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Determination of microbiological and chemical characteristics of kefir consumed in Bursa

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This study was conducted to determine of microbiological quality and some chemical characteristics of 50 kefir samples purchased from different retail markets in Bursa province. In the samples investigated, the mean numbers of lactobacilli, lactococci, enterococci, Enterobacteriacea, Staphylococcus aureus and yeast were determined as 3.6×10 7 cfu/ml, 1.8×10 8 cfu/ml, 4.8×10 4 cfu/ml, 7.3×10 3 cfu/ml, 2.4×10 2 cfu/ml and 7.7×10 4 cfu/ml, respectively. 26 of 50 kefir samples were found to have <0.30 MPN/ml of total coliform while 5 samples had numbers of >110 MPN/ml. For remaining 19 samples, the average bacteria count was 5.3 MPN/ml. Escherichia coli was isolated from 11 (22%) of the samples. Mean acidity, fat and dry matter contents of kefir samples were found as 0.8 L.A.%, 2.3% and 11.3%, respectively, while the pH values varied between 3.9 and 4.7. Consequently, the microbiological findings showed the contamination of kefir samples with microorganisms including E. coli and S. aureus, well known as a remarkable bacterial pathogens, and thus possible health risks for consumers.
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Ankara Üniv Vet Fak Derg, 59, 217-221, 2012
Determination of microbiological and chemical characteristics of kefir
consumed in Bursa
Figen CETINKAYA, Tulay ELAL MUS
University of Uludag, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Gorukle-Bursa, Turkey.
Summary: This study was conducted to determine of microbiological quality and some chemical characteristics of 50 kefir
samples purchased from different retail markets in Bursa province. In the samples investigated, the mean numbers of lactobacilli,
lactococci, enterococci, Enterobacteriacea, Staphylococcus aureus and yeast were determined as 3.6x107 cfu/ml, 1.8x108 cfu/ml,
4.8x104 cfu/ml, 7.3x103 cfu/ml, 2.4x102 cfu/ml and 7.7x104 cfu/ml, respectively. 26 of 50 kefir samples were found to have <0.30
MPN/ml of total coliform while 5 samples had numbers of >110 MPN/ml. For remaining 19 samples, the average bacteria count was
5.3 MPN/ml. Escherichia coli was isolated from 11 (22%) of the samples. Mean acidity, fat and dry matter contents of kefir samples
were found as 0.8 L.A.%, 2.3 % and 11.3 %, respectively, while the pH values varied between 3.9 and 4.7. Consequently, the
microbiological findings showed the contamination of kefir samples with microorganisms including E. coli and S. aureus, well
known as a remarkable bacterial pathogens, and thus possible health risks for consumers.
Key words: Chemical characteristic, kefir, microorganism and pathogen bacteria.
Bursa’da tüketime sunulan kefirlerin mikrobiyolojik ve kimyasal özelliklerinin belirlenmesi
Özet: Çalışma, Bursa ilindeki farklı perakende satış yerlerinden satın alınan 50 adet kefir örneğinin mikrobiyolojik kalitesini
ve bazı kimyasal özelliklerini belirlemek amacıyla gerçekleştirildi. İncelenen örneklerde laktobasil, laktokok, enterokok,
enterobakteri, Staphylococcus aureus ve maya sayıları sırasıyla ortalama 3.6x107 kob/ml, 1.8x108 kob/ml, 4.8x104 kob/ml, 7.3x103
kob/ml, 2.4x102 kob/ml ve 7.7x104 kob/ml olarak belirlendi. 50 kefir örneğinden 26’sının <0.30 MPN/ml düzeyinde, buna karşılık 5
örneğin >110 MPN/ml seviyelerinde koliform bakterileri içerdiği tespit edildi. Kalan 19 örnekte ise, ortalama bakteri sayısı 5.3
MPN/ml’ydi. Örneklerin 11’inden (% 22) Escherichia coli izole edildi. Kefir örneklerinde pH değerleri 3.9 ve 4.7 arasında
değişirken; ortalama asitlik, yağ ve kurumadde içeriklerinin sırasıyla %0.8 L.A., %2.3 ve %11.3 olduğu tespit edildi. Sonuç olarak,
mikrobiyolojik bulgular kefir örneklerinin önemli bakteriyel patojenler arasında yer alan E. coli ve S. aureus gibi
mikroorganizmalarla kontamine olduğunu ve dolayısıyla tüketiciye yönelik olası sağlık risklerini ortaya koydu.
Anahtar sözcükler: Kefir, kimyasal özellik, mikroorganizma ve patojen bakteri.
Introduction
Fermented milks or beverages made with co-
cultures of lactic acid bacteria and yeasts are widely
produced in many countries in the region between
Eastern and Mongolia. Some typical examples are
acidophilin, kefir and koumiss (2). Kefir is a natural
probiotic containing live bacteria, yeasts and the products
that these microorganisms produce (22), and has a sharp
acidic taste and yeasty flavour (13).
