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Microbiological study of white-brined cheese made from raw goat milk
Food Microbiology,
1992,9,
Microbiological raw goat milk
study
E. Litopoulou-Tzanetaki*
13-19
of white-brined
cheese
made from
and N. Tzanetakis
Laboratory of Dairy Technology, Faculty of Agriculture, Thessaloniki, Thessaloniki 54006, Greece Received 16 October 1990
University
of
The microflora of a white-brined cheese made from raw goat milk was studied during a go-day ripening period. High counts of aerobic bacteria, lactic acid bacteria, psychrotrophs, proteolytic and lipolytic bacteria were recorded throughout ripening of the cheese. After 15 days, lactic acid bacteria predominated. Low pH levels (4.5) and high NaCl content (brine concentration 5.8-6.270) after 75 days affected the growth of most microbial groups resulting in considerably lower counts at 3 months. Leuconostocs were the most frequently found group in curd, lactococci dominated at 15 days and following this lactobacilli dominated over the other lactic acid bacteria. Lactobacillus plantarum, Lactococcus lactis subsp. lactis and Enterococcus faecium were the predominant species.
Introduction Goat milk produced in Greece (440-460 million 1 year’) is either used mixed with ewe’s milk for cheese production or for the manufacture of a traditional type of white-pickled cheese from raw goat milk at small creameries between December and June. The use of raw milk leads to unpredictable chemical or biological changes during manufacture and ripening. In addition, because the cheese is made without a starter culture quality is frequently indifferent. The natural lactic microflora of raw goat milk used for cheese production may play an important role in the manufacture of white-brined cheese. The main species of lactic acid bacteria isolated from raw goat cheese milk are Enterococcus durans, Lactobacillus plantarum and Leuconostoc paramesenteroides. In addition, enterococci are the most frequently isolated group in spring and summer, together with a high population of lactobacilli (Tzanetakis and 0740-0020/92/010013
+ 07 $02.00/O
Litopoulou-Tzanetaki 1989). This natural lactic microflora along with other contaminants may contribute to ripening changes in the cheese through synergistic influences (Law et al. 1976). This study describes microbiological changes that occured during manufacture and ripening of a white-brined cheese made from raw goat’s milk with special reference to changes of lactic acid bacteria.
Materials
and Methods
Cheese Cheese obtained from two small creameries in Northern Greece was manufactured as follows: The milk was filtered through cheese cloths and was then renneted at 32°C with 30 ml of calf rennet per 100 1 of milk as usual for white brined cheeses (Haddadin, 1982). The coagulum was taken from the vat in thin slices with a ladle and put into a deep metal mould lined with cheese cloth, where it was left to drain until the next morning; the curd was then turned and left overnight. The drained curd was cut into pieces (8 x 15 0 1992 Academic
Press
Limited
14
E. Litopoulou-Tzanetaki
and N. Tzanetakis
x 15 ems) and these were put into tins. The tins were filled with 15% brine and were then transferred in cool rooms (about 15°C) for 1 month; cheese ripening was completed in cold stores at 2-3°C. Four lots of cheese were manufactured and examined from February to June; curd and cheese at 15, 75 and 90 days of ripening were analyzed. Microbiological and chemical analyses Cheese samples were diluted by the dilution pour-plate method. Total aerobic counts were made on plate count agar (Oxoid Ltd, UK) after incubation at 30°C for 72 h, and coliform counts on violet red bile agar toxoid Ltd.) after incubation at 30°C for 24 h. Staphylococci were enumerated on BairdParker medium (Oxoid Ltd) after incubation at 37°C for 48 h, yeasts on acidified potato dextrose agar (Oxoid Ltd) after incubation at 22°C for 5 days, psychrotrophs on plate count agar incubated at 7°C for 10 days, proteolytic bacteria on plate count agar, supplemented with 10% reconstituted skim-milk, incubated at 22°C for 5 days, and lipolytic bacteria on tributyrin agar (Oxoid Ltd) after incubation at 30°C for 72 h. Lactic acid bacteria were enumerated on MRS agar (Oxoid Ltd) adjusted to pH5.5 so that the growth of other organisms could be supressed (Kitchell and Shaw 1975; Garcia et al. 1987) and lactobacilli on acetate agar (Rogosa et al. 1951) after incubation at 30°C for 5 days in anaerobic jars (Gas-Pack anaerobic system, BBL). Enterococci were enumerated on citrate azide agar (Saraswat et al. 1963) after incubation at 37°C for 72 h and lactococci were grown on the arginine medium of Turner et al. (1962) incubated at 30°C for 48 h. The pH of the cheese was determined electrometrically, moisture by heating at 102°C to constant weight and NaCl content according to the International Dairy Federation method (1972). Salt content was expressed as brine concentration. Analyses of variance were performed on data obtained at different stages after a log transformation for bacterial counts. Significance of differences between means was assessed by the Student-Newman-Keul’s multiple range test (Steel and Torrie 1980). Isoktion and identification of the isolates Colonies (15-30 per sample) were picked at random from acetate agar, arginine medium
and citrate azide agar plates after purification maintained in yeast dextrose litmus milk + chalk (Sharpe and Fryer 1965) at 4°C. Gram-positive, catalase-negative cocci in pairs and chains were identified using the methods and criteria of Sharpe and Fryer (1965), Schleifer et al. (1985), Schleifer and Kilpper-Balz (1984), Collins et al. (1984) and Garvie (1985). Gram-positive, catalase-negative rods were characterized according to the methods and criteria of Sharpe and Fryer (1965), Sharpe (1981) and Collins et al. (1989). Results
and Discussion
High counts of aerobic bacteria, lactic acid bacteria, psychrotrophs, proteolytic and lipolytic bacteria were recorded throughout ripening of the cheese (Table 1). Low incidences of coliforms as well as yeasts were detected in the curd of the cheese (mean log counts 3.04 and I.99, respectively), although it was made from raw goat’s milk and incidence were well below (respective means 5.65 and 5.14) those reported by Del Pozo et al. (1988) for curd from raw ewe’s milk. Levels of staphylococci and enterococci were also lower by 1 and 2 log units, respectively, than those reported for curd from ewe’s milk (Del Pozo et al. 1988). The significant decrease in pH during the first 15 days of ripening (Table 2) may be considered as a consequence of acid production by the increasing microbial populations. Levels of most microbial groups increased by 1.6-2.9 log units during ripening for 15 days, when coliforms, staphylococci and lipolytic bacteria reached their peak growth (respective mean log counts 5.95, 5.36 and 7.46). Staphylococci increased by only 0.3 log units, probably due to the combined inhibitory effect of pH and brine concentration (Litopoulou-Tzanetaki 1977). Numbers of yeasts also increased slightly (0.2 log units), even
Microbiology, Table 1. Mean log plate counts of different and ripening of white-brined cheese made
microbial from raw
groups during goat milk.
