Lait (1989) 69, 537-546 537 © Elsevier/INRA Original article
Evolution of the microflora of Kopanisti chee se during ripening. Study of the yeast flora
S.E. Kaminarides and E.M. Anifantakis
Agricultural University of Athens, Athens, Greece
(received 25 May 1988, accepted 5 July 1989)
Summary - The microflora of Kopanisti cheese during its ripening was studied. For this, samples were taken from the surface and the interior of the cheese 1, 8, 16, 32 and 46 days after its manu- facture and the total streptococci, lactobacilli, yeast and molds counts as weil as coliforms were de- termined. The results showed the changes in these microbial parameters during cheese ripening as weil as the differences between the surface and the interior of the cheese. 86 strains of yeast from the cheese sampies were also isolated and identified out of which the following species were found : Trichosporon cutaneum (T. beigelil), Kluyveromyces lactis (KI. marxianus var. lactis), Saccharomyc- es exiguus, Saccharomyces cerevisiae, Rhodotorula rubra, (Rh. mudlaginosa), Trichosporon penici/- latum (Geotrichum penicillatum), Candida lusitaniae (Clavispora lusitaniae) and Debaryomyces han- senii.
Kopanisti cheese - maturation - identification - yeasts
Résumé - Evolution de la flore microbienne du fromage Kopanisti au cours de la maturation. Étude des levures. Nous avons étudié la flore microbienne qui se développe à la surface et à /'inté- rieur du fromage Kopanisti durant sa maturation. Dans ce but, des échantillons ont été examinés, 1, B, 16, 32 et 46 j après la fabrication. Nous avons dénombré la flore microbienne totale, les streptoco- ques, les lactobacilles, les levures, les moisissures et les bactéries coliformes. Les résultats obtenus ont démontré, d'une part l'évolution de ces groupes microbiens durant la maturation du fromage, d'autre part la différence entre la surface et la partie interne. De ces échantillons, nous avons isolé B6 souches de levures parmi lesquelles nous avons identifié les espèces suivantes: Trichosporon cutaneum (T. beigelii), Kluyveromyces lactis (KI. marxianus var. lactis), Saccharomyces exiguus, Saccharomyces cerevisiae, Rhodotorula rubra, (Rh. mucilaginosa), Trichosporon penicillatum (Geo- trichum penicillatum), Candida lusitaniae (Clavispora lusitaniae) et Debaryomyces hansenii.
fromage Kopanisti - maturation - identification - levures
INTRODUCTION with good spreadability and a strong fla- vour similar to that of blue cheeses. These interesting properties of the cheese are at- Kopanisti is a weil known traditional Greek tributed to the intensive changes during its cheese made of cow's, ewe's and goat's ripening. The procedure of manufacture milk or mixtures of them (Zygouris, 1952; and ripening promotes an abundant Davis, 1976; Anifantakis, 1987). It is soft, growth of a great variety of microorgan- 538 S.E. Kaminarides and E.M. Anifantakis
isms which are considered responsible for Cheese manufacture these changes. The present work, which is part of a wider project, was carried out The cheese was manufactured according to the at the Agricultural University of Athens in method described by Anifantakis (1987). The order to study the microflora of Kopanisti main stages of this method are the addition of cheese and its changes during ripening as 4% sodium chloride te drained acid curd (pH weil as the yeast flora. 4.9, moisture 53.5%), mixing it with 1% of ripe Kopanisti cheese of good quality, as usually practiced and keeping the mixture in open ba- sins at room temperature for ripening. During the ripening the mass of the cheese is mixed 3-4 times in order to distribute the microflora MATERIAL AND METHODS grown on the surface of the cheese into the whole mass. This practice permits the growth of an abundant microflora to which the flavour of the cheese is attributed. Five cheese prepara- Milk tions were made in total.
