Yeast are detected and isolated from all sorts of cheeses (Bockelmann and Hoppe-Seyler 2001; de Boer and Kuik 1987; Nooitgedagt and Hartog 1988; Roostita and Fleet 1996a; Schmidt and Lenoir 1980; van den Tempel and Jakobsen 1998; Tzanetakis et al. 1998; Vivier et al. 1994; Welthagen and Viljoen 1998). The number of yeast can be in the range 104cfu/g or even higher. The most common yeast species in cheese are D. hansenii, Y. lipolytica, G. geotrichum, K. lactis, K. maxianus, and S. cerevisiae with D. hansenii as the most predominant. The development of yeast in cheese occurs spontaneously while controlled use of yeast as starter cultures in cheese is used for production of some kind of mold ripened cheeses and smear ripened cheeses, but seldom for other types of cheese.
Until recently it was assumed that the yeast in cheese primarily originated from the cheese brine. Cheeses are often salted in brines containing 22-25% NaCl (Hansen et al. 2001) and often the brine is not changed or pasteurized between the salting of different batches leading to accumulation of salt tolerant yeast (Tudor and Board 1993) in the range of 102-106cfu/g (Devoyod and Sponem 1970; Rohm et al. 1992; Seiler and Busse 1990; van den Tempel and Jakobsen 1998). It has also been suggested that the yeast in cheese originated from raw milk because of the survival of yeast through pasteurization (van den Tempel 2000; Vadillo et al. 1987). In a study carried out by Petersen et al. (2002) the successions of yeast on the surface of Danbo was followed using mtDNA RFLP. The investigation showed that the dominating flora after 4 days belongs to D. hansenii and that the dominating strain did not originate from raw milk, brine, or the starter culture, but from the dairy "house microflora," which also includes yeast from air in the ripening room, ripening pads and humans. This indicates that cheese quality depends on an intact "house microflora" where the balance easily can be disturbed because the cheese surface in many ways is exposed to the environment of the dairy with its inherent population of micro-organisms (Bockelmann and Hoppe-Seyler 2001).
Examinations of different kinds of cheese showed that the yeast population often develops from a heterogeneous to a homogeneous population during the ripening period (Bockelmann and Hoppe-seyler 2001; Hansen et al. 2001; Petersen et al. 2002; van den Tempel and Jakobsen 1998). Yeast need an energy source to grow, therefore the ability to assimilate or ferment residual carbohydrates and acids is important for the yeasts in order to survive in the cheese and to compete with other micro-organisms during ripening (Roostita and Fleet 1996b). But also the general tolerance towards NaCl of yeasts and the effect of NaCl on the uptake of lactate and other carbohydrates is important (Petersen et al. 2002). The assimilation of different carbohydrates seems to be affected by the microenvironment in the cheese. Investigations of D. hansenii showed that the assimilation of lactate (Petersen et al. 2002; van den Tempel and Jakobsen 2000) and citrate (van den Tempel and Jakobsen 2000) was strain specific and that inhibitions for some strains occurred at 6% (w/v) NaCl while other strains could assimilate both citrate and lactate in the presence of 14% (w/v) NaCl (van den Tempel and Jakobsen 2000). Similar results were seen for assimilation of lactose and galactose. The same pattern was observed for Y. lipolytica though inhibition already occurred at low levels of salt, i.e., 2 % (w/v) NaCl for some of the strains examined (van den Tempel and Jakobsen 2000).
Semi-soft and soft cheeses that develop a smear of microbial growth on the surface during maturation are called surface ripened cheese. These cheeses have maturation times that vary from several days to month and at temperature ranging from 10 to 20°C. Some examples of these cheeses are Limburger, Danbo, Brick, and Tilsiter (Fleet 1990; Bockelmann and Hoppe-Seyler 2001; Petersen et al. 2002). The microbial smear on the surface of these cheeses is very important for the maturation process and play a major role for the final cheese quality (Bockelmann 2002; Bockelmann and Hoppe-Seyler 2001; Fleet 1990; Jakobsen and Narvhus 1996). The cheese smear is a bio-mass of yeast and bacteria (Bockelmann and Hoppe-Seyler 2001; Fleet 1990; Fleet and Mian 1987; Masoud and Jakobsen 2003). The yeast species D. hansenii seems to be dominant and also the most important yeast during the whole maturation time but depending on the type of cheese other yeast such as Trichosporon spp., Y. lipolytica, K. lactis, and Candida spp. have been detected during the first day of ripening (Bockelmann 2002; Fleet 1990; Petersen et al. 2002). The bacterial flora consists of Brevibacterium lines, Arthobacter spp., Corynebacterium spp., Micrococcus spp., and Staphylococcus spp. (Bockelmann 2002; Bockelmann and Hoppe-Seyler 2001; Fleet 1990).
