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Microbiology of Kopanisti, a traditional Greek cheese
Food Microbiology, 1987, 4, 251-256
Microbiology of Kopanisti, a traditional Greek cheese N. Tzanetakis, E. Litopoulou-Tzanetaki* and K. Manolkidis
Laboratory of Dairy Technology, School of Agriculture, University of T h e s s a l o n i k i , 5 4 0 0 6 T h e s s a l o n i k i , Greece Received 7 A u g u s t 1987 Samples (50) of Kopanisti cheese were analyzed for total count, coliforms, lactic acid bacteria, micrococcaceae, yeasts and moulds. Total counts and lactobaciUi were >105 g- i in 82% and86% of the samples respectively. Yeasts were >103 g -1 in 78%, moulds ranged between 101 and 103 g- i in 58% of the samples, while coliforms were absent in 60% and present in low numbers in 32% of the samples. In this cheese pH (in 74% of the samples <5.0), moisture (in 72% of the samples >60.0%) and salt (in 52% of the samples <4.0%) ranged greatly. Lactobacillus plantarum and L. casei subsp, casei were the dominant lactobacillus species. Enterococci and P. pentosaceus were also isolated. Micrococci were found in one sample only. Among yeasts Pichia membranefasciens predominated while moulds were characterized as Penicillium commune.
Introduction Kopanisti is a cheese variety with a characteristic peppery taste produced mainly in the Greek islands of the Aegean sea. Even though its manufacture is not yet standardized, there are some common steps t ha t are generally followed by the producers. Thus, whole cow's milk is used, which is clotted, with a small quantity of rennet over a period of a few hours. The curd is drained in a cheese cloth after which a quantity of older cheese is added and the curd kneaded by hand and formed into balls the size of a small orange. The cheese is then left for several days until a green or blue green mould forms on the surface. The cheese balls are then sprinkled with fine salt and kneaded again so th at the mould spreads evenly throughout the mass of the cheese. The cheese ripens for 1-2 months during which it is kneaded several times. *Two whom correspondence should addressed. 0740-0020/87/030251 + 06 $02.00/0
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Although there is some information on its technology (Davies 1976, EekhofStork 1976, Zygouris 1952) there are no published data on either the microbiology or the chemistry of this cheese. The purpose of the present investigation was to study the numbers and principal types of microflora of this cheese. Thus, results of this study would probably help us to prepare a specific starter appropriate for industrial manufacture.
Methods Cheese samples Cheese samples (10/month) were obtained from retail shops over a period of five months and were derived from four islands and Peloponnesos.
Microbiological analyses The microorganism counts were made by the dilution pour-plate method. The following culture media and incubation temperatures were used: (a) total viable count: plate count agar (Oxoid Ltd) and incubation at 30°C for 72 h; (b) lactic acid bacteria: MRS agar (Oxoid Ltd) and incubation at 30°C for 5 days; (c) enterococci: citrate azide agar, Saraswat's O 1987 AcademicPress Limited
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modification (Saraswat et al. 1963) and incubation at 37°C for 72 h; (d) staphylococci and micrococci: mannitol salt agar (Oxoid Ltd) and incubation at 37°C for 48 h; and (e) yeasts and moulds: potato dextrose agar (Oxoid Ltd) and incubation at 25°C for 5 days.
Isolation and identification of the isolates After the end of the incubation period colonies (ten]sample) were picked at random from MRS agar plates and, after purification, maintained in yeast dextrose litmus milk + chalk (Sharpe and Fryer 1965) at 4°C. The isolates thus obtained were characterized according to Sharpe (1979) using the techniques described by Sharpe and Fryer (1965), Deibel (1964), Facklam (1972), Gunther and White (1961) and Whittenbury (1965). Every colony growing on mannitol salt agar plates was picked, purified and Gram stained thereafter. Gram-positive, catalase-positive cocci were maintained on nutrient agar slopes at 4°C and were afterwards submitted to further characterization according to their Oxidation Fermentation (O/F) reaction, acid production of glucose aerobically, nitrate reduction and oxidase activity following the methods of Cowan and Steel (1975) and the scheme of Baird-Parker (1979). Five colonies of yeasts (or every colony from low count plates) were picked, purified and maintained on malt extract agar slopes at 4°C. Yeasts were characterized according to Lodder (1971) following the methods suggested by Lodder (1971) and Devoyod and Sponem (1970). Moulds were also picked similarly and were characterized according to Raper and Thom (1949).
Chemical analyses The pH of the cheese was determined electrometrically, the NaC1 content according to the method of F.I.L. (1972) and the moisture content by heating at 102°C until constant weight. Salt was finally expressed as brine concentration.
