1-ol) showed that the investigated D. hansenii strains varied significantly with respect to production of these flavor compounds. Contrary to the alcohols (2-Methyl-1-propanol, 3-Methyl-1-butanol, and 3-Methyl-3-buten-1-ol), the aldehydes (2-Methylpropanal, 3-Methylbutanal) were produced by the D. hansenii strains in concentrations higher than their sensory threshold values, and thus seemed more important than alcohols for cheese flavor. These results show that D. hansenii strains may have potential to be applied as cultures for increasing the nutty/malty flavor of cheese due to their production of aldehydes. However, due to large strain variations, production of flavor compounds has to be taken into consideration for selection of D. hansenii strains as starter cultures for cheese production.
Varieties of blue veined cheese were analyzed regularly during different stages of ma-nufacturing and ripening to determine the origin of contaminating the yeasts present in them, their population diversity and development until the end of the storage. Yeast di-versity and development in the inner and outer core of the cheeses during ripening were also compared. Air samples revealed few if any yeasts whereas the samples in contact with the equip-ment and the surroundings revealed high number of yeasts, implicating it as the possible main source of post-pasteurization contamination, as very few yeasts were isolated from the milk and cheese making process itself. Samples from the inner and outer core of the maturing cheeses had typical survival curves. The number of yeasts on the outer core was about a 100-fold more than of those in the inner core. The most abundant yeasts isolated from the environment and ripening cheeses were identified as Debaryomyces hansenii, Saccharomyces cerevisiae, Torulaspora delbrueckii, Tricho-sporon beigelii, Candida versatilis and Cryptococcus albidus, while the yeasts Candida zeylanoi-des and Dekkera anomala were additionally isolated from the environment. Yeasts were pre-sent in high number, making their occurrence in blue-veined cheeses meaningful.
The use of yeasts and filamentous fungi in the production of bread, cheese and meat products is described within a historic
perspective. Recent research activities mainly on the impact of microbial interactions and molecular techniques are given,
with the main emphasis on cheese. Molecular techniques are addressed, in particular proteomics, for a description of yeast
starter cultures and the mechanisms underlying their adaptation to relevant stress conditions. Furthermore, the most recent
taxonomy of the yeasts Debaryomyces hansenii, Yarrowia lipolytica, Geotrichum candidum and Saccharomyces cerevisiae is described. Finally, the formation of aroma components by filamentous fungi is highlighted.
A major diversity of microbial flora is associated with cheese ripening. This flora may result from deliberate addition or through adventitious colonisation. The application of molecular techniques to the study of cheese microbiology is providing a valuable insight into the behaviour of individual strains and populations during ripening. Further characterisation of metabolic potential of the cheese flora is required to elucidate the methods by which these micro-organisms influence cheese quality. The diversity of the flora at species and strain level provides a major biotechnological resource that offers the potential for manipulation in the development of new and innovative cheese products.
Six strains of Debaryomyces hansenii var. hansenii and Yarrowia lipolytica, respectively, originating from blue mould cheeses were examined for their potential use as starter cultures for the production of Danablu in laboratory studies. D. hansenii showed strong growth and assimilation of lactose, galactose, lactate and five out of six strains assimilated citric acid under the environmental conditions prevailing in Danablu during maturation at 10°C. Y. lipolytica was more sensitive to NaCl and did not assimilate lactose and galactose. Both yeasts hydrolysed tributyrin with the highest activity observed for Y. lipolytica. D. hansenii showed little if any release of free fatty acids from butterfat at 10°C. Y. lipolytica was strongly lipolytic. The strains of D. hansenii were not able to hydrolyse casein at 10°C whereas 4 of the 6 strains of Y. lipolytica degraded all components of the casein. Strain-specific interactions, in cheese agar resulting in inhibition of mycelial growth and sporulation of P. roqueforti was observed, especially for Y. lipolytica.