Cow's milk was utilized without antibiotics and pasteurised at 68 oCfor 10 min. Sampling
Samples from the 2-cm thick surface layer and Starters the interior of the cheese were taken under aseptic conditions 1, 8, 16, 32 and 46 days after Lyophilized starters of Streptoeoccus tectis and manufacture of cheese. The sampies of the 1st Laetobaeillus easei (Chr. Hansen's Laboratory, day were taken before salting and mixing the Copenhagen, Denmark) were used at 2% and curd with the ripe Kopanisti. 0.5%, respectively. These starters were grown overnight at 30 "C in skim milk three times be- 1 fore use. Microbial examination of samples 1
The total streptococci, lactobacilli, yeast and Rennet 1 mold counts were determined using the meth- ods of the American Public Health Association
Calf rennet powder (trade name HA-LA, Chr. (1967). The coliforms were enumerated accord- 1 Hansen's Laboratory, Copenhagen, Denmark) ing to the International Dairy Federation (1974). 0.3 9 for 100 kg of milk was used. The rennet The media and conditions used were, for total was added to the milk at 30 oCafter an increase count : milk agar (Oxoid), incubation for 3 days 1 in its acidity of 2-3 °D produced by the start- at 30 oC; for streptococci : M-17 agar (Merck), ers. Under these conditions the curd was ready incubation for 2 days at 37 "O under CO2 + H2 1 for cutting 3 h after the addition of the rennet. atmosphere (Gas-Pak system); for lactobacilli : MRS Agar at pH 5.7 (Oxoid), incubation for 2 days at 37 oC under CO2 + H2 atmosphere; for 1 yeasts and molds : chloramphenicol yeast ex- Ripe Kopanisti cheese tract glucose agar (IDF 1980), incubation for 2 1 days at 37 -c. Good quality of mature Kopanisti cheese was Yeasts were isolated from chloramphenicol used at 1%. This ripe cheese was weil mixed yeast extract glucose agar medium, purified by 1 with fresh curd after it had been drained. streaking on the same medium and maintained
1 Veast flora of Kopanisti cheeses 539
at 4 "C on the surface of potato dextrose agar the cheese during ripening ranged from (Oxoid). 107-109fg of cheese and that the maxi- The idenfication of yeasts was made accord- mum values were obtained at the 1st day ing to the taxonomy of Barnett et al. (1979) and of the ripening. Between the 1st and the Lodder & Kreger-van Rij (1970) on the basis of 8th day, a small decrease of these values their morphological and biochemical characteris- was observed. Afterwards the counts re- tics. Microscopie appearance of yeasts on siide cultures, anaerobic fermentation of 10 sugars mained nearly constant. Similar results (D-glucose, D-galactose, D-maltose, sucrose, (J.- were also obtained for Camembert ~iku> --- D-trehalose; milibiose, lactose, D-cellobiose, chi, 1966) and Roquefort (Devoyod et al., melezitose and raffinose), resistance to actidi- 1968). one (0.01% and 0.1%) and assimilation of etha- nol were carried out as described by Harrigan & The number of the streptococci and Mc Canee (1976), while formation of ascospores their changes during ripening of Kopanisti or other spores in Gorodkowa agar (Van Der cheese were similar to that of the total col- Walt, 1970), with growth at 37 oC and assimila- ony counts (Fig. 2). This was also ob- tion of nitrate in Bacto yeast base (Difco) were served in Camembert cheese (Lenoir, performed according to Wickerham (1951). 1962). Also, assimilation of carbon sources was investi- gated using the API system' 50 CH. Contrary to the total colony and strepto- The enzyme pattern of the yeasts was done cocci counts, the numbers of lactobacilli using the rapid miniaturized system API-ZVM. showed a significant increase from the 1st to the 8th day and remained nearly con- stant thereafter throughout the ripening.
RESULTS AND DISCUSSION 1010
_-0 Evolution of the microflora of Kopanisti _----.0- _ cheese during ripening
The evolution of the total streptococci, lac- tobacilli, yeast and mold counts grown on 1 the surface and in the interior of the Kopa- nisti cheese after 1, 8, 16, 32 and 46 days
of ripening is presented in Figures 1, 2, 3 1 and 4 respectively. It can be easily seen that the values at each stage of the ripen- 1 1 1 • 8 16 32 46 1 ing for the total streptococci and lactobacilli Days of ripening counts were similar for the surface and the Fig. 1. Changes in total counts of microorga- interior of the cheese. By contrast, yeasts nisms during ripening of Kopanisti cheese (e--e 1 and molds were found to grow significantly interior of cheese, 0-0 surface layer of faster on the surface. cheese). Evolution de la flore microbienne totale du fro- 1 Figure 1 shows that the total colony mage Kopanisti durant la maturation (e--e inté- counts for the surface and the interior of rieur du fromage, 0-0 extérieur du fromage). 1 !;.API system 'La BalmO-I'~~O Montalleu-Vercieu, Francs. 1
1
1 540 S.E. Kaminarides and E.M. Anifantakis
Significant increase of lactobacilli during the first days of ripening was also ob- served in Manchego (Ordonez et ..al., 1978) and Kefalotyri (Anifantakis &~.'!1~> arides, 1987) and was attributed ta the higher resistance of the lactobacilli ta the low acidities (Harrigan & McCance, 1976). The changes in yeasts and molds counts of Kopanisti cheese during ripening are shawn in Figure 4. Comparing these results ta those presented in the Figures 1, 2 and 3 it can be seen that yeasts and molds grow in a different way from the oth- er groups of microorganisms. Their num- ber was significantly higher on the surface than in the interior of the cheese and in- 8 16 32 46 creased gradually up ta the 16th and 32nd Days of ripening day, respectively. Then a small decrease Fig. 3. Changes in lactobacilli counts during ripe- was observed. This group of microorgan- ning of Kopanisti cheese (...... interior of isms was found ta show a similar growth cheese, 0-0 surface layer of cheese). tendency in Camembert cheese. In this Evolution des lactobacilles du fromage Kopanisti case, the maximum values were observed durant la maturation ( intérieur du fromage, at the 10th day for the surface and 15th 0-0 extérieur du fromage). day for the interior of the cheese (Schmidt & Lenoir, 1978).