The microbial ecology of the smear is very complex, but during the first days of cheese ripening a natural selection takes place (Petersen et al. 2002). The most suitable group of yeast in the surface ripened cheese, seems to adapt easily to the microenvironment with high NaCl concentration, low pH, and lactate as a main carbon source. Among the yeast D. hansenii in most cases grow fast and become the dominant species. It has been observed a particular subspecies out range the other strains and become dominant of D. hansenii present (Petersen et al. 2002).
The growth of D. hansenii on the surface of the cheese enhances the growth of the smear bacteria (Valdes-Staber et al. 1997) by metabolizing the lactate (Le Clercq-Perlat et al. 1999). After 4-7 days the pH increases from 5.2 to 5.7 allowing more acid-sensitive bacteria like B. linens to grow (Bockelmann and Hoppe-Seyler 2001; Petersen et al. 2002; Valdes-Staber et al. 1997). The number of yeast begin to decrease after one or two weeks of ripening while the bacteria dominates on the cheese surface during the last part of the ripening period (Bockelmann 2002; Petersen et al. 2002). Furthermore, yeast appears to support the bacterial growth by release of vitamins and amino acids (Viljoen 2001). Investigations carried out by Masoud and Jakobsen (2003) showed that D. hansenii had a significant effect on the intensity of the reddish pigment produced by the bacteria flora. Significant differences were observed between the D. hansenii strains examined but also the NaCl content and pH played a important role in the pigment production.
Blue mold cheeses are semi-soft cheeses primarily ripened by growth of the mold P. roqueforti. Blue cheeses normally have a significally higher content of NaCl compared to surface ripened cheeses and white mold cheeses. The NaCl concentration in blue mold cheeses after brining is 0.2% (w/w) in the core and 7% (w/w) in the surface layer. After eight weeks of maturation NaCl concentration is approximately 2.0% (w/w) in the core and 4.0% (w/w) in the surface layer. The pH after 24 hours is at the level pH 4.6-4.7. During ripening, pH in the core increases to about 6.5 and to 5.9 in the surface layer (Gobbetti et al. 1997; Godinho and Fox 1982; Hansen et al. 2001).
To permit air into enter the cheese center and carbon dioxide to escape the cheeses are pierced before maturation which also affect yeast growth. In blue veined cheese, yeasts are detected in high levels, without affecting cheese quality negatively (Fleet 1992; Hansen et al. 2001; van den Tempel 2000). The number of yeasts detected in blue mold cheese is in the order of 107-108cfu/g on the surface and 105-106cfu/g in the core, but higher concentrations have been observed (Hansen et al. 2001; van den Tempel and Jakobsen 1998). The most common yeasts isolated from raw milk cheeses like Roquefort are D. hanseniilC. famata, C. catenulata, Y. lipolytica/ C. lipolytica, C. kruseii, T. cutaneum, and K. lactis/ C. spherica (Besancon et al. 1992; Roostita and Fleet 1996a). In Mycella, which is made from pasteurized milk, the predominant yeasts isolated from both the core and the surface is D. hanseniilC. famata but in the beginning of the maturation other yeast like Zygosaccharomyces spp., G. geotrichum Y. lipolytica, and C. rugosa are seen, in low numbers (Hansen et al. 2001). In four-week old Danablu, the most common yeast are D. hansenii (van den Tempel and Jakobsen 1998). Examinations of Danablu produced at four different dairies and the Danish blue cheese Mycella showed that the yeast population developed from a heterogeneous flora to a homogeneous flora of D. hansenii during the ripening period (Hansen et al. 2001; van den Tempel and Jakobsen 1998).