Microorganisms are an essential component of all natural cheese varieties and play important roles during both cheese manufacture and ripening. They can be divided into two main groups; starters and secondary flora. The starter flora, Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii used either individually or in various combinations depending on the cheese variety, are responsible for acid development during cheese production. Starters may be either blends of defined strains or, as in the case of many cheeses manufactured by traditional methods, composed of undefined mixtures of strains which are either added at the beginning of manufacture or are naturally present in the cheese milk. During cheese ripening, the starter culture, along with the secondary flora promote a complex series of biochemical reactions which are vital for proper development of both flavour and texture. The secondary flora is composed of complex mixtures of bacteria, yeasts and moulds, and is generally specifically associated with particular cheese varieties. In many cheese varieties, the action of the secondary flora contributes significantly to the specific characteristics of that particular variety. The secondary flora may be added in the form of defined cultures, but in many situations is composed of adventitious microorganisms gaining access to the cheese either from ingredients or the environment. During cheese manufacture and ripening, complex interactions occur between individual components of the cheese flora. Environmental factors within the cheese also contribute to these interactions. Elucidation of such interactions would greatly add to our understanding of the cheese ripening process and would enable a more targeted approach to starter/adjunct selection for cheese quality improvement. In the past, research in this area was dependent on classical microbiological techniques, which are very time consuming, not suitable for handling large numbers of isolates and generally not suitable to studies at sub species levels. However, developments in this area have recently undergone a major revolution through the development of a range of molecular techniques, which enable rapid identification of individual isolates to species and strain level. Application of such techniques to the study of cheese microbiology should lead to major advances in understanding this complex microbial ecosystem and its impact on cheese ripening and quality in the coming years.
The predominant microorganisms in the Sudanese traditional fermented dairy product Rob from cows’ milkwere isolated and
identified by using morphological, physiological and molecular typing techniques. From seven production sites in Khartoum,
samples of Rob used as inocula for fermentation were analysed by the determination of the cfumL�1 on MRS, M17, potato dextrose
agar and plate count agar. Samples of inocula from two production sites were used for Rob fermentation in the laboratory followed
by similar microbiological analysis. The results obtained showed that the Rob inocula contained lactococci and lactobacilli in the
range of 107–108 and yeasts in the range of 106–107 cfumL�1. During the course of the two laboratory fermentations, the lactococci
and lactobacilli increased to 108 cfumL�1, the yeasts to 107 cfumL�1 and the aerobic mesophilic counts decreased to 105 cfumL�1.
The pH of the inocula ranged between 3.4 and 3.6 and the pH of Rob produced in the laboratory was 4.2–4.5.
Using the internal transcribed spacer-polymerase chain reaction (PCR) technique, 189 representative strains of lactic acid bacteria
and 109 representative isolates of yeasts were divided into nine and two groups, respectively, according to the size of the PCR
product formed. Representative members of all groups were identified by the API 50 CH and the ID 32 API kits for lactic acid
bacteria and yeasts, respectively. Confirmation of identification of lactic acid bacteria species was achieved by sequencing the 16S
rRNA gene. The predominant lactic acid bacteria in Rob fermentation were found to be Lactobacillus fermentum, Lactobacillus
acidophilus, Lactococcus lactis, and Streptococcus salivarius. The predominant yeasts were found to be Saccharomyces cerevisiae and
Candida kefyr.
Yak milk is a type of milk that people are less familiar with due to its remote geographical location, the particular geographical environment and climatic conditions in Tibet, which may have significant effects on composition, microbiota and fermentation outcome. To investigate the chemical composition and microbiota of fresh and fermented yak milk, and to isolate and characterize the predominant microorganisms in the fermented milk, yak milk (24 fresh and 30 fermented milk samples) was collected from four areas of different altitudes in Tibet, and their microbiological profile and chemical composition were investigated. Yak milk had a higher fat, crude protein, lactose and dry matter content than cow milk. The fermented yak milk showed a great diversity in fat and dry matter levels due to the different ways of processing in different localities, and lower pH and higher lactic acid content compared with commercial cow milk yogurt. Fermented yak milk had a better sanitary quality than fresh yak milk. Three species of lactobacilli (Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus curvatus) and five species of yeast (Saccharomyces cerevisiae, Candida kefyr, Candida lambica, Candida famat and Candida holmii) were identified phenotypically and encountered as predominant fermentation microbiota. The predominant lactic species in fermented milk was L. fermentum
The lipolytic activity of 30 strains of Penicillium roqueforti was investigated by agar diffusion tests on tributyrin (esterase activity) and olive oil agar (lipase activity), by titration of the free fatty acids (FFA) produced and by gas chromatographic analysis of the individual FFA released after growth at 25 or 10 degrees C in butterfat emulsions containing 0, 2 or 7% NaCl. All strains investigated by the agar diffusion tests possessed esterase activity and 23 strains were also able to hydrolyse olive oil, but differences in esterase activity were seen. The agar diffusion tests and the titration of FFA showed that the amount of FFA released by a strain of P. roqueforti is determined by both esterase and lipase activity. A large release of FFA was only seen for strains with the ability to hydrolyse both short- and long-chained fatty acids, while strains with esterase activity produced smaller amounts of FFA. Between 7 and 14 days of incubation a steep increase in the release of FFA was observed both by the titration and by GC analysis, and then a decline from 14 to 21 days, probably caused by conversion of FFA to methyl ketones. Identical FFA profiles were found for two strains with different lipolytic activity. Long-chained fatty acids dominated the profile, while the short-chained fatty acids only were detected in small amounts and mainly in the end of incubation. Both strains were stimulated by NaCl in the emulsions.