ID 108 8 16 32 46 8 16 32 46 Days of ripening Days of ripening Fig. 2. Changes in streptococci counts during ri- Fig. 4. Changes in yeasts and molds counts du- pening of Kopanisti cheese (...... interior of ring ripening of Kopanisti cheese (...... interior cheese, 0-0 surface layer of cheese). of cheese, 0-0 surface layer of cheese). Evolution des streptocoques du fromage Kopa- Evolution des levures et des champignons du fro- nisti durant la maturation (...... intérieur du fro- mage Kopanisti durant la maturation ( inté- mage, 0-0 extérieur du fromage). rieur du fromage, 0-0 extérieur du fromage). Yeastfloraof Kopanisticheeses 541 A small number of coliforms ir(Ko-paois~------to this species. Out of these 61 strains, 47 ti cheese was observed on the surface at were found in ripe and 14 in unripe the beginning of ripening. They may be cheese. The high frequency of Trichospo- considered as contaminants. These micro- ron cutaneum in Kopanisti cheese could organisms disappeared during the process be attributed to the wide occurrence of of ripening and were not found in ripe this species in nature and to the fact that it cheese. The presence of coliforms in fresh is frequently associated with humans~ cheese as contaminants, and their de- der & Kreger-Van Rij, 1970). Kluyveromyc- crease or disappearance in ripe cheese es lactis (KI. marxianus var. lactis) was was also observed in other types of chees- also isolated from the cheese samples tak- es such as Cottage, Roquefort, Cheddar, en at the middle and the end of ripening. Feta, Camembert, Manchego, Telemes, This yeast, which was also found to domi- Gruyère, Halloumi, Kefalotyri, etc. ~/ nate at the end of ripening in Camembert ze & Oison, 1960; Devoyod & Bret, 1966; (Schmidt & Lenoir, 1978) and Trichospo- Dommet, 1970; Veinoglou et al., 1974; ron cutaneum seem to be the most impor- Mourgues et el., 1977; Ordonez et al., tant microbes for the ripening of Kopanisti 1978; Veinoglou et al., 1980; Zerfiridis et cheese. al., 1984; Kaminarides & Anifantakis, 1985; Anifantakis & Kaminarides, 1987). The other species of yeasts that were The low pH of the cheese inhibits the isolated, in order of frequency, were : Sac- growth of the acid sensitive coliform bacte- charomyces exiguus, Rhodotorula rubra ria and leads to their disappearance from (Rh. mucilaginosa), Saccharomyces cere v- ripe cheese (Kosikowski & Fox, 1968; isiae, Trichosporon penicillatum (Geotri- Reamer et al., 1974). chum penicillatum), Candida lusitaniae (Clavispora lusitaniae) and Debaryomyces hansenii. The yeasts Kluyveromyces marx- ianus var lactis were also isolated from Study of the yeast ttore Camembert (Schmidt & Lenoir, 1978; 1980), Roquefort (Devoyod & Sponem, 1970), Saint-Nectaire (Vergeade et sl., The yeasts represent a substantial part of 1976), Cantal (Millet et al., 1974) blue- the flora of Kopanisti cheese mainly attrib- uted to the special handling during the veined cheeses (De Boer & Kuik, 1987), cheese manufacture. and Cabrales (Nunez et al., 1981), Sac- charomyces cerevisiae from Camembert The study of the yeast flora was carried (Schmidt & Lenoir, 1978; 1980), Saint- out to identify the major species of yeasts Paulin (Ducastelle & Lenoir, 1965) and and their enzymatic activities. blue-veined cheeses (De Boer & Kuik, Eighty-six strains of yeasts taken from 1987), Debaryomyces hansenii from Tel- samples of different ages were isolated emes (Georgantas, 1979), Camembert and identified. The results of yeast identifi- (Schmidt & Lenoir, 1978; 1980), blue- cation as weil as some biochemical activi- veined cheeses (De Boer & Kuik, 1987), ties which were not included in taxonomy Roquefort (Devoyod & Sponem, 1970) system used and that couId be new useful Saint-Nectaire (Dale, 1972; Vergeade