The beverage is commonly manufactured by
fermenting milk with kefir grains by a complex microbial
symbiotic mixture of bacteria Lactobacillus, Lactococcus,
Leuconostoc and Streptococcus, and yeasts
Kluyveromyces, Torula and Saccharomyces (11, 14). So
that according to Turkish Food Codex, kefir is defined as
the product in potable consistency, obtained by
fermentation of milk by kefir grains containing lactic
acid bacteria, acetic acid bacteria and Torula yeast (18).
Kefir can be produced by fermenting milk with
commercial freeze-dried kefir starter cultures or
traditional kefir grains (2). The grains are insoluble in
water and irregular in shape and size varying from 0.3-
3.5 cm in diameter (3, 16). The product is mainly
produced from bovine milk as well as caprine and sheep
milks (2, 4). For traditional kefir production, kefir grains
are added to cow’s milk in a 1:20 ratio, and left to
ferment at 18-20ºC for about 20 h. At the end of the
fermentation, the kefir grains are retrieved by sieving and
re-used for new fermentations (7).
Lactobacilli are present as the largest portion (65-
80%) of the microbial population, with lactococci and
yeasts making up the remaining portion of the microbes
present in the kefir grain (20). Both the bacteria and
yeasts are surrounded by a polysaccharide matrix, called
kefiran, which is a water-soluble branched glucogalactan,
and which has been reported to possess antibacterial,
Figen Cetinkaya - Tulay Elal Mus
218
antimycotic and antitumor activity (12). In in vitro tests
with cell-free extracts of kefir, the growth of
Staphylococcus aureus, Bacillus cereus, Escherichia coli,
Clostridium tyrobutyricum and Listeria monocytogenes
was inhibited. In general, the antimicrobial activity of the
beverage is atributed to lactic acid, volatile acids,
hydrogen peroxide, carbondioxide, diacetyl, acetaldehyde,
and/or bacteriocins produced by LAB (16). Yeasts are
also important in kefir fermentation because of the
production of ethanol and carbondioxide. Kefir grains
usually contain lactose-fermenting yeasts (Kluyveromyces
lactis, Kluyveromyces marxianus, Torula kefir), as well
as nonlactose-fermenting yeasts (Saccharomyces cerevisiae)
(10).
The microbial community composition of kefir can
change by several factors such as the source and
microbial load of the grains, the fermentation process,
and storage conditions (4, 5, 16). The aim of this study
was to evaluate the microbiological quality and
physicochemical characteristics of kefir samples sold in
Bursa province and to manifest health hazards for the
consumer.
Materials and Methods
Samples: A total of 50 kefir samples were collected
from different retail markets in Bursa province (Turkey).
The samples were transported to the laboratory under
refrigerated conditions, and all analyses were carried out
on the same day.
Microbiological analysis: 10 ml of the kefir samples
were homogenized with 90 ml of a sterile 0.1% peptone
water solution using a Lab-Blender 80 Stomacher
(London, UK) for at least 2 min. Serial dilutions of the
homogenate were made with sterile peptone water and
plated in duplicates on specific media. Lactobacilli
counts were performed on deMan Rogosa and Sharpe
medium (MRS, Oxoid CM361) at incubation temperature
of 30ºC under anaerobic conditions (5% C02) for 3 days.
Lactococci counts were carried out on M17 medium
(Oxoid CM785) at incubation of 30ºC under anaerobic
conditions for 2 days (10). Dilutions were plated on
Slanetz-Bartley (SB, Oxoid CM377) and Violet Red Bile
Glucose (VRBG, Oxoid CM485) mediums for
enterococci and Enterobacteriaceae, respectively. Plates
were incubated aerobically at 37ºC for 24-48 h. Yeasts
were enumerated in Potato Dextrose Agar (PDA, Oxoid
CM139) (pH 3.5) with 10% added tartaric acid, after
incubation at 22ºC for 5 days. S. aureus cultures was
grown on Baird Parker agar (Oxoid CM0275)
supplemented with egg yolk-tellurite emulsion (Merck
1.03785) at 35ºC for 48 h. The coagulase activity was
performed by using Staphaurex test (Remel ZL30) (9).