The starter culture Penicillium roqueforti, undesired cultures Penicillium caseifulvum and Geotrichum candidum and the potential starter culture Debaryomyces hansenii were examined for their growth and interactions at environmental conditions similar to the Danish blue cheese Danablu. The combined effect of low oxygen (0.3%) and high level of carbon dioxide (25%) at 4% NaCl w/v (a(w) 0.97) and pH 4.5 and 6.5 on radial growth was examined on a cheese medium at 10 degrees C. P. roqueforti and G. candidum were well adapted to growth at low levels of oxygen and high levels of carbon dioxide but G. candidum was not able to grow in the presence of 4% NaCl (w/v). Growth of P. caseifulvum was strongly inhibited at atmospheric conditions comprising 25% carbon dioxide, especially in combination with 0.3% oxygen. Generally D. hansenii showed strong growth at all environmental conditions examined, except at 0.3% oxygen combined with 25% carbon dioxide and 4% NaCl (w/v). Growth and sporulation of P. roqueforti was highly affected in the presence of G. candidum at 25% carbon dioxide irrespective of levels of oxygen and NaCl in the cheese media. P. caseifulvum caused a pronounced inhibitory effect towards growth of P. roqueforti and D. hansenii at 21% oxygen. D. hansenii caused weak inhibition of P. roqueforti at 21% oxygen, while positive interactions between the two species were indicated at 25% carbon dioxide and 0.3% oxygen.
In blue veined cheeses, the dominant yeast species in most cases is Debaryomyces hansenii. Saccharomyces spp. occurs less frequently, but they can be found in some blue veined cheeses. In the present study, the taxonomy of Saccharomyces spp. associated to blue veined cheeses was studied and comparisons made to type strains of Saccharomyces spp. and starter cultures of Saccharomyces spp. used in other food fermentations. Phenotypically, the cheese strains were referred to the Saccharomyces sensu stricto complex and were further identified as S. cerevisiae. Genotypically, the Saccharomyces spp. investigated were similar although chromosomal polymorphism were observed. Concerning the technological characteristics, they were similar in assimilation and fermentation of the residual sugars and organic acids naturally found in cheese. The investigated yeasts were also similar in their lipolytic activity being able to hydrolyse tributyrin and low chain (C:8), but not C:14 fatty acids. However, they differed in their tolerance to NaCl with the blue cheese strains showing a higher tolerance. The cheese strain S. cerevisiae FB 7 was the only yeast capable of degrading casein. It mainly degraded the alpha(s1)-casein and the beta(alpha2)-casein components. It was also the only isolate stimulating the development of Penicillium roqueforti in cheese agar imitating the conditions in blue veined cheese. The stimulation of P. roqueforti was most pronounced for the least proteolytic strain of P. roqueforti examined.