et characteristics are given in Table 1. al., 1976) and Cabrales (Nunez et al., lt is obvious that Trichosporon cutan- 1981), Rhodotorula sp. from Telemes eum (T. beigelli) is the yeast which domi- (Georgantas, 1979), Cantal (Millet et al., nates in the cheese. From the 86 identified 1974), Roquefort (Devoyod & Sponem, strains of yeasts, 61 were found to belong 1970) and Camembert (Lenoir, 1962) and ~01 1\) Tableau 1. Classification of 86 strains of yeasts isolated from Kopanisti cheese during ripening and sorne of their biochemical characteristics. Classification des 86 souches de levures isolées du fromage Kopanisti durant l'affinage, et quelques-unes de leurs caractéristiques biochimiques. Species of Utilization of carbon source yeasts D-Fruc-D-Man- Dulci- D-Sor- Amyg- Escu- Glyco- Xyli- Gentio- D-Tura- D-Ly- D-Taga- D-Fu -L-Fu- D-Arabi-L-Arabi- G1u- tose nase toi bitai dalin lin gen toI biose nase xose tose case case toi toi conate Trichosporon cutaneum + + V - - V - - V V - - - V - - + (Trischosporon beigelii) Kluyveromyces Candida lusitaniae + + - + + + - + + + - - - - + (Clavispora lusitaniae) Debaryomyces hansenii + + - + - - - - - + ------~01 Ct.) -: Negative reaction; +/-: weak reaction; + : positive reaction; ++: intense reaction; V: variable reaction. - : réaction négative. +/- : réaction faible; + : réaction positive; ++ : réaction intense; V : réaction variable. 544 S.E. Kaminarides and E.M. Anifantakis Candida sp. from Camembert (Lenoir, cheese changes during cheese ripening. 1962; Schmidt & Lenoir, 1978), blue- Lactobacilli and streptococci were found to veined cheeses (De Boer & Kuik, 1987), be the predominant groups,. especially at Saint-Nectaire (Dale, 1972; Vergeade et the beginning of ripening. Yeasts and sl., 1976), Saint-Paulin (Ducastelle &~ molds were also found in high numbers in noir, 1965) and Roquefort (Devoyod & Kopanisti, especially on its surface and Sponem, 1970). therefore their contribution to the chemical The microscopic appearance and repro- and organoleptic characteristics of the duction of isolated yeasts are given in Fig- cheese may be important. From the yeasts ure 5. which were isolated from the cheese, Tri- chosporon cutaneum (T. beigeli) and Kluy- The enzymatic activities of the isolated veromyces lactis (KI. marxianus var. lactis) microorganisms were examined by using the miniaturized API-ZYM test that allows were the predominant species. the determination of 19 characteristics. The hygienic quality of the experimental The most interesting activities that might Kopanisti cheese, judged by the coliform play an important role during ripening - presence, was found to be satisfactory. On esterase, lipase and aminopeptidase - the contrary, Kopanisti cheese made from are given in Table 1. The following conclu- raw milk, without using starters and uncon- sions can be summarized : trolled inoculum of ripe Kopanisti was not Trichosporon cutaneum (T. beigelii), Tri- always satisfactory (Kaminarides, 1986). chosporon penicillatum (Geotrichum peni- cillatum), Kluyveromyces lactis (KI. marxia- nus var lactis), Rhodotorula rubra (Rh. mucilaginosa), Saccharomyces cerevisiae ACKNOWLEDGMENTS and Candida lusitaniae (Clavispora lusitan- iae) showed an intensive production of leu- The authors thank Ors Kalatzopoulos, O. Xena- cine aminopeptidase and a weak to re- kis and Mrs. O. Leousi for their assistance. markable production of valine amino- peptidase. Trichosporon cutaneum (T. beigelii) and Saccharomyces exiguus presented a re- REFERENCES markable production of esterase C4 while the remaining species of yeasts had a American Public Health Association (1967) Standard Methods for the Examination of Dairy weak production. 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