Mean
15
manufacture
log plate counts
Ripening Microbial
goat milk cheese
group
Curd
15
Total aerobic count Coliforms Staphylococci Lactic acid bacteria Lactobacilli Enterococci Yeasts Psychrotrophs Proteolytic Lipolytic
5.82a 3.04 5.07 6.12 6.04 4.26” 1.99= 5.96 5.83” 5.61
8.61b 5.95 5.36 8.20 7.66 6.88b 2.22b 7.66 7.45b 7.46
time (days) 75
90
8.74b 3.50 3.56 8.55 7.82 6.92” 3.84b 7.49 7.78b 7.38
6.95ab 1.96 2.35 6.65 5.96 6.69” 3.98” 6.96 6.59” 5.97
Values are the means of four lots of cheese. ilJlMeans
for the same organism
with
different
superscripts
though pH decreased to levels favourable for their growth; this suggests the possible presence of salt sensitive species. Numbers of total counts, coliforms, enterococci and micro-organisms on MRS agar form the cheese at 15 days were similar to those previously reported for a goat milk cheese at 10 days (Fatichenti et al. 1979); yeasts, however, were recorded at considerably lower levels in our cheese. A mean pH value in the range 4.15-450, from 15 to 90 days and an increasing brine concentration up to
differ
significantly
P
6.21% in go-day-old cheese (Table 2) may be considered unfavourable for most microbial groups (Table 1). The combined effect of pH and brine concentration caused a rapid reduction in levels of staphylococci (Litopoulou-Tzanetaki 1977). It also seems that high lactic acid levels affected the growth of coliforms; these organisms are unaffected by the salt concentration in white brined cheese (Yanai et al. 1977). In contrast, total aerobic counts reached their maxima at 75 days (mean log count 8.74) and the same was observed
Table 2. Mean values of pH, moisture and brine concentration during ture and ripening of white-brined cheese made from raw goat milk. Ripening Characteristic PH Moisture NaCl (c7r) Brine concentration
f%)
time (days)
Curd
15
75
90
6.30a -
5aib 58.0 3.1 5.1
4.53b 59.4 3.7 5.8
4.5ob 56.8 3.8 6.2
NaCl %
Values are the mean of four lots of cheese. Brine concentration
=
i’J]Means
NaCl % + moisture Q differ significantly (P
of the same characteristic
with
manufac-
different
superscripts
x 100.
16
E. Litopoulou-Tzanetaki
and N. Tzanetakis
with lactic acid bacteria (mean log counts: lactic acid bacteria, 855; lactobacilli, 7.82; enterococci, 6.92;) and proteolytic bacteria (mean count 7.78). Numbers of yeasts increased and reached their highest levels at 90 days and it seems possible that this microbial group was not adversely affected by the high salt levels of the cheese, probably due to the predominance of salt-resistant species (Mansour and Alais 1973). Nevertheless, yeasts did not belong to the predominant microflora in go-dayold cheese which contrasts with results previously obtained for white brined cheese (Mansour and Alais 1973). Psychrotrophs were not severely affected by the inhibitory effects of pH and salt content, though from 15 days onwards a Table 3. Changes in species cheese made from raw goat
of lactic milk.
Species
acid bacteria
during
Curd
15 days
strains (%)
strains (%)
No. L. casei L. rhamnosus L. casei subsp.alactosus L. paracacei subsp.paracasei L. paracacei subsp.tolerans L. plantarum L. confusus L. brevis L. buchneri L. cellobiosus Lact. lactis subsp.lactis Lact. lactis subsp.lactis
slight decrease in their number was observed. However, their high counts were not accompanied by any defect in the cheese, suggesting the presence of biochemically active, Gram-negative bacteria at high levels. Lactic acid bacteria were the predominant microbial group throughout ripening of the cheese (Table 1). Although leuconostocs predominated in curd, lactococci (Lactococcus Zactis) were found more frequently (31.1% of the isolates) than lactobacilli, enterococci and/or leuconostocs in 15day-old cheese; however, their proportion diminished rapidly with ageing (Table 3). At the end of the ripening period, 61.5% of the isolates were lactobacilli, 1.6% lactococci, 28.7% enterococci and 8.2% leuconostocs.