In the present work, the occurrence of yeasts in different types of typical Sardinian ewe's cheeses (32 samples of pecorino, 32 of caciotta, 40 of feta, 56 of ricotta) was determined. For the strains isolated the following properties were studied: proteolytic and lipolytic activities, the ability to grow at different temperatures, different concentrations of salt, and to assimilate and/or ferment compounds like lactate, citrate, lactose, glucose, galactose, lactic acid. Of 160 samples analysed, 76.2% yielded growth of yeasts. Yeast counts showed a certain variability among the samples. The highest levels were observed in caciotta and feta cheeses. A total of 281 strains belonging to 16 genera and 25 species were identified. In general, Debaryomyces hansenii was the dominant species, representing 28.8% of the total isolates. Other frequently appearing species were Geotrichum candidum, Kluyveromyces lactis and K. marxianus. Other genera encountered were Pichia, Candida, Dekkera, Yarrowia and Rhodotorula. With regard to the biochemical and technological properties of the yeasts, only K. lactis, K. marxianus and Dek. anomala assimilated and fermented lactose, whereas the majority of the species assimilated lactic acid. The assimilation of citrate was a characteristic of D. hansenii, R. rubra and Y. lipolytica. On the whole, the yeasts were weakly proteolytic while lipolytic activity was present in several species. A high percentage of strains showed a certain tolerance to low temperatures while only some strains of D. hansenii and K. lactis were able to grow at a 10% NaCl concentration.
In this study, the yeast populations in feta cheese from two different Sardinian dairies were examined. Samples of good quality feta (32) and samples of feta with a slimy surface defect (10) were examined from Dairy A. Similar, samples of good quality feta (23), feta with slimy surface defects (14) and samples with swelling defects (6) were examined from Dairy B. Kluyveromyces lactis was the dominating species in feta from Dairy A (95.2% of samples) followed by Debaryomyces hansenii (76.2%), Dekkera anomala (28.6%) and Dek. bruxellensis (19%). D. hansenii was dominant in samples from Dairy B (93%), followed by K. lactis (23.3%), Geotrichum candidum (23.3%) and Dek. anomala (18.6%). No significant difference was observed between the occurrence of yeast species in feta of good quality and in feta with slimy surface defects, thus confirming that slimy production is not associated with yeast contaminations. The swelling of samples observed in Dairy B seems to be caused by Dek. anomala. In fact, this strong fermenting species was present in all swelled samples in numbers exceeding 10(6) CFU g(-1), while it was isolated in very low concentration in only 5.4% of good samples.
Yeast microflora on the surface and interior of Rokpol cheese was examined for cheeses produced at three dairies located in Lower Silesia, Poland. Yeast populations on the surface of the cheeses ranged from 10(5)-10(9) cfu/g, but were 10-100 times lower for interior samples. The occurrence and proportions of yeast species varied, depending on the dairy plant and cheese sample. The most frequently isolated species were: Candida famata and C. spherica, followed by C. intermedia and Geotrichum sp. Other species such as Saccharomyces kluyveri, C. kefyr and C. lipolytica were found occasionally. Extracellular and intracellular proteolytic and lipolytic activities were examined for 39 isolates of C. famata.
The potential use of Saccharomyces cerevisiae FB7 as an additional starter culture for the production of Mycella, a Danish Gorgonzola type cheese, was investigated. Two dairy productions of Mycella, each containing batches of experimental cheeses with S. cerevisiae added and reference cheeses without yeast added were carried out. For both experimental and reference cheeses, chemical analysis (pH, a(w), NaCl, water and fat content) were carried out during the ripening period, but no significant differences were found. The evolution of lactic acid bacteria was almost identical in both the experimental and reference cheeses and similar results were found for the number of yeast. S. cerevisiae FB7 was found to be predominant in the core of the experimental cheeses throughout the ripening period, while Debaryomyces hansenii dominated in the reference cheese and on the surface of the experimental cheeses. In the cheeses with S. cerevisiae FB7, an earlier sporulation and an improved growth of Penicillium roqueforti was observed compared to the reference cheeses. Furthermore, in the experimental cheese, synergistic interactions were also found in the aroma analysis, the degradation of casein and by the sensory analysis. The observed differences indicate a positive contribution to the overall quality of Mycella by S. cerevisiae FB7.