105 and 10 v g-1 (mean 2.4 x 106 g - D in 26 samples, while 15 samples contained >10 v g-1 (mean 1.6 x l 0 s g - D total counts. The highest and lowest levels of total counts were 1-6 x 109 g-1 and 8.0 x 102 g-1 respectively. Coliform organisms were not detected in 30 samples while 16 out of 20 coliform positive samples contained low counts in the r a n g e of 101-10 s g-1 (mean 2.0 x 102 g-D. The highest coliform count observed was 2.0 x 103 g-1 High levels of lactic acid bacteria (>107 g - l , m e a n 6.1 x 10 v g - D were counted in 20 samples. In 23 samples their n u m b e r s r a n g e d between 105 and 10 v g-1 (mean 3.8 x 10 e g-D, while 7 samples contained <105 g-1 lactic acid bacteria. The lowest and highest counts were 6.4 x 103 g-1 and 1.7 x l0 s g-1 respectively. Enterococci were p r e s e n t in relatively low numbers: in the 22 enterococci positive samples the highest count observed was 1.9 x 104 g-1. Microorganisms growing on m a n n i t o l salt a g a r were present in 45 samples, of which 28 contained counts in the r a n g e of 103-105 g-1. Yeasts and moulds were a b s e n t in 8 and 4 samples respectively and found in relatively low n u m b e r s (101-103 g - l ) in 13 and 29 samples respectively. In 18 samples yeasts r a n g e d between 10 s and 105 g-1 (mean 3.0 x 104 g - l ) while 11 samples contained t h e m in r e l a t i v e l y high n u m b e r s (10~-10 v g - l , m e a n 5.9 x 105 g-D. In 16 cheese samples mould count r a n g e d between 103 and 105 g-1 (mean 3-7 x 104 g-l). The highest yeast and mould counts were 7.4 x 105 g-1 and 1.1 x 108 g-1 respectively.
Results
Levels of microorganisms in Kopanisti cheese Ranges in n u m b e r s of microorganisms present in the 50 cheese samples are presented in Table 1. Total viable counts r a n g e d between
Chemical analysis of the cheese E s t i m a t i o n of pH values, NaC1 and moisture contents of cheese samples gave results p r e s e n t e d in Table 2. According to our results, pH, NaC1 and moisture contents r a n g e d greatly.
1 16 0 15
15 13 29
0 30 0 28
5 8 4
101-103
3.1 × 102 1.9 × 102 1-3 x 102
8.0 x 102 2-0 × 102 0 2.0 x 102
Mean
28 18 16
8 4 7 7
103-105
x x × x
104 103 104 103
7.5 × 103 3.0 × 104 3.7 × 104
2.6 1.5 2.5 8.3
Mean
1 11 1
26 0 23 0
I05-i07
1.1 × 105 5-9 x l 0 s 1.1 x 105
2.4 x 106 0 3.8 × 106 0
Mean
No. of samples h a v i n g counts/g in the r an ge of
1 0 0
15 0 20 0
>107
1 24 12 13
<4.0 4.0 -4.5 4"51-5.0 >5-0
3-9 4.2 + 0.1 4-8_+ 0.1 5"15 + 0"05
Mean a
a Mean and standarddeviation.
No. of samples
Range ofpH <4.0 4-0-5.0 5.1-7.0 >7-0
Range of NaCl(%) 26 9 11 4
No. of samples 3.15 4-56 6.03 8.27
+ + + +
0.49 <60-0 0.33 60.0-65.0 0-59 65.1-70.0 0.88 >70-0
Mean(%)
Range of moisture (%)
11 18 15 6
No. of samples
55.30 +__6-45 63.07 + 1.44 67.62_+ 1.38 71.35 + 0.72
<5"5 5-5-6-5 6"5-7.5 >7.5
22 6 6 16
Range of No. of Mean(%) b r ine c onc 'n samples
T a b l e 2. R a n g e s o f pH , s a l t c o n t e n t , m o i s t u r e c o n t e n t a n d b r i n e c o n c e n t r a t i o n in K o p a n i s t i c h e e s e s a m p l e s .
Total viable count Coliforms Lactic acid bacteria Enterococci Microorganisms growing on Mannitol Salt A g a r Yeasts Moulds
Microorganisms
No. of samples negative
T a b l e 1. N u m b e r s o f m i c r o o r g a n i s m s in K o p a n i s t i c h e e s e .