No.
-
1 1
t 13 1 2 1 7 -
1.1 1.1 3.4 14.8 1.1 2.3 1.1 8.0
ripening
of white-brined
75 days No.
No.
strains (c/c1
0.8
4 1 1 7
5.1
90 days
2.9 0.8 0.8 5.1
strains (%) 1
0.8 10.7 0.8 47.6
-
%6 1
30.3 0.8
;O -
14.5
13 1 58 2
7 15 4 8 4
5.9 12.6 3.4 6.7 3.4
13 8 18
9.5 5.8 13.1
8 15 12
6.6 12.3 9.8
5
3.7
2
1.6
8
6.7 1.7
5 8
3.7 5.9
4 4
3.3 3.3
7
0.8
2
1.6
2 25 1 -
21.0 0.8
45 2
32.6 1.6
1.6
biov. diacetylactis Ent. faecalis Ent. faecium Ent. durans Leuc. lactis Leuc. paramesenteroides Leuc. mesenteroides subsp. mesenteroides Leuc. dextranicum Leuc. cremoris Unidentified lactobacilli
1 18 9 7 6
Total
88
1.1 20.5 10.2 8.0 6.8 9.1 8.0 2.3 1.1
2
-
1
Microbiology, Thus, it seems that salt-resistant lactic acid bacteria predominated in the cheese as ripening progressed. Lactococci and the leuconostocs lactis and creare salt-sensitive organisms moris (Nunez and Medina 1979; Sharpe 1979) and it seems that pH and NaCl contributed to their rapid decline in the cheese as ripening progressed. Shehata et al. (1975) also observed that Lact. Zactis disappeared after 30 days of ripening in Domiati cheese. In contrast, Lombadze et al. (1976) found that lactic streptococci constituted 95-989 of the lactic microflora during ripening of Domiati cheese. Lactobacilli predominated in 75day-old cheese (Table 1) and they may play an important role in the manufacture of white-brined cheeses (Rasic 1962a); salt-tolerant species multiply and predominate in mature brined cheese (Yanay et al. 1977). Lactobacillus plantarum, which was the dominant lactobacillus species throughout ripening of goat milk cheese, is salt resistant (Rasic 1962b) and represented 14.8%, 21.0%, 32.6 and 47.6% of the isolates in curd from 15, 75 and go-day-old cheese, respectively (Table 3). The same species was also found to predominate in Domiati cheese (Shehata et al. 1975) and in Bulgarian white-brined cheeses made from raw cow’s and ewe’s milk during the first month of ripening (Haddadin 1982). L. paracasei subsp. paracasei was also isolated with increased frequency as ripening progressed and constituted
goat milk cheese
17
10.7% of the isolates at the end of the ripening period (Table 3). Enterococci appeared to be an important group in the microflora of our cheese (Table 1); this agrees with previously reported results on white-brined cheese (Yanai et al. 1977). Enterococcus faecium was the most frequently isolated species in curd (20.5% of the isolates) and dominated over E. faecalis and E. durans in curd from 15 and go-day-old cheese (Table 3). Results from the present study suggest that lactic acid bacteria predominate over the other microbial groups during ripening of a white-brined cheese made from raw goat milk and thus may regulate the ripening changes in the cheese. Non-uniform quality, flavour abnormalities and texture problems arising from the use of traditional methods for cheese production could be solved effectively by the use of a specific starter. The results of this study suggest that combinations of lactococci, which will ensure mainly acidification of Lactobacillus casei and E. durans can be tried as starter for cheese production. Considering open texture as a problem for this cheese, heterofermentative lactic acid bacteria (leuconostocs) could probably be used, but at very low levels in starter combinations. Acknowledgements The authors acknowledge the financial assistance provided by the EEC.
References Collins, M. D., Jones, D., Far-row, J. A. E., Kilpper-Balz, R. and Schleifer, K. H. (1984) Enterococcus avium nom, rev. comb. nov.; E. casseliflavus nom. rev. comb. nov., E. durans nom. rev. comb. nov.; E. gallinarum comb. nov. and E. malodoratus sp nov. Znt. J. Syst. Bacterial. 34, 220-223. Collins, M. D., Phillips, B. A. and Zanoni, P. (1989) Deoxyribonucleic acid homology studies of Lactobacillus casei. Lactobacillus paracasei sp. nov., subsp. paracasei and subsp. tolerans, and Lactobacillus rhamnosus sp. nov., comb nov. Int. J. Syst. Bacterial. 39, 105-108. Del Pozo, B. F., Gaya, P., Medina, M., Rodriquez-Marin, M. A. and Nunez, M. (1988) Changes in the microflora of La Serena ewe’s milk cheese during ripening. J. Dairy Res. 55.449-455.