Yeast isolates collected from various Hungarian dairy products were identified using simplified identification system (SIM) and restriction fragment analysis of PCR-amplified 18S rDNA with the neighbouring ITS1 region (ITS-PCR; ribotyping). The isolates were grouped into 26 species; Geotrichum candidum, Debaryomyces hansenii, Yarrowia lipolytica, Kluyveromyces lactis and Candida catenulata were found as the predominant species. SIM and ITS-PCR proved to be useful and convenient taxonomic tools for rapid identification at species level. Two most frequent species, G. candidum and D. hansenii, were further characterized by randomly amplified polymorphic DNA (RAPD-PCR) analysis. RAPD-PCR using M13 primer resulted in discrimination between most strains of the same species and allowed a certain degree of intraspecific typing.
Yeasts are present in indigenous African fermented milks in numbers up to log 8 cfu g(-1), together with a varied lactic acid bacteria (LAB) flora, and therefore potentially contribute to product characteristics. However, interaction between yeasts and LAB in these products has received little notice. In studies of indigenous fermented milk in Zimbabwe and Uganda, many samples contained more than one species of yeast, but Saccharomyces cerevisiae was most commonly isolated. Other frequent isolates were other species of Saccharomyces and several species of Candida. Most yeast isolates were lactose-negative but usually galactose-positive. Some strains assimilated lactate and citrate. The growth in milk of strains of yeasts and LAB, isolated from naturally soured milk in Zimbabwe, and their interaction when selected pairs of strains were grown together has been studied. Interactions were shown by the significantly different amounts of certain metabolites produced, such as acetaldehyde and malty aldehydes, when co-cultures were compared to pure cultures. Preliminary sensory acceptance tests did not show, however, that milks made from a co-culture with Candida kefyr and LAB were preferable to the pure LAB culture. Further work is still needed to elucidate the reactions that may be taking place in fermented milk between varying LAB and yeast populations. The potential for use as starter cultures depends on various aspects, including the final product being prepared. The role of other microorganisms in naturally fermented milk also needs to be studied.
The microflora of downgraded Danish bulk tank milk was examined to identify the main causes of increased microbial counts. Seventy-five representative samples with a microbial count exceeding 3.0 x 10(4) cfu/mL were selected for a more detailed microbial examination. A total of 1237 isolates from these samples were identified. Gram-negative, oxidase-positive bacteria were found in 72% of the samples. Coliforms were found in 20% of the samples, and non-coliforms were found in 49% of the samples. Coryneforms, other gram-positive rods, Lactococcus spp., Micrococcus spp., and coagulase-negative Staphylococcus spp. were found in 28 to 53% of the samples. Bacillus spp., Enterococcus spp., Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis, and yeasts were found in <25% of the samples. Additionally, the isolates were divided into 3 groups, based on the main cause of an elevated microbial count. Microorganisms primarily associated with poor hygiene dominated the microflora in 64% of the samples; bacteria also related to poor hygiene, but in addition associated with growth at low temperatures (psychrotrophic bacteria) dominated the microflora in 28% of the samples; and bacteria mainly associated with mastitis dominated the microflora in 8% of the samples. A bulk tank milk storage period of 48 h instead of 24 h did not affect the proportion of downgraded milk samples and could not be associated with a specific group of microorganisms. Further, no relationship was found between somatic cell counts and the presence of mastitis bacteria.
The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.
The mycoflora of a Spanish blue cheese made in a natural way (Valdeón cheese) was investigated. Moulds and yeasts isolated in cheeses at different stages of manufacture and at the stage of consumption were identified to species level and the proteolytic and lipolytic activities of the major strains estimated. Penicillium was the main genus identified, with P. roqueforti, P. verrucosum, P. chrysogenum, the morphologically closely related species P. aurantiogriseum/P. solitum/P. commune, and P. expansum being the major species. Other genera found were Cladosporium, Mucor and to a lesser extent Paecilomyces, Acremonium, Alternaria and Geotrichum. The major species of yeasts found were Debaryomyces hansenii, Kluyveromyces lactis and Yarrowia lipolytica. On the surface of the finished cheese P. aurantiogriseum/P. solitum/P. commune and D. hansenii were dominant. The most lipolytic species were P. roqueforti, P. verrucosum and Y. lipolytica. P. chrysogenum was the most proteolytic. None of the P. roqueforti strains showed proteolytic activity.