Total coliform counts were determined by the standard
most probable number (MPN) method (19). Lauryl
Sulphate Tryptose Broth (Oxoid CM451) containing
Durham’s tube was used as the media for presumptive
test. 10 ml, 1 ml and 0.1 ml of aliquots sample was
inoculated into 3 Lauryl Sulphate Tryptose tubes and all
tubes were incubated at 37ºC for 24 to 48 h. Any gas
formation in Durham’s tubes with slight turbidity in the
medium was regarded as positive and coliform numbers
were estimated using the MPN tables to determine the
MPN index per millilitre. For the determination of E.
coli, a loopful from each positive Lauryl Sulphate
Tryptose tube was streaked on Eosin Methylene Blue
agar (EMB, Oxoid CM69) and incubated at 37ºC for 24-
48 h. Suspicious colonies (dark centered with or without
a green metallic sheen) of E. coli on EMB agar were
subjected to confirmation tests (IMVIC). Indole (+),
methyl red (+), Voges Proskauer (–) and citrate (–)
cultures after 24 to 48 h incubation at 37ºC were assessed
as E. coli type 1.
Physicochemical analysis: For pH measurements, a
Mettler Toledo pH meter with a glass electrode was used.
The acidity was determined by titration with N/10 NaOH
in the presence of phenolphthalein and expressed as per
cent lactic acid (LA). The total fat and dry matter
contents in kefir samples were performed according to
Tekinsen et al. (17).
Results
Lactobacilli, lactococci, enterococci, Entero-
bacteriaceae, S. aureus and yeast counts obtained from
kefir samples and frequency distribution are presented in
Table 1 and Table 2, respectively. Table 3 summarizes
the results obtained for coliform bacteria in this study.
The pH values and some chemical characteristics of kefir
are shown in Table 4.
Table 1. Mean counts (cfu/ml) of microorganisms in kefir samples
Tablo 1. Kefir örneklerindeki ortalama mikroorganizma sayıları (kob/ml)
Microorganisms Mean SEM Minimum Maximum
Lactobacilli 3.6x107 1.4x107 1x101 5.9x108
Lactococci 1.8x108 2.1x107 1.0x105 6.3x108
Enterococci 4.8x104 2.6x104 <1x102 9.1x105
Enterobacteriaceae 7.3x103 7.2x103 <1x101 3.6x105
S. aureus 2.4x102 2.0x102 <1x102 1x104
Yeast 7.7x104 2.9x104 <1x102 1.1x106
Ankara Üniv Vet Fak Derg, 59, 2012 219
Table 4. The physicochemical characteristics of kefir samples
Tablo 4. Kefir örneklerinin fizikokimyasal özellikleri
Parameter Mean ± SEM Minimum Maximum
pH 4.3 ± 0.02 3.9 4.7
Acidity (L.A.), % 0.8 ± 0.02 0.7 1.4
Dry matter, % 11.3 ± 0.36 8.0 16.5
Fat, % 2.3 ± 0.14 0.3 4.5
Discussion and Conclusion
As seen in Table 1, the mean counts (3.6x107
cfu/ml) of lactobacilli in samples purchased from retail
markets in Bursa were similar to Irigoyen et al. (10),
Fontan et al. (6) and Witthuhn et al. (20) These authors
found counts of 8 log cfu/ml, 7.2 log cfu/ml and 1.2x107
cfu/ml, respectively. The lactobacilli levels (9.42 log
cfu/ml and 8.33 log cfu/ml) detected by Gulmez et al. (8)
and Dinc (4), respectively, were also higher than the ones
described in this study. On the other hand, lower levels
of lactobacilli (1.5 to 3.48 log cfu/ml) were obtained by
Wszolek et al. (21) in experimental kefir samples.
In the present study, the mean counts of lactococci
were 1.8x108 cfu/ml (Table 1). Similar results were
reported by Irigoyen et al. (10) and Fontan et al. (6) who
found the mean counts of lactococci as 8 log cfu/ml and
7.8 log cfu/ml, respectively. On the other hand, lower or
higher values of lactococci loads than that of the current
work have been suggested by other some authors in kefir.
Wszolek et al. (21) examined kefir samples made from
ovine milk using kefir grains in Poland and detected
counts of 9 log cfu/ml. Witthuhn et al. (20) obtained
counts of 1.2x107 cfu/ml for lactococci in kefir grains
after 20 day of traditional kefir production.
In this investigation, enterococci and Entero-
bacteriaceae were found in 34% and 10% of the samples,
with mean counts of 4.8x104 and 7.3x103 cfu/ml,
respectively. The results are illustrated in Table 1. A
study conducted by Dinc (4) suggested that average
counts of enterococci and Enterobacteriaceae in 120
kefir samples purchased from supermarkets in Ankara
were 2.5 log cfu/ml and 2.1 log cfu/ml, respectively.
These levels of contamination were lower than those
obtained in our study. In Poland, Molska et al. (15)
recorded 29% and 17% of kefir samples harbored
enterococci at levels of 101-103 cfu/ml and >103 cfu/ml,
respectively.
Eight of kefir samples analysed were found positive
for S aureus ranging from 1x102 to 1x104 cfu/ml, with a
mean of 2.4x102 cfu/ml (Table 1). Among them, one
isolate showed coagulase-positive activity and had counts
of 2x102 cfu/ml (data