18
E. Litopoulou-Tzanetaki
and N. Tzanetakis
Fatjchenti, F., Deiana, P., Farris, G. A. and Soggia, G. (1979) Etudes microbiologiques sur le lait et le fromage de chevre en Sardaigne. Note II: streptocoques, lactobacilles et leuconostot. Le Lait 587, 387-400. Garcia, M. C., Oteco, A., Garcia, M. L. and Moreno, B. (1987) Microbiological quality and compositionof two types of Spanish sheep’s milk cheeses (Manchego and Burgos varieties). J. Dairy Res. 54, 551-557. Garvie, E. I. (1985) Genus Leuconostoc. In Bergey’s manual of systematic bacteriology (Eds Sneath, P. H. A., Mair, S., Sharpe, M. E. and Holt, J. C.) 9th edn, pp. 1071-1075. Baltimore, The Williams and Wilkins Co. Haddadin, M. S. Y. (1982). Microbiology of white-brined cheeses. In Decelopments in food microbiology-2 (Ea. Robinson, R. K.) pp. 67-89. London, Elsevier Applied Science publishers. International Dairy Federation (1972) Cheese: determination of chloride content (reference method) FIGIDF Standard 17A. Brussels. Kitchell, A. G. and Shaw, B. (1975) Classification problems ecology and some biochemical activities of lactobacilli of meat products. In Lactic acid bacteria in beverages and foods. (Eds Carr, J. G., Cutting, C. V. and Whiting, G. C.) pp. 209-220. London, Academic Press. Law, B. A., Castanon, M. and Sharpe, M. E. (1976) The effect of non-starter bacteria on the chemical composition and the flavour of Cheddar cheese. J. Dairy Res. 43, 117-125. Litopoulou-Tzanetaki, E. (1977) Staphylococci and micrococci in Kefalotryri cheese. Milchwissenschaft 32, 211-213. Lombadze, R. N., Mamatelashvili, G. S., Purtseladze, N. G., De Murishvili, L. I. and Mandzhavidze, N. P. (1976) Molochnaya Promyshlennost 12, 13-15. From Haddadin, S. Y. (1986) Microbiology of white-brined cheeses. In Developments in food microbiology-2 (Ed. R. K. Robinson) pp. 67-69. London, Elsevier Applied Science Publishers. Mansour, A. and Alais C. (1973) Etude du salage et de I’affinage du fromage en saumure. III. Aspect Bacteriologique. Le Lait 523-524, 137-145. Nunez, M. and Medina, M. (1979) La flore lactique du fromage bleu de Cabrales. Le‘Lait 59, 497-513. Rasic, J. (1962a) Trends of bacterial population during the manufacture and ripening of white cheese. XVI Int. Dairy Cong. Section IV, 2, 840-845. Rasic, J. (196213) A study of the resistance of lactic acid bacteria to sodium chloride. XVI In t. Dairy Cong. B, 881-886. Rogosa, M., Mitchell, J. A. and Wiseman, R. F. (1951) A selective medium for the isolation of oral and faecal lactobacilli. J. Bacterial. 62, 132-133. 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. Milh Food Technol. 26, 114-118. Schleifer, K. H. and Kilpper-Balz, R. ( 1984 I Transfer of Streptococcus faecalis and StreptoCOCCUS faeciurn to the Genus Enterococcus nom. rev. as Enterococcus faecalis comb. no\‘. and Enterococcus faecium comb. nov. Int. J. Syst. Bacterial. 34, 31-34. Schleifer, K. H., Kraus, J., Dvorak, C., Kilpper-Balz, R., Collins, M. D. and Fisher, W. (1985) Transfer of Streptococcus lactis and related streptococci to the genus Lactococcus gen. nov. Syst. Appl. Microbial. 6, 183-195. Sharpe, M. E. (1979) Identification of lactic acid bacteria. In Identification methocls for microbiologists, 2nd edn (Eds Skinner, F. A. and Lovelock, D. W.). 233 pp. London, Academic Press. Shave, M. E. (1981) The genus Lactobacillus. In The prokaryotes, Vol. II. (Eds Stan, M. P., Stolp, H., Truper, H. G. Balouis, J. A. and Schlegel, H. G.). pp. 1614-1630. New York, Springer-Verlag. Sharpe, M. E. and Fryer, T. F. ( 1965) Media for lactic acid bacteria. Lab. Practice 697-701. Shehata, A. E., El-Sadek, G. M., Khalafalia, S. M. and El-Magdoub, M. N. (1975) Egyptian J. Dairy Sci. 3, 139-144. From Haddadin, S. Y. C. (1986) Microbiology of white-brined cheese. In Developments in food microbiology-2 (Ed. Robinson, R. K.) pp. 67-89. London, Elsevier Applied Science Publishers. Steel, R. G. D. and Torrie, J. H. (1980) Principles and procedures of statistics. 2nd ed. New York, N. Y., McGraw-Hill Book Co., Inc.
Microbiology,
goat milk cheese
19
Turner, N., Sandine, W. E. and Elliker, P. R. (1962) An agar medium for differentiating Streptococcus lactis and Streptococcus cremoris. J. Dairy Sci. 45, 665. Tzanetakis, N. and Litopoulou-Tzanetaki, E. (1988) Lactic acid bacteria in raw goat milk and some of their biochemical properties. Microbial.-Aliments-Nutr. 7, 73-80. Yanai, Y., Rosen, B., Pinoky, A. and Sklan, D. (1977) The microbiology of pickled cheese during manufacture and maturation. J. Dairy Res. 44, 149-153.