Cheese texture and flavour are obtained through a series of chemical changes which occur in the curd during the early stages of ripening. The lowering of pH and Eh, a result of lactic bacteria metabolism, greatly influences texture through water and mineral contents, but has also further repercussions on some chemical changes. Lipid hydrolysis leads to free fatty acids which serve as a substrate for further reactions. Proteolysis influences texture, but mainly flavour, as it results in the formation of peptides and amino acids which, for flavour, leads to aroma compounds through enzymatic and, perhaps, purely chemical reactions.
Enzymes either from milk itself or added in the form of rennet or microorganisms play the dominant role in the conversion of milk in cheese. Of the enzymic reactions the breakdown of caseins is by far the most important reaction. Five main systems contribute to the hydrolysis of caseins: Rennet, indigenous milk proteinase, proteinases and peptidases produced by the starter cultures, enzymes of the secondary starters, enzymes of non‐starter cultures. The relative contributions of these systems in the ripening of various types of cheese will be discussed in detail. Some attention will be paid to secondary reactions like amine formation and the action of lipases during cheese ripening.
Lipolytic enzymes from various microorganisms have been studied for lipolysis, a recognized priority in biotechnology research. For the screening of lipolytic fungi we employed different classical methods which caused some difficulties in the interpretation of results. For this reason, we improved a screening method based in depth of clearing measurement of tributyrin medium opaque column. The best conditions for the assay were 1% agar and, 0.1 or 1% tributyrin incubated for 120 h. This new method, based on depth of clearing, can not only be used as a presumptive screening of lipolytic fungi but can also give quantifiable results.
An extract of Kluyveromyces lactis 416 and a β-galactosidase preparation (Maxilact 40000) contaminated with proteinase, showed similar pH profiles of caseinolytic activity. Similar modes of casein hydrolysis (κ-, > αs-, ≥ β-) were observed at pH 5·0 (the pH of Cheddar cheese), without detection of bitterness. The contaminated Maxilact preparation contained similar proteinase types to those detected in an autolysate of K. lactis. Both the autolysate and the Maxilact preparation contained acid endopeptidase (proteinase A), serine endopeptidase (proteinase B) and serine exopeptidase (carboxypeptidase Y) activities. Some aminopeptidase activity was also detected in both preparations. There were some differences in apparent molecular weight and charge properties between proteinase A and B and carboxypeptidase Y from the 2 proteinase sources.
The microflora of Serra cheese was monitored during a 35 d ripening period at three different periods within the ewe's lactation season. After 7 d ripening, the numbers of micro-organisms reached their maximum, and lactic acid bacteria (LAB) and coliforms were the predominant groups. Pseudomonads were not detected after 1 week of ripening. At all stages of ripening, cheeses manufactured in spring exhibited the lowest numbers of LAB and yeasts, whereas cheeses manufactured in winter showed the lowest numbers of coliforms and staphylococci.
Leuconostoc lactis was the most abundant LAB found in Serra cheese whereas Enterococcus faecium and Lactococcus lactis spp. lactis exhibited the highest decrease in percentage composition. Numbers of both Leuc. mesenteroides and Lactobacillus paracasei tended to increase throughout ripening. The most abundant coliform was Hafnia alvei. Klebsiella oxytoca was found in curd but declined in number during ripening. Staphylococcal flora of curd was mainly composed of Staphylococcus xylosus, Staph. aureus and Staph. epidermidis. Staphylococcus xylosus was the major species found at the end of ripening. Pseudomonas fluorescens, was the only Pseudomonas species isolated from the curd. Although a broad spectrum of yeasts were found in Serra cheese, Sporobolomyces roseus was the most abundant yeast isolated.
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.
In a study of the halotolerant yeast Debarymyces hansenii cultured in 4 mM and 2.7 M NaCl the intracellular ATP pool, the heat production, the oxygen uptake, and, in the high culture salinity also, the intracellular glycerol concentration were found to be correlated. The intracellular ATP in the 2.7 M NaCl culture had a constant concentration of 3.5 mM ATP during the second half of the lag phase, while in 4 mM NaCl it rose to a maximum of 3.1 mM during the late log phase. The intracellular glycerol concentration in 2.7 M NaCl was about 1.3M during the entire exponential growth phase. Sine the glycerol concentration of the medium was not more than 0.23 mM, glycerol must contribute to the osmotic balance of the cells in high salinity. The corresponding maximum values for the 4 mM NaCl culture were 0.16 M and 0.08 mM. The experimental enthalpy changes were approximately the same for the two salinities, viz. about-1200 kJ per mole consumed glucose. The Y
m-values for the 4 mM and 2.7 M NaCl cultures were 91 and 59, respectively, the difference being a consequence of the decreased efficiency of growth in high salinity.