4.30 6.00 7.14 9-6
+ + + +
2.55 0.13 0.31 3.77
Mean
3.3 × 10 s 0 0
1-6 × l 0 s 0 6.1 × 107 0
Mean
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254
N. Tzanetakis et al.
as shown in Table 3. Lactobacillus plantarum was the dominant Lactobacillus species (174 isolates), followed by L. casei subsp, casei (53 isolates), L. casei subsp. rhamnosus (35 isolates) and L. curvatus (28 isolates) and these species were present in 42, 24, 19 and 17 samples respectively. Isolates classified as L. xylosus, L. brevis, L. buchneri, L. viridescens and L. cellobiosus were found less frequently. Twenty-two strains of lactobacilli, 12 of which were streptobacteria and 10 betabacteria, could not be classified at the species level and remained unclassified. The 69 isolates of enterococci were classified as Streptococcus faecalis (6 isolates), S. faecium (18 isolates), S. faecium subsp, casseliflavus Table 3. Lactic acid bacteria isolated (19 isolates) and S. durans (26 isolates). from Kopanisti c h e e s e samples. The isolates of pediococci were classified as P. pentosaceus. No. of Gram positive catalase positive cocci samples No. of carrying were found only in one cheese sample Species isolates the species and the two isolated strains were characterized as Micrococcus luteus. In addiL. casei subsp. tion, 849 Gram positive catalase positive casei 53 24/50a rods were also isolated from MSA plates L. casei subsp. rhamnosus 35 19/50 but they were not submitted to further L. plantarum 174 42/50 characterization. L. curvatus 28 17/50 Yeasts were classified in species, as L. xylosus 12 5/50 shown in Table 4. Pichia membraneL. brevis 9 6/50 fasciens was predominant (58 isolates) L. buchneri 6 4/50 L. cellobiosus 4 4/50 followed by P. fermentans (14 isolates) L. viridescens 4 4/50 and these species were present in 33 and Unclassified 10 samples respectively. Strains of Canlactobacilli 22 11/50 dida kefyr, Saccharomyces sp., KluyveroS. faecalis 6 4/50 myces sp., Rhodotorula rubra, Pichia S. faecium 18 7/50 S. faecium subsp. homerii, Hansenula sp. and Torulopsis casseliflavus 19 5/50 sp. were also isolated. S. durans 26 8/50 The 82 isolates of moulds were penicilP. pentosaceus 19 8/50 lin which were classified as PeniciUium Number of samples carrying the species/number commune. Nevertheless, in about 50% of the cheese samples pH ranged between 4.0-4-5 (mean 4.2 + 0.1) and the NaC1 content was <4.0% (mean 3.15 + 0.36). Generally the pH values were quite low, with minimum and m a xi m um values of 3.9 and 5.2 respectively. In 66% of the samples the values for moisture ranged between 60.0 and 70.0%. The low NaC1 and the high moisture content of certain cheese samples resulted in low (<5.5) brine concentration values in 22 samples (mean 4.30 __ 2.55). Whilst relatively high (>7.5) brine concentration values were observed in 16 samples (mean 9-6 + 3.77) due to the high NaC1 and the low moisture content of certain samples.
of samples examined.
Discussion
Identification of the isolates Isolates from MRS agar were classified as lactobacilli (347 isolates), pediococci (19 isolates) and enterococci (69 isolates)
The wide range of values in pH, % NaC1 and % moisture of Kopanisti cheese samples is due to the fact t hat the manufacturers do not follow the same
Microbiology of Kopanisti 255 Table 4. Yeast species isolated from Kopanisti cheese samples.
disappeared during cheese ripening due to unfavourable conditions in the cheese. LactobaciUus plantarum and L. casei, No. of which were the most frequently isolated samples strains, are known as salt resistant No. of carrying Species isolates the species lactobacilli (Rasic 1962) and the same applies to enterococci. The predominant Pichia membranespecies of lactic acid bacteria of Kopanfasciens 58 33/42a isti cheese are usually found in various Pichia fermentans 14 10/42 cheese varieties. Thus, L. casei, L. planCandida kefyr 3 2/42 Saccharomyces sp. 3 2/42 tarum and S. faecalis were previously Kluyveromyces sp. 2 1/42 found in Roquefort cheese (Devoyod Rhodotorula rubra 1 1/42 1969, Devoyod 1970). In addition, L. Pichia homerii 1 1/42 plantarum, L. casei, L. casei subsp. Hansenula sp. 1 1/42 rhamnosus L. brevis, S. faecalis and S. Torulopsis sp. 1 1/42 faecium were also isolated from Cabrales - Number of samples carrying the species/number cheese (Nunez and Medina 1979), where of yeast positive samples. L. plantarum predominated as in Kopanisti cheese. Yeasts were also probably favoured by method of production. Although there was a wide range of pH values, 75% of the cheese environment and their numthese were <5.0. This also applies to % bers were >103 g-1 in about 70% of the NaC1 and % moisture, where 70 and 88% positive samples. Pichia membraneof the values were <5.0 and <70-0% fasciens and P. fermentans were the predominant species accounting for 69% respectively. The combined effect of the low pH and 17% of the yeast isolates respecvalues (<5.2) and brine concentration tively. Both species were previously (>6-5% for the 44% of the samples) might found in Cabrales cheese (Nunez et al. 1981). Pichia membranefasciens was also be expected to influence the numbers and types of microorganisms t h a t finally isolated from Roquefort cheese (Devoyod occur in the cheese. Thus, coliforms were and Sponem 1970). Moulds were detected in relatively low not found in the 60% of the samples and numbers (101-103 g-l) in 63% of the their number in the positive samples was positive samples. In addition to lactic quite low. Micrococci and staphylococci acid bacteria and yeasts, the presence of were practically absent, since only one PeniciUium commune moulds in 92% of sample carried 2 Micrococcus luteus strains. In addition to the inhibitory samples suggests that they also belong to effect of pH and brine concentration, the essential microflora of Kopanisti antibacterial substances produced by cheese and t h a t they possibly contribute moulds possibly contributed to the dis- to ripening changes and flavour characteristics of this cheese. appearance of these microorganisms. Thus the results of the present study The cheese environment probably did not adversely affect the growth of lactic suggest t h a t a standard method of manuacid bacteria, since these organisms facture should be adopted for the production of this cheese. Experiments should were present in high numbers (>105 g-l) in 86% of the samples. Nevertheless, salt be conducted on the steps of its manufacsensitive lactic streptococci, which were ture so that the cheese can be stanprobably present in the fresh cheese, dardized and improved, but will not loose
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its traditional character. In addition, the use of a starter culture consisting of lactic acid bacteria, a penicillium and possibly a yeast is considered necessary. Thus, combinations of mesophilic streptococci (Streptococcus lactis, S. cremoris, S. diacetylactis), which will ensure mainly acidification and Lactobacillus casei, S. durans and Pichia membranefasciens which belong to the predominan t microflora of the cheese and possibly play an important role in its ripen-
ing and flavour characteristics should be preferred. Heterofermentative lactic acid bacteria (leuconostocs) which would provide the cheese with an open texture by production of gas, thus providing favourable conditions for the fungus, should also be tried. The possible role of the above microorganisms and Penicillium, possibly P. roqueforti, in cheese ripening and flavour compounds formation need to be studied.
References Baird-Parker, A. C. (1979) Methods for identifying staphylococci and micrococci. In Idehtification methods for microbiologists, 2nd edn. (Ed. Skinner and Lovelock) 201pp. London, Academic Press. Cowan, S. T. (Ed.) (1974) Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd edn. London, Cambridge University Press. Davis, J. G. (1976) Cheese, Vol. III, 879 pp. New York, Churchill Livingstone. Deibel, R. H. (1964) The group D streptococci. Bacteriol. Rev. 28, 330-366. Devoyod, J. J. (1969) La flore microbienne du fromage de Roquefort. IV Les enterocoques. Le Lait 49, 637-650. Devoyod, J. J. (1970) La flore microbienne du fromage de Roquefort. V Les lactobacilles. Le Lait 50, 277-284. Devoyod, J. J. and Sponem, D. (1970) La flore microbienne du fromage de Roquefort. VI Les levures. Le Lait 498, 524-543. Eekhof-Stork, N. (1976) The world atlas of cheese (Ed. Bailey, A.) 136pp. Paddington Press Ltd, USA. Facklam, R. R. (1972) Recognition of group D streptococcal species of human origin by biochemical and physiological tests. Appl. Microbiol. 23, 1131-1139. Federation International de Laiterie. FIL-IDF Normes Internationales 1972, 17A. Giinther, H. L. and White, H. R. (1961) The cultural and physiological characters of the pediococci. J. Gen. Microbiol. 26, 185-197. Lodder, J. (1971) The yeasts. A taxonomic study, 2nd edn. Amsterdam and London, North-Holland Publishing Company. Nunez, M. and Medina, M. (1979) La flore lactique du fromage bleu de Cabrales. Le Lait 59, 497-513. Nunez, M., Medina, M., Gaya, P. and Diaz-Amado, C. (1981) Les levures et les moisissures dans le fromage bleu de Cabrales. Le Lait 601/602, 62-79. Raper, K. and Thorn, C. (1949) A manual of the Penicillia. Baltimore, Williams & Wilkins Co. Rasic, J. (1962) A study of the resistance of lactic acid bacteria to sodium chloride. XVI International Dairy Congress B, 881-886. Saraswat, D. S., Clark, W. S., Jr. and Reinbold, G. W. (1963) Selection of a medium for the isolation and enumeration of enterococci in dairy products. J. Milk Food Technol. 26, 114-118. Sharpe, M. E. (1979) Identification of lactic acid bacteria. In Identification methods for microbiologists, 2nd edn. (Ed. Skinner and Lovelock) 233pp. London, Academic Press. Sharpe, M. E. and Fryer, T. F. (1965) Media for lactic acid bacteria. Lab. Practice, 697-701. Whittenbury, R. (1965) A study of some pediococci and their relationship to Aerococcus viridans and the enterococi. J. Gen. Microbiol. 40, 97-106. Zygouris, N. P. (1952) Milk industry, 2nd edn. (Ed. Ministry of Agriculture) 430pp. Athens.