1992,9,
Microbiological raw goat milk
study
E. Litopoulou-Tzanetaki*
13-19
of white-brined
cheese
made from
and N. Tzanetakis
Laboratory of Dairy Technology, Faculty of Agriculture, Thessaloniki, Thessaloniki 54006, Greece Received 16 October 1990
University
of
The microflora of a white-brined cheese made from raw goat milk was studied during a go-day ripening period. High counts of aerobic bacteria, lactic acid bacteria, psychrotrophs, proteolytic and lipolytic bacteria were recorded throughout ripening of the cheese. After 15 days, lactic acid bacteria predominated. Low pH levels (4.5) and high NaCl content (brine concentration 5.8-6.270) after 75 days affected the growth of most microbial groups resulting in considerably lower counts at 3 months. Leuconostocs were the most frequently found group in curd, lactococci dominated at 15 days and following this lactobacilli dominated over the other lactic acid bacteria. Lactobacillus plantarum, Lactococcus lactis subsp. lactis and Enterococcus faecium were the predominant species.
Introduction Goat milk produced in Greece (440-460 million 1 year’) is either used mixed with ewe’s milk for cheese production or for the manufacture of a traditional type of white-pickled cheese from raw goat milk at small creameries between December and June. The use of raw milk leads to unpredictable chemical or biological changes during manufacture and ripening. In addition, because the cheese is made without a starter culture quality is frequently indifferent. The natural lactic microflora of raw goat milk used for cheese production may play an important role in the manufacture of white-brined cheese. The main species of lactic acid bacteria isolated from raw goat cheese milk are Enterococcus durans, Lactobacillus plantarum and Leuconostoc paramesenteroides. In addition, enterococci are the most frequently isolated group in spring and summer, together with a high population of lactobacilli (Tzanetakis and 0740-0020/92/010013
+ 07 $02.00/O
Litopoulou-Tzanetaki 1989). This natural lactic microflora along with other contaminants may contribute to ripening changes in the cheese through synergistic influences (Law et al. 1976). This study describes microbiological changes that occured during manufacture and ripening of a white-brined cheese made from raw goat’s milk with special reference to changes of lactic acid bacteria.
Materials
and Methods
Cheese Cheese obtained from two small creameries in Northern Greece was manufactured as follows: The milk was filtered through cheese cloths and was then renneted at 32°C with 30 ml of calf rennet per 100 1 of milk as usual for white brined cheeses (Haddadin, 1982). The coagulum was taken from the vat in thin slices with a ladle and put into a deep metal mould lined with cheese cloth, where it was left to drain until the next morning; the curd was then turned and left overnight. The drained curd was cut into pieces (8 x 15 0 1992 Academic
Press
Limited
14
E. Litopoulou-Tzanetaki
and N. Tzanetakis
x 15 ems) and these were put into tins. The tins were filled with 15% brine and were then transferred in cool rooms (about 15°C) for 1 month; cheese ripening was completed in cold stores at 2-3°C. Four lots of cheese were manufactured and examined from February to June; curd and cheese at 15, 75 and 90 days of ripening were analyzed. Microbiological and chemical analyses Cheese samples were diluted by the dilution pour-plate method. Total aerobic counts were made on plate count agar (Oxoid Ltd, UK) after incubation at 30°C for 72 h, and coliform counts on violet red bile agar toxoid Ltd.) after incubation at 30°C for 24 h. Staphylococci were enumerated on BairdParker medium (Oxoid Ltd) after incubation at 37°C for 48 h, yeasts on acidified potato dextrose agar (Oxoid Ltd) after incubation at 22°C for 5 days, psychrotrophs on plate count agar incubated at 7°C for 10 days, proteolytic bacteria on plate count agar, supplemented with 10% reconstituted skim-milk, incubated at 22°C for 5 days, and lipolytic bacteria on tributyrin agar (Oxoid Ltd) after incubation at 30°C for 72 h. Lactic acid bacteria were enumerated on MRS agar (Oxoid Ltd) adjusted to pH5.5 so that the growth of other organisms could be supressed (Kitchell and Shaw 1975; Garcia et al. 1987) and lactobacilli on acetate agar (Rogosa et al. 1951) after incubation at 30°C for 5 days in anaerobic jars (Gas-Pack anaerobic system, BBL). Enterococci were enumerated on citrate azide agar (Saraswat et al. 1963) after incubation at 37°C for 72 h and lactococci were grown on the arginine medium of Turner et al. (1962) incubated at 30°C for 48 h. The pH of the cheese was determined electrometrically, moisture by heating at 102°C to constant weight and NaCl content according to the International Dairy Federation method (1972). Salt content was expressed as brine concentration. Analyses of variance were performed on data obtained at different stages after a log transformation for bacterial counts. Significance of differences between means was assessed by the Student-Newman-Keul’s multiple range test (Steel and Torrie 1980). Isoktion and identification of the isolates Colonies (15-30 per sample) were picked at random from acetate agar, arginine medium
and citrate azide agar plates after purification maintained in yeast dextrose litmus milk + chalk (Sharpe and Fryer 1965) at 4°C. Gram-positive, catalase-negative cocci in pairs and chains were identified using the methods and criteria of Sharpe and Fryer (1965), Schleifer et al. (1985), Schleifer and Kilpper-Balz (1984), Collins et al. (1984) and Garvie (1985). Gram-positive, catalase-negative rods were characterized according to the methods and criteria of Sharpe and Fryer (1965), Sharpe (1981) and Collins et al. (1989). Results
and Discussion
High counts of aerobic bacteria, lactic acid bacteria, psychrotrophs, proteolytic and lipolytic bacteria were recorded throughout ripening of the cheese (Table 1). Low incidences of coliforms as well as yeasts were detected in the curd of the cheese (mean log counts 3.04 and I.99, respectively), although it was made from raw goat’s milk and incidence were well below (respective means 5.65 and 5.14) those reported by Del Pozo et al. (1988) for curd from raw ewe’s milk. Levels of staphylococci and enterococci were also lower by 1 and 2 log units, respectively, than those reported for curd from ewe’s milk (Del Pozo et al. 1988). The significant decrease in pH during the first 15 days of ripening (Table 2) may be considered as a consequence of acid production by the increasing microbial populations. Levels of most microbial groups increased by 1.6-2.9 log units during ripening for 15 days, when coliforms, staphylococci and lipolytic bacteria reached their peak growth (respective mean log counts 5.95, 5.36 and 7.46). Staphylococci increased by only 0.3 log units, probably due to the combined inhibitory effect of pH and brine concentration (Litopoulou-Tzanetaki 1977). Numbers of yeasts also increased slightly (0.2 log units), even
Microbiology, Table 1. Mean log plate counts of different and ripening of white-brined cheese made
microbial from raw
groups during goat milk.