This review deals with yeasts and their potential use as starter cultures in dairy products as well as their role as spoilage organisms. The taxonomy of relevant yeasts is described, with emphasis on molecular techniques and simplified identification systems applicable to industry. Quantitative assessment of undesirable yeast contamination is discussed with regard to present requirements for quality assurance in dairies. Special attention is given to the role of yeasts in the complex microbial interactions prevailing in several dairy products and to controlling fermentation, maturation and important product characteristics. Antimicrobial activities of yeasts, including secretion of ‘killer factors’ with broad antimicrobial spectra are linked with the reported probiotic starter cultures. The desired properties of yeasts to be used as starter cultures in the dairy industry are summarized and areas of research are proposed to gain the insight required to obtain full advantage of the active use of yeast cultures in a wider range of products.
Yeasts were isolated, identified and enumerated from 161 samples of retail dairy products. Highest yeast populations (up to 106–107 cells/g) were found in yogurt and cheese samples while lower counts occurred in samples of pasteurized milk, cream, butter and ice cream. Candida famata, Kluyveromyces marxianus, Candida diffluens and Rhodotorula glutinis were the most frequency isolated species. The growth of these and other species was demonstrated during the refrigerated storage of cream, butter and cheese samples and by their inoculation and incubation in milk and yogurt samples. The predominance and growth species was probably related to their production of protein and fat hydrolysing enzymes and the ability to grow at 5°C.
Interactions between lactic acid starter cultures and Penicillium roqueforti used in the production of Danablu have been investigated. Based upon screening of 20 strains of Penicillium roqueforti and 15 strains of Leuconostoc ssp, Lactobacillus ssp Streptococcus ssp and Lactococcus ssp negative and positive interactions, ie, inhibition or stimulation of P roqueforti were demonstrated in laboratory media and a cheese-based model system. It was observed that the interactions were highly affected by the media used. In general positive interactions were stronger and seen more frequently in the cheese model system than in laboratory media. For P roqueforti as well as the lactic acid starter cultures the interactions were strain specific. The positive interactions were expressed as faster growth rate, increase of sporulation, a more intense blue/green colour and a thicker and more velvet mycelial growth. For combinations of NaCl, pH and temperatures relevant to the production of Danablu the relative importance of positive interactions increased with increasing NaCl concentrations. This applied for growth as well as sporulation. Detailed studies on the amino acid composition of the cheese based model systems inoculated with lactic acid starter cultures, P roqueforti or the two together indicate that the lactic acid bacteria stimulate P roqueforti by releasing amino acids like arginine and leucine.
The change in yeast flora on the surface of two batches of Roquefort cheese was monitored over a period of 6 months. 401 isolates were determined and their technological properties were investigated. The main species isolated were: Debaryomyces hansenii and its non sporulating form Candida famata, Kluyveromyces lactis and its non sporulating form Candida sphaerica and Candida species. The species Debaryomyces hansenii inoculated on the surface of the cheese in one of the batches just before the salting phase was abundant throughout the ripening phases but never exceeded 50% of the yeast count. About 80% of the isolates of each species were resistant to 15% (w/v) of sodium chloride. Most of the species were able to assimilate lactose and lactic acid. 50-90% of the isolates of each species were able to hydrolyze rapeseed oil and glycerol tributyrate. Ten isolates among 401 hydrolyzed gelatin. Most of them were able to assimilate cadaverine, histamine, putrescine and tyramine.
A total of 365 yeasts were isolated from the brines of soft, semihard and hard cheeses from different manufacturers. Identification was based on 131 characteristics, primarily employing a method with microtitration plates. Most brines exhibited a characteristic yeast flora. The predominant strains proved to be mainly Debaryomyces hansenii and Candida versatilis. In a few brines Trichosporon beigelii, C. rugosa, C. intermedia, Kluyveromyces marxianus, Saccharomyces sp. and C. tenuis/polymorpha were predominant. Also of importance were C. tropicalis, C. parapsilosis, C. zeylanoides, Issatchenkia orientalis and Geotrichum klebahnii. Not all strains could be clearly identified. Lists of characters are provided for subdividing D. hansenii and T. beigelii. The specificity of the yeast flora of brines is assumed to contribute to the sensory variety of cheeses.