Microbiology of Kopanisti, a traditional Greek cheese N. Tzanetakis, E. Litopoulou-Tzanetaki* and K. Manolkidis
Laboratory of Dairy Technology, School of Agriculture, University of T h e s s a l o n i k i , 5 4 0 0 6 T h e s s a l o n i k i , Greece Received 7 A u g u s t 1987 Samples (50) of Kopanisti cheese were analyzed for total count, coliforms, lactic acid bacteria, micrococcaceae, yeasts and moulds. Total counts and lactobaciUi were >105 g- i in 82% and86% of the samples respectively. Yeasts were >103 g -1 in 78%, moulds ranged between 101 and 103 g- i in 58% of the samples, while coliforms were absent in 60% and present in low numbers in 32% of the samples. In this cheese pH (in 74% of the samples <5.0), moisture (in 72% of the samples >60.0%) and salt (in 52% of the samples <4.0%) ranged greatly. Lactobacillus plantarum and L. casei subsp, casei were the dominant lactobacillus species. Enterococci and P. pentosaceus were also isolated. Micrococci were found in one sample only. Among yeasts Pichia membranefasciens predominated while moulds were characterized as Penicillium commune.
Introduction Kopanisti is a cheese variety with a characteristic peppery taste produced mainly in the Greek islands of the Aegean sea. Even though its manufacture is not yet standardized, there are some common steps t ha t are generally followed by the producers. Thus, whole cow's milk is used, which is clotted, with a small quantity of rennet over a period of a few hours. The curd is drained in a cheese cloth after which a quantity of older cheese is added and the curd kneaded by hand and formed into balls the size of a small orange. The cheese is then left for several days until a green or blue green mould forms on the surface. The cheese balls are then sprinkled with fine salt and kneaded again so th at the mould spreads evenly throughout the mass of the cheese. The cheese ripens for 1-2 months during which it is kneaded several times. *Two whom correspondence should addressed. 0740-0020/87/030251 + 06 $02.00/0
be
Although there is some information on its technology (Davies 1976, EekhofStork 1976, Zygouris 1952) there are no published data on either the microbiology or the chemistry of this cheese. The purpose of the present investigation was to study the numbers and principal types of microflora of this cheese. Thus, results of this study would probably help us to prepare a specific starter appropriate for industrial manufacture.
Methods Cheese samples Cheese samples (10/month) were obtained from retail shops over a period of five months and were derived from four islands and Peloponnesos.
Microbiological analyses The microorganism counts were made by the dilution pour-plate method. The following culture media and incubation temperatures were used: (a) total viable count: plate count agar (Oxoid Ltd) and incubation at 30°C for 72 h; (b) lactic acid bacteria: MRS agar (Oxoid Ltd) and incubation at 30°C for 5 days; (c) enterococci: citrate azide agar, Saraswat's O 1987 AcademicPress Limited
252
N. Tzanetakis et al.
modification (Saraswat et al. 1963) and incubation at 37°C for 72 h; (d) staphylococci and micrococci: mannitol salt agar (Oxoid Ltd) and incubation at 37°C for 48 h; and (e) yeasts and moulds: potato dextrose agar (Oxoid Ltd) and incubation at 25°C for 5 days.
Isolation and identification of the isolates After the end of the incubation period colonies (ten]sample) were picked at random from MRS agar plates and, after purification, maintained in yeast dextrose litmus milk + chalk (Sharpe and Fryer 1965) at 4°C. The isolates thus obtained were characterized according to Sharpe (1979) using the techniques described by Sharpe and Fryer (1965), Deibel (1964), Facklam (1972), Gunther and White (1961) and Whittenbury (1965). Every colony growing on mannitol salt agar plates was picked, purified and Gram stained thereafter. Gram-positive, catalase-positive cocci were maintained on nutrient agar slopes at 4°C and were afterwards submitted to further characterization according to their Oxidation Fermentation (O/F) reaction, acid production of glucose aerobically, nitrate reduction and oxidase activity following the methods of Cowan and Steel (1975) and the scheme of Baird-Parker (1979). Five colonies of yeasts (or every colony from low count plates) were picked, purified and maintained on malt extract agar slopes at 4°C. Yeasts were characterized according to Lodder (1971) following the methods suggested by Lodder (1971) and Devoyod and Sponem (1970). Moulds were also picked similarly and were characterized according to Raper and Thom (1949).
Chemical analyses The pH of the cheese was determined electrometrically, the NaC1 content according to the method of F.I.L. (1972) and the moisture content by heating at 102°C until constant weight. Salt was finally expressed as brine concentration.