Mean
15
manufacture
log plate counts
Ripening Microbial
goat milk cheese
group
Curd
15
Total aerobic count Coliforms Staphylococci Lactic acid bacteria Lactobacilli Enterococci Yeasts Psychrotrophs Proteolytic Lipolytic
5.82a 3.04 5.07 6.12 6.04 4.26” 1.99= 5.96 5.83” 5.61
8.61b 5.95 5.36 8.20 7.66 6.88b 2.22b 7.66 7.45b 7.46
time (days) 75
90
8.74b 3.50 3.56 8.55 7.82 6.92” 3.84b 7.49 7.78b 7.38
6.95ab 1.96 2.35 6.65 5.96 6.69” 3.98” 6.96 6.59” 5.97
Values are the means of four lots of cheese. ilJlMeans
for the same organism
with
different
superscripts
though pH decreased to levels favourable for their growth; this suggests the possible presence of salt sensitive species. Numbers of total counts, coliforms, enterococci and micro-organisms on MRS agar form the cheese at 15 days were similar to those previously reported for a goat milk cheese at 10 days (Fatichenti et al. 1979); yeasts, however, were recorded at considerably lower levels in our cheese. A mean pH value in the range 4.15-450, from 15 to 90 days and an increasing brine concentration up to
differ
significantly
P
6.21% in go-day-old cheese (Table 2) may be considered unfavourable for most microbial groups (Table 1). The combined effect of pH and brine concentration caused a rapid reduction in levels of staphylococci (Litopoulou-Tzanetaki 1977). It also seems that high lactic acid levels affected the growth of coliforms; these organisms are unaffected by the salt concentration in white brined cheese (Yanai et al. 1977). In contrast, total aerobic counts reached their maxima at 75 days (mean log count 8.74) and the same was observed
Table 2. Mean values of pH, moisture and brine concentration during ture and ripening of white-brined cheese made from raw goat milk. Ripening Characteristic PH Moisture NaCl (c7r) Brine concentration
f%)
time (days)
Curd
15
75
90
6.30a -
5aib 58.0 3.1 5.1
4.53b 59.4 3.7 5.8
4.5ob 56.8 3.8 6.2
NaCl %
Values are the mean of four lots of cheese. Brine concentration
=
i’J]Means
NaCl % + moisture Q differ significantly (P
of the same characteristic
with
manufac-
different
superscripts
x 100.
16
E. Litopoulou-Tzanetaki
and N. Tzanetakis
with lactic acid bacteria (mean log counts: lactic acid bacteria, 855; lactobacilli, 7.82; enterococci, 6.92;) and proteolytic bacteria (mean count 7.78). Numbers of yeasts increased and reached their highest levels at 90 days and it seems possible that this microbial group was not adversely affected by the high salt levels of the cheese, probably due to the predominance of salt-resistant species (Mansour and Alais 1973). Nevertheless, yeasts did not belong to the predominant microflora in go-dayold cheese which contrasts with results previously obtained for white brined cheese (Mansour and Alais 1973). Psychrotrophs were not severely affected by the inhibitory effects of pH and salt content, though from 15 days onwards a Table 3. Changes in species cheese made from raw goat
of lactic milk.
Species
acid bacteria
during
Curd
15 days
strains (%)
strains (%)
No. L. casei L. rhamnosus L. casei subsp.alactosus L. paracacei subsp.paracasei L. paracacei subsp.tolerans L. plantarum L. confusus L. brevis L. buchneri L. cellobiosus Lact. lactis subsp.lactis Lact. lactis subsp.lactis
slight decrease in their number was observed. However, their high counts were not accompanied by any defect in the cheese, suggesting the presence of biochemically active, Gram-negative bacteria at high levels. Lactic acid bacteria were the predominant microbial group throughout ripening of the cheese (Table 1). Although leuconostocs predominated in curd, lactococci (Lactococcus Zactis) were found more frequently (31.1% of the isolates) than lactobacilli, enterococci and/or leuconostocs in 15day-old cheese; however, their proportion diminished rapidly with ageing (Table 3). At the end of the ripening period, 61.5% of the isolates were lactobacilli, 1.6% lactococci, 28.7% enterococci and 8.2% leuconostocs.