. An arbitrary parameter ‘rejection time’, i.e. the time required for a fungal inoculum to form a 2 mm diam. colony, was used to express the shelf life of jam after unsealing and exposure of the contents to airborne contamination. Individual and combined effects of water activity (aw), pH value and temperature on rejection time of low sugar jam were estimated from the radial growth on agar of colonies of 9 fungi. The decreases in aw (0·94–0·90) and temperature (25–15°) practicable for low sugar jam were more effective in increasing rejection time than the feasible decrease in pH value (3·7–2·9). The interaction between aw and temperature was significant. The effect of the changes in aw, temperature and pH on rejection time was broadly similar for media adjusted by either sucrose or glycerol. At a given aw, moulds were slightly more tolerant of glycerol than sucrose but yeasts, except for the osmophile Saccharomyces rouxii, were markedly more tolerant of glycerol than sucrose.
Yeast populations greater than 10(6) cfu/g were found in approximately 54% and 36%, respectively in surface samples of retail Camembert (85 samples) and Blue-veined (45 samples) cheeses. The most predominant species isolated were Debaryomyces hansenii, Candida catenulata, C. lipolytica, C. kefyr, C. intermedia, Saccharomyces cerevisiae, Cryptococcus albidus and Kluyveromyces marxianus. The salt concentration of the surface samples of the cheeses varied between 2.5-5.5% (w/w) (Camembert) and 7.5-8.3 (Blue-veined), depending upon brand, and influenced the yeast ecology, especially the presence of S. cerevisiae. Yeasts grew to populations of 10(6)-10(8) cfu/g when cheeses were stored at either 25 degrees C or 10 degrees C. These populations decreased on continued storage at 25 degrees C, but such decreases were not so evident on storage at 10 degrees C. The properties of yeasts influencing their occurrence and growth in cheese were: fermentation/assimilation of lactose; production of extracellular lipolytic and proteolytic enzymes, utilisation of lactic and citric acids; and growth at 10 degrees C.
Sources of yeast contamination which may lead to contamination of the curd during Cheddar and Gouda cheese making, were examined in a single cheese factory. A total of 187 representative yeast isolates present in the factory environment, on working surfaces, the brine and on workers' hands and aprons were identified according to conventional methods and cellular long-chain fatty acid analyses. Product line samples were taken at critical control points in the manufacturing process and analysed after incubation at 25 degrees C for 96 h. The most prevalent isolates belonged to the genera Debaryomyces and Candida. Other genera encountered were Cryptococcus, Rhodotorula, Yarrowia, Pichia, Trichosporon, Torulaspora, Issatchenkia, Saccharomyces and Zygosaccharomyces. Characterization of the predominant yeast isolates indicated that the cheese brine was responsible for the largest variety and number of yeast isolates yielding a total of 64 yeast strains belonging to nine different genera.
The growth of several yeast species in milk containing added sodium chloride (0-15%, w/v) at 25 degrees C and 10 degrees C was examined in conjunction with yeast metabolism of milk constituents. Depending on conditions, all yeasts grew to maximum populations of 10(7)-10(8) cfu/ml. Kluyveromyces marxianus gave strong utilisation of lactose and weak metabolism of citrate, protein and fat with the production of ethanol, glycerol, lactic acid and propionic acid. As measured by the production of free amino acids and free fatty acids, Candida lipolytica and Candida catenulata gave strong proteolytic and lipolytic reactions, the specificities of which appeared to be influenced by temperature and the presence of NaCl. These species also metabolised organic acids. Although giving strong growth responses, Debaryomyces hansenii and Saccharomyces cerevisiae did not metabolise lactose and gave only very weak lipolytic and proteolytic reactions. Citrate was metabolised by D. hansenii but not by S. cerevisiae. Both species produced small amounts of ethanol, glycerol and lactic acid.
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