105 and 10 v g-1 (mean 2.4 x 106 g - D in 26 samples, while 15 samples contained >10 v g-1 (mean 1.6 x l 0 s g - D total counts. The highest and lowest levels of total counts were 1-6 x 109 g-1 and 8.0 x 102 g-1 respectively. Coliform organisms were not detected in 30 samples while 16 out of 20 coliform positive samples contained low counts in the r a n g e of 101-10 s g-1 (mean 2.0 x 102 g-D. The highest coliform count observed was 2.0 x 103 g-1 High levels of lactic acid bacteria (>107 g - l , m e a n 6.1 x 10 v g - D were counted in 20 samples. In 23 samples their n u m b e r s r a n g e d between 105 and 10 v g-1 (mean 3.8 x 10 e g-D, while 7 samples contained <105 g-1 lactic acid bacteria. The lowest and highest counts were 6.4 x 103 g-1 and 1.7 x l0 s g-1 respectively. Enterococci were p r e s e n t in relatively low numbers: in the 22 enterococci positive samples the highest count observed was 1.9 x 104 g-1. Microorganisms growing on m a n n i t o l salt a g a r were present in 45 samples, of which 28 contained counts in the r a n g e of 103-105 g-1. Yeasts and moulds were a b s e n t in 8 and 4 samples respectively and found in relatively low n u m b e r s (101-103 g - l ) in 13 and 29 samples respectively. In 18 samples yeasts r a n g e d between 10 s and 105 g-1 (mean 3.0 x 104 g - l ) while 11 samples contained t h e m in r e l a t i v e l y high n u m b e r s (10~-10 v g - l , m e a n 5.9 x 105 g-D. In 16 cheese samples mould count r a n g e d between 103 and 105 g-1 (mean 3-7 x 104 g-l). The highest yeast and mould counts were 7.4 x 105 g-1 and 1.1 x 108 g-1 respectively.
Results
Levels of microorganisms in Kopanisti cheese Ranges in n u m b e r s of microorganisms present in the 50 cheese samples are presented in Table 1. Total viable counts r a n g e d between
Chemical analysis of the cheese E s t i m a t i o n of pH values, NaC1 and moisture contents of cheese samples gave results p r e s e n t e d in Table 2. According to our results, pH, NaC1 and moisture contents r a n g e d greatly.
1 16 0 15
15 13 29
0 30 0 28
5 8 4
101-103
3.1 × 102 1.9 × 102 1-3 x 102
8.0 x 102 2-0 × 102 0 2.0 x 102
Mean
28 18 16
8 4 7 7
103-105
x x × x
104 103 104 103
7.5 × 103 3.0 × 104 3.7 × 104
2.6 1.5 2.5 8.3
Mean
1 11 1
26 0 23 0
I05-i07
1.1 × 105 5-9 x l 0 s 1.1 x 105
2.4 x 106 0 3.8 × 106 0
Mean
No. of samples h a v i n g counts/g in the r an ge of
1 0 0
15 0 20 0
>107
1 24 12 13
<4.0 4.0 -4.5 4"51-5.0 >5-0
3-9 4.2 + 0.1 4-8_+ 0.1 5"15 + 0"05
Mean a
a Mean and standarddeviation.
No. of samples
Range ofpH <4.0 4-0-5.0 5.1-7.0 >7-0
Range of NaCl(%) 26 9 11 4
No. of samples 3.15 4-56 6.03 8.27
+ + + +
0.49 <60-0 0.33 60.0-65.0 0-59 65.1-70.0 0.88 >70-0
Mean(%)
Range of moisture (%)
11 18 15 6
No. of samples
55.30 +__6-45 63.07 + 1.44 67.62_+ 1.38 71.35 + 0.72
<5"5 5-5-6-5 6"5-7.5 >7.5
22 6 6 16
Range of No. of Mean(%) b r ine c onc 'n samples
T a b l e 2. R a n g e s o f pH , s a l t c o n t e n t , m o i s t u r e c o n t e n t a n d b r i n e c o n c e n t r a t i o n in K o p a n i s t i c h e e s e s a m p l e s .
Total viable count Coliforms Lactic acid bacteria Enterococci Microorganisms growing on Mannitol Salt A g a r Yeasts Moulds
Microorganisms
No. of samples negative
T a b l e 1. N u m b e r s o f m i c r o o r g a n i s m s in K o p a n i s t i c h e e s e .