No.
-
1 1
t 13 1 2 1 7 -
1.1 1.1 3.4 14.8 1.1 2.3 1.1 8.0
ripening
of white-brined
75 days No.
No.
strains (c/c1
0.8
4 1 1 7
5.1
90 days
2.9 0.8 0.8 5.1
strains (%) 1
0.8 10.7 0.8 47.6
-
%6 1
30.3 0.8
;O -
14.5
13 1 58 2
7 15 4 8 4
5.9 12.6 3.4 6.7 3.4
13 8 18
9.5 5.8 13.1
8 15 12
6.6 12.3 9.8
5
3.7
2
1.6
8
6.7 1.7
5 8
3.7 5.9
4 4
3.3 3.3
7
0.8
2
1.6
2 25 1 -
21.0 0.8
45 2
32.6 1.6
1.6
biov. diacetylactis Ent. faecalis Ent. faecium Ent. durans Leuc. lactis Leuc. paramesenteroides Leuc. mesenteroides subsp. mesenteroides Leuc. dextranicum Leuc. cremoris Unidentified lactobacilli
1 18 9 7 6
Total
88
1.1 20.5 10.2 8.0 6.8 9.1 8.0 2.3 1.1
2
-
1
Microbiology, Thus, it seems that salt-resistant lactic acid bacteria predominated in the cheese as ripening progressed. Lactococci and the leuconostocs lactis and creare salt-sensitive organisms moris (Nunez and Medina 1979; Sharpe 1979) and it seems that pH and NaCl contributed to their rapid decline in the cheese as ripening progressed. Shehata et al. (1975) also observed that Lact. Zactis disappeared after 30 days of ripening in Domiati cheese. In contrast, Lombadze et al. (1976) found that lactic streptococci constituted 95-989 of the lactic microflora during ripening of Domiati cheese. Lactobacilli predominated in 75day-old cheese (Table 1) and they may play an important role in the manufacture of white-brined cheeses (Rasic 1962a); salt-tolerant species multiply and predominate in mature brined cheese (Yanay et al. 1977). Lactobacillus plantarum, which was the dominant lactobacillus species throughout ripening of goat milk cheese, is salt resistant (Rasic 1962b) and represented 14.8%, 21.0%, 32.6 and 47.6% of the isolates in curd from 15, 75 and go-day-old cheese, respectively (Table 3). The same species was also found to predominate in Domiati cheese (Shehata et al. 1975) and in Bulgarian white-brined cheeses made from raw cow’s and ewe’s milk during the first month of ripening (Haddadin 1982). L. paracasei subsp. paracasei was also isolated with increased frequency as ripening progressed and constituted
goat milk cheese
17
10.7% of the isolates at the end of the ripening period (Table 3). Enterococci appeared to be an important group in the microflora of our cheese (Table 1); this agrees with previously reported results on white-brined cheese (Yanai et al. 1977). Enterococcus faecium was the most frequently isolated species in curd (20.5% of the isolates) and dominated over E. faecalis and E. durans in curd from 15 and go-day-old cheese (Table 3). Results from the present study suggest that lactic acid bacteria predominate over the other microbial groups during ripening of a white-brined cheese made from raw goat milk and thus may regulate the ripening changes in the cheese. Non-uniform quality, flavour abnormalities and texture problems arising from the use of traditional methods for cheese production could be solved effectively by the use of a specific starter. The results of this study suggest that combinations of lactococci, which will ensure mainly acidification of Lactobacillus casei and E. durans can be tried as starter for cheese production. Considering open texture as a problem for this cheese, heterofermentative lactic acid bacteria (leuconostocs) could probably be used, but at very low levels in starter combinations. Acknowledgements The authors acknowledge the financial assistance provided by the EEC.
References Collins, M. D., Jones, D., Far-row, J. A. E., Kilpper-Balz, R. and Schleifer, K. H. (1984) Enterococcus avium nom, rev. comb. nov.; E. casseliflavus nom. rev. comb. nov., E. durans nom. rev. comb. nov.; E. gallinarum comb. nov. and E. malodoratus sp nov. Znt. J. Syst. Bacterial. 34, 220-223. Collins, M. D., Phillips, B. A. and Zanoni, P. (1989) Deoxyribonucleic acid homology studies of Lactobacillus casei. Lactobacillus paracasei sp. nov., subsp. paracasei and subsp. tolerans, and Lactobacillus rhamnosus sp. nov., comb nov. Int. J. Syst. Bacterial. 39, 105-108. Del Pozo, B. F., Gaya, P., Medina, M., Rodriquez-Marin, M. A. and Nunez, M. (1988) Changes in the microflora of La Serena ewe’s milk cheese during ripening. J. Dairy Res. 55.449-455.