Yeast are considered to play an important role in the ripening of blue mold cheese and seems to contribute positively to microbial environment by assimilation of the residual carbohydrates and acids. Yeast are assumed to create a stable microenvironment, which prevent undesired microbial growth. The gas produced in the curd during fermentation is likely to create minor fissures and chinks in the cheese curd, which is assumed to promote the development of P. roqueforti (Coghill 1979). Positive interactions have been detected between D. hansenii and P. roqueforti under conditions simulating the environment in Danablu (van den Tempel and Nielsen 2000) The yeast S. cerevisiae is known to have a positive affect on growth and sporulation of P. roqueforti (Hansen and Jakobsen 2001; Hansen et al. 2001). S. cerevisiae is also found to stimulate the release of free fatty acids (FFA) by P. roqueforti and a synergistic effect between P. roqueforti and S. cerevisiae has been demonstrated in the degradation of casein (Hansen and Jakobsen 2001; Hansen et al. 2001). The positive interactions between P. roqueforti and S. cerevisiae were verified in a dairy trial. In the cheeses added S. cerevisiae improved growth and earlier sporulation of P. roqueforti was observed compared to the reference cheeses. Furthermore, positive contribution from S. cerevisae were also found in the aroma analysis, the degradation of casein, and by sensory analysis. The observed differences indicate the potential use of S. cerevisiae as an additional starter culture for production of Mycella (Hansen et al. 2001).
In laboratory studies inhibition of P. roqueforti by Y. lipolytica, G. geotrichum and K. lactis have been observed. A negative effect of the yeast on the growth of P. roqueforti has not been verified in cheese, but should be kept in mind if the development of P. roqueforti is slow or absent. It should also be keep in mind that some of these yeast, e.g., D. hansenii and Y. lipolytica are known to produce reddish pigment on the cheese surface primarily from oxidation of tyrosine to melanin (Carreira et al. 1998; van den Tempel and Jakobsen 2000).
White mold cheeses are semi-soft cheeses with growth of P. camemberti creating a white greyish mycelium on the surface on the cheese. The most famous variants are Brie and Camembert. In these cheeses pH is reduced to about 4.7 during the first 24 hours by the primary lactic acid starter culture. P. camemberti metabolizes the lactate to water and CO2 resulting in an increasing pH, most pronounced on the surface of the cheese. A pH gradient of decreasing values will be established towards the center of the cheese causing lactate to migrate towards the surface where it is used as a carbon source for P. camemberti. When all the lactate is depleted, casein will be degraded into amino acids and ammonia causing pH to increase further and the gradient to become stronger while the pH is still low in the center. The acid condition in the center causes soluble calcium phosphate to migrate towards the surface where it precipitates as a result of the higher pH (Karahadian and Linsa 1987; Vassel et al. 1986). The establishment of the pH gradient caused by P. camemberti indirectly is the key factor in the maturation process (Lawrence et al. 1987), but the desired soft texture of white mold cheeses is a direct result of the depletion of calcium phosphate in the center and the proteolytic activity of rennet, plasmin, and enzymes from the lactic acid bacteria and yeast. Yeast has been detected in several types of white mold cheeses and the positive role of yeast in the maturation and aroma formation of white mold cheese has been proposed (Schmidt and Lenoir 1980; Siewert 1986). However, the use of yeast as starter culture is still the exception rather than the rule in white mold cheeses. During ripening the yeast population increases to a level of 105-107 at the center and 106-108 at the surface. Several different yeast species have been isolated from white mold cheese and at the surface K. lactis, K. marxianus, and G. geotrichum have been the dominant cultures while D. hansenii has been predominant in the center. S. cerevisiae and Zygosaccharomyces rouxii are also found, but less frequently (Baroiller and Schmidt 1990). Except from G. geotrichum the role of yeast in the maturation process is still not clear, but it is assumed that their lipolytic and proteolytic activity and the capability to metabolize lactate and galactose, glucose and lactose play a role in the maturation. Further yeast have been mentioned to have an inhibitory effect on the undesired growth of Mucor spp. on the surface of Camembert cheese (Siewert 1986).
a. The Role of Galactomyces Geothricum in White Mold Cheese. Galactomyces geotrichum is closely related to the production of white mold cheeses. G. geotrichum is often used as a co-culture together with P. camemberti in the production of white mold cheese (Addis et al. 2001; Molimard et al. 1995) and in a few variants of these cheeses G. geotrichum is used as the only culture. G. geotrichum is able to assimilate lactate and it grows faster on the surface of the cheese than P. camemberti. It seems to contribute strongly, along with different sulfides, to the characteristic aroma profile. Methanethiol and dimethyl is produced from methionine by two distinct pathways (Demerigny et al. 2000). The formation of dimethyl disulfide, dimethyl trisulfide, and S-methyl thioesters are also well known for this yeast (Berger et al. 1999). Further, G. geotrichum is able to produce volatile compounds like methylketones, alcohols, esters, and fatty acids (Jollivet et al. 1994).