4.30 6.00 7.14 9-6
+ + + +
2.55 0.13 0.31 3.77
Mean
3.3 × 10 s 0 0
1-6 × l 0 s 0 6.1 × 107 0
Mean
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b~ c,u
w
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M
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as shown in Table 3. Lactobacillus plantarum was the dominant Lactobacillus species (174 isolates), followed by L. casei subsp, casei (53 isolates), L. casei subsp. rhamnosus (35 isolates) and L. curvatus (28 isolates) and these species were present in 42, 24, 19 and 17 samples respectively. Isolates classified as L. xylosus, L. brevis, L. buchneri, L. viridescens and L. cellobiosus were found less frequently. Twenty-two strains of lactobacilli, 12 of which were streptobacteria and 10 betabacteria, could not be classified at the species level and remained unclassified. The 69 isolates of enterococci were classified as Streptococcus faecalis (6 isolates), S. faecium (18 isolates), S. faecium subsp, casseliflavus Table 3. Lactic acid bacteria isolated (19 isolates) and S. durans (26 isolates). from Kopanisti c h e e s e samples. The isolates of pediococci were classified as P. pentosaceus. No. of Gram positive catalase positive cocci samples No. of carrying were found only in one cheese sample Species isolates the species and the two isolated strains were characterized as Micrococcus luteus. In addiL. casei subsp. tion, 849 Gram positive catalase positive casei 53 24/50a rods were also isolated from MSA plates L. casei subsp. rhamnosus 35 19/50 but they were not submitted to further L. plantarum 174 42/50 characterization. L. curvatus 28 17/50 Yeasts were classified in species, as L. xylosus 12 5/50 shown in Table 4. Pichia membraneL. brevis 9 6/50 fasciens was predominant (58 isolates) L. buchneri 6 4/50 L. cellobiosus 4 4/50 followed by P. fermentans (14 isolates) L. viridescens 4 4/50 and these species were present in 33 and Unclassified 10 samples respectively. Strains of Canlactobacilli 22 11/50 dida kefyr, Saccharomyces sp., KluyveroS. faecalis 6 4/50 myces sp., Rhodotorula rubra, Pichia S. faecium 18 7/50 S. faecium subsp. homerii, Hansenula sp. and Torulopsis casseliflavus 19 5/50 sp. were also isolated. S. durans 26 8/50 The 82 isolates of moulds were penicilP. pentosaceus 19 8/50 lin which were classified as PeniciUium Number of samples carrying the species/number commune. Nevertheless, in about 50% of the cheese samples pH ranged between 4.0-4-5 (mean 4.2 + 0.1) and the NaC1 content was <4.0% (mean 3.15 + 0.36). Generally the pH values were quite low, with minimum and m a xi m um values of 3.9 and 5.2 respectively. In 66% of the samples the values for moisture ranged between 60.0 and 70.0%. The low NaC1 and the high moisture content of certain cheese samples resulted in low (<5.5) brine concentration values in 22 samples (mean 4.30 __ 2.55). Whilst relatively high (>7.5) brine concentration values were observed in 16 samples (mean 9-6 + 3.77) due to the high NaC1 and the low moisture content of certain samples.
of samples examined.
Discussion
Identification of the isolates Isolates from MRS agar were classified as lactobacilli (347 isolates), pediococci (19 isolates) and enterococci (69 isolates)
The wide range of values in pH, % NaC1 and % moisture of Kopanisti cheese samples is due to the fact t hat the manufacturers do not follow the same
Microbiology of Kopanisti 255 Table 4. Yeast species isolated from Kopanisti cheese samples.
disappeared during cheese ripening due to unfavourable conditions in the cheese. LactobaciUus plantarum and L. casei, No. of which were the most frequently isolated samples strains, are known as salt resistant No. of carrying Species isolates the species lactobacilli (Rasic 1962) and the same applies to enterococci. The predominant Pichia membranespecies of lactic acid bacteria of Kopanfasciens 58 33/42a isti cheese are usually found in various Pichia fermentans 14 10/42 cheese varieties. Thus, L. casei, L. planCandida kefyr 3 2/42 Saccharomyces sp. 3 2/42 tarum and S. faecalis were previously Kluyveromyces sp. 2 1/42 found in Roquefort cheese (Devoyod Rhodotorula rubra 1 1/42 1969, Devoyod 1970). In addition, L. Pichia homerii 1 1/42 plantarum, L. casei, L. casei subsp. Hansenula sp. 1 1/42 rhamnosus L. brevis, S. faecalis and S. Torulopsis sp. 1 1/42 faecium were also isolated from Cabrales - Number of samples carrying the species/number cheese (Nunez and Medina 1979), where of yeast positive samples. L. plantarum predominated as in Kopanisti cheese. Yeasts were also probably favoured by method of production. Although there was a wide range of pH values, 75% of the cheese environment and their numthese were <5.0. This also applies to % bers were >103 g-1 in about 70% of the NaC1 and % moisture, where 70 and 88% positive samples. Pichia membraneof the values were <5.0 and <70-0% fasciens and P. fermentans were the predominant species accounting for 69% respectively. The combined effect of the low pH and 17% of the yeast isolates respecvalues (<5.2) and brine concentration tively. Both species were previously (>6-5% for the 