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E. Litopoulou-Tzanetaki
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Fatjchenti, F., Deiana, P., Farris, G. A. and Soggia, G. (1979) Etudes microbiologiques sur le lait et le fromage de chevre en Sardaigne. Note II: streptocoques, lactobacilles et leuconostot. Le Lait 587, 387-400. Garcia, M. C., Oteco, A., Garcia, M. L. and Moreno, B. (1987) Microbiological quality and compositionof two types of Spanish sheep’s milk cheeses (Manchego and Burgos varieties). J. Dairy Res. 54, 551-557. Garvie, E. I. (1985) Genus Leuconostoc. In Bergey’s manual of systematic bacteriology (Eds Sneath, P. H. A., Mair, S., Sharpe, M. E. and Holt, J. C.) 9th edn, pp. 1071-1075. Baltimore, The Williams and Wilkins Co. Haddadin, M. S. Y. (1982). Microbiology of white-brined cheeses. In Decelopments in food microbiology-2 (Ea. Robinson, R. K.) pp. 67-89. London, Elsevier Applied Science publishers. International Dairy Federation (1972) Cheese: determination of chloride content (reference method) FIGIDF Standard 17A. Brussels. Kitchell, A. G. and Shaw, B. (1975) Classification problems ecology and some biochemical activities of lactobacilli of meat products. In Lactic acid bacteria in beverages and foods. (Eds Carr, J. G., Cutting, C. V. and Whiting, G. C.) pp. 209-220. London, Academic Press. Law, B. A., Castanon, M. and Sharpe, M. E. (1976) The effect of non-starter bacteria on the chemical composition and the flavour of Cheddar cheese. J. Dairy Res. 43, 117-125. Litopoulou-Tzanetaki, E. (1977) Staphylococci and micrococci in Kefalotryri cheese. Milchwissenschaft 32, 211-213. Lombadze, R. N., Mamatelashvili, G. S., Purtseladze, N. G., De Murishvili, L. I. and Mandzhavidze, N. P. (1976) Molochnaya Promyshlennost 12, 13-15. From Haddadin, S. Y. (1986) Microbiology of white-brined cheeses. In Developments in food microbiology-2 (Ed. R. K. Robinson) pp. 67-69. London, Elsevier Applied Science Publishers. Mansour, A. and Alais C. (1973) Etude du salage et de I’affinage du fromage en saumure. III. Aspect Bacteriologique. Le Lait 523-524, 137-145. Nunez, M. and Medina, M. (1979) La flore lactique du fromage bleu de Cabrales. Le‘Lait 59, 497-513. Rasic, J. (1962a) Trends of bacterial population during the manufacture and ripening of white cheese. XVI Int. Dairy Cong. Section IV, 2, 840-845. Rasic, J. (196213) A study of the resistance of lactic acid bacteria to sodium chloride. XVI In t. Dairy Cong. B, 881-886. Rogosa, M., Mitchell, J. A. and Wiseman, R. F. (1951) A selective medium for the isolation of oral and faecal lactobacilli. J. Bacterial. 62, 132-133. 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. Milh Food Technol. 26, 114-118. Schleifer, K. H. and Kilpper-Balz, R. ( 1984 I Transfer of Streptococcus faecalis and StreptoCOCCUS faeciurn to the Genus Enterococcus nom. rev. as Enterococcus faecalis comb. no\‘. and Enterococcus faecium comb. nov. Int. J. Syst. Bacterial. 34, 31-34. Schleifer, K. H., Kraus, J., Dvorak, C., Kilpper-Balz, R., Collins, M. D. and Fisher, W. (1985) Transfer of Streptococcus lactis and related streptococci to the genus Lactococcus gen. nov. Syst. Appl. Microbial. 6, 183-195. Sharpe, M. E. (1979) Identification of lactic acid bacteria. In Identification methocls for microbiologists, 2nd edn (Eds Skinner, F. A. and Lovelock, D. W.). 233 pp. London, Academic Press. Shave, M. E. (1981) The genus Lactobacillus. In The prokaryotes, Vol. II. (Eds Stan, M. P., Stolp, H., Truper, H. G. Balouis, J. A. and Schlegel, H. G.). pp. 1614-1630. New York, Springer-Verlag. Sharpe, M. E. and Fryer, T. F. ( 1965) Media for lactic acid bacteria. Lab. Practice 697-701. Shehata, A. E., El-Sadek, G. M., Khalafalia, S. M. and El-Magdoub, M. N. (1975) Egyptian J. Dairy Sci. 3, 139-144. From Haddadin, S. Y. C. (1986) Microbiology of white-brined cheese. In Developments in food microbiology-2 (Ed. Robinson, R. K.) pp. 67-89. London, Elsevier Applied Science Publishers. Steel, R. G. D. and Torrie, J. H. (1980) Principles and procedures of statistics. 2nd ed. New York, N. Y., McGraw-Hill Book Co., Inc.
Microbiology,
goat milk cheese
19
Turner, N., Sandine, W. E. and Elliker, P. R. (1962) An agar medium for differentiating Streptococcus lactis and Streptococcus cremoris. J. Dairy Sci. 45, 665. Tzanetakis, N. and Litopoulou-Tzanetaki, E. (1988) Lactic acid bacteria in raw goat milk and some of their biochemical properties. Microbial.-Aliments-Nutr. 7, 73-80. Yanai, Y., Rosen, B., Pinoky, A. and Sklan, D. (1977) The microbiology of pickled cheese during manufacture and maturation. J. Dairy Res. 44, 149-153.