Some strains of G. geotrichum have anti-microbial activities. Production and excretion of 2-hydroxy-3-phenyl-propanoic acid, which have a broad anti-bacterial effect
(Dieuleveux et al. 1998) and D-3-phenyllactic acids inhibiting Listeria monocytogenes (Dieuleveux et al. 1998) have been reported. G. geotrichum is known to inhibit contaminating molds on the surface of mold cheese and in studies where G. geotrichum was cultured together with P. commune, P. caseifulvum, P. verrucosum, P. solitum, and Aspergillus versicolor; it was found that mycotoxin were produced in significantly lower concentration by the five molds compared to growth of the mold as single cultures (Nielsen et al. 1998a,b). All together, it indicates that G. geotrichum plays an important role in the inhibition of undesired microorganisms in mold ripened cheese (Nielsen et al. 1998a,b). Many strains of G. geotrichum have been described and the diversity among the strains is very pronounced with regard to all their technological characteristics (Spinnler et al. 2001).
4.6.4 Contribution and Spoilage of Yeast in Other Types of Cheese
Besides the important role yeast play in surface-ripened and mold-ripened cheese, they may also have a desired influence on the maturation and final quality of hard and semi-hard cheeses like Cheddar, Gouda, and Parmesan (Deiana et al. 1984; Viljoen and Greyling 1995). Yeast are mentioned to accelerate the ripening process (Deiana et al. 1984), but the role of yeast in these types of cheeses is not clear. A variety of yeast including, e.g., D. hansenii, Y. lipolytica, S. cerevisiae, K. marxianus, C. catanulata, T. delbruckii, and R. glutinis have been isolated from these types of cheese and seems primarily to be connected to spoilage, e.g., uncontrolled maturation, flavor defects and blowing of the cheese, and undesired pigment formation. On the other hand, D. hansenii has been reported to have an inhibitory effect on the growth of Clostridium tyrobutyricum and C. butyricum, which is a well known spoilage organism in theses types of cheeses (Deiana et al. 1984; Fatichenti et al. 1983).
In unripened cheese yeast only seems to cause spoilage. In cottage cheese, spoilage caused by, e.g., Y. lipolytica/ C. lipolytica, Candidum sake, C. spherica, and K. maxianus has been reported (Brockelhurst and Lund 1985; Fleet 1990). The spoilage appears as visible colonies on the surface of the cheese, flavor and aroma defects and undesired production of gas (Brockelhurst and Lund 1985).
Several investigations have been reported on yeast in Feta cheese (Kaminarides and Laskos 1992; Tzanetakis et al. 1998; Vivier et al. 1994). The dominant yeast in Greek Feta and the brine of Greek Feta is D. hansenii/C. famata, S. cerevisiae, Torulaspora delbrueckii, Pichia farinosa, and Pichia membranaefaciens (Kaminarides and Laskos 1992; Tzanetakis et al. 1998). In Danish and Sardinian Feta, uncontrolled gas production by yeast caused swelling defects in the final product (Fadda et al. 2001; Westall and Filtenborg 1998). In Danish Feta with swelling defects Torulaspora delbrueckii was predominant while the responsible yeast in Sadinien Feta was found to be Dekkera anomala. Several other yeast species such as Y. lipolytica, D. hansenii, C. sake, K. marxianus, C. bututyri, and G. candidum, Dekkerar bruxellensis, K. lactis were isolated from Feta cheese in these investigations. The yeast population in the Sardinian Feta was the same in the cheese from the two dairies investigated while the occurrence of yeast in Danish Feta seems to vary from dairy to dairy. The high concentration of yeast in the environment of the production area in the dairies indicated that the occurrence of yeast in these types of Feta was due to recontamination.
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