44% of the samples) might found in Cabrales cheese (Nunez et al. 1981). Pichia membranefasciens was also be expected to influence the numbers and types of microorganisms t h a t finally isolated from Roquefort cheese (Devoyod occur in the cheese. Thus, coliforms were and Sponem 1970). Moulds were detected in relatively low not found in the 60% of the samples and numbers (101-103 g-l) in 63% of the their number in the positive samples was positive samples. In addition to lactic quite low. Micrococci and staphylococci acid bacteria and yeasts, the presence of were practically absent, since only one PeniciUium commune moulds in 92% of sample carried 2 Micrococcus luteus strains. In addition to the inhibitory samples suggests that they also belong to effect of pH and brine concentration, the essential microflora of Kopanisti antibacterial substances produced by cheese and t h a t they possibly contribute moulds possibly contributed to the dis- to ripening changes and flavour characteristics of this cheese. appearance of these microorganisms. Thus the results of the present study The cheese environment probably did not adversely affect the growth of lactic suggest t h a t a standard method of manuacid bacteria, since these organisms facture should be adopted for the production of this cheese. Experiments should were present in high numbers (>105 g-l) in 86% of the samples. Nevertheless, salt be conducted on the steps of its manufacsensitive lactic streptococci, which were ture so that the cheese can be stanprobably present in the fresh cheese, dardized and improved, but will not loose
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its traditional character. In addition, the use of a starter culture consisting of lactic acid bacteria, a penicillium and possibly a yeast is considered necessary. Thus, combinations of mesophilic streptococci (Streptococcus lactis, S. cremoris, S. diacetylactis), which will ensure mainly acidification and Lactobacillus casei, S. durans and Pichia membranefasciens which belong to the predominan t microflora of the cheese and possibly play an important role in its ripen-
ing and flavour characteristics should be preferred. Heterofermentative lactic acid bacteria (leuconostocs) which would provide the cheese with an open texture by production of gas, thus providing favourable conditions for the fungus, should also be tried. The possible role of the above microorganisms and Penicillium, possibly P. roqueforti, in cheese ripening and flavour compounds formation need to be studied.
References Baird-Parker, A. C. (1979) Methods for identifying staphylococci and micrococci. In Idehtification methods for microbiologists, 2nd edn. (Ed. Skinner and Lovelock) 201pp. London, Academic Press. Cowan, S. T. (Ed.) (1974) Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd edn. London, Cambridge University Press. Davis, J. G. (1976) Cheese, Vol. III, 879 pp. New York, Churchill Livingstone. Deibel, R. H. (1964) The group D streptococci. Bacteriol. Rev. 28, 330-366. Devoyod, J. J. (1969) La flore microbienne du fromage de Roquefort. IV Les enterocoques. Le Lait 49, 637-650. Devoyod, J. J. (1970) La flore microbienne du fromage de Roquefort. V Les lactobacilles. Le Lait 50, 277-284. Devoyod, J. J. and Sponem, D. (1970) La flore microbienne du fromage de Roquefort. VI Les levures. Le Lait 498, 524-543. Eekhof-Stork, N. (1976) The world atlas of cheese (Ed. Bailey, A.) 136pp. Paddington Press Ltd, USA. Facklam, R. R. (1972) Recognition of group D streptococcal species of human origin by biochemical and physiological tests. Appl. Microbiol. 23, 1131-1139. Federation International de Laiterie. FIL-IDF Normes Internationales 1972, 17A. Giinther, H. L. and White, H. R. (1961) The cultural and physiological characters of the pediococci. J. Gen. Microbiol. 26, 185-197. Lodder, J. (1971) The yeasts. A taxonomic study, 2nd edn. Amsterdam and London, North-Holland Publishing Company. Nunez, M. and Medina, M. (1979) La flore lactique du fromage bleu de Cabrales. Le Lait 59, 497-513. Nunez, M., Medina, M., Gaya, P. and Diaz-Amado, C. (1981) Les levures et les moisissures dans le fromage bleu de Cabrales. Le Lait 601/602, 62-79. Raper, K. and Thorn, C. (1949) A manual of the Penicillia. Baltimore, Williams & Wilkins Co. Rasic, J. (1962) A study of the resistance of lactic acid bacteria to sodium chloride. XVI International Dairy Congress B, 881-886. Saraswat, D. S., Clark, W. S., Jr. and Reinbold, G. W. (1963) Selection of a medium for the isolation and enumeration of enterococci in dairy products. J. Milk Food Technol. 26, 114-118. Sharpe, M. E. (1979) Identification of lactic acid bacteria. In Identification methods for microbiologists, 2nd edn. (Ed. Skinner and Lovelock) 233pp. London, Academic Press. Sharpe, M. E. and Fryer, T. F. (1965) Media for lactic acid bacteria. Lab. Practice, 697-701. Whittenbury, R. (1965) A study of some pediococci and their relationship to Aerococcus viridans and the enterococi. J. Gen. Microbiol. 40, 97-106. Zygouris, N. P. (1952) Milk industry, 2nd edn. (Ed. Ministry of Agriculture) 430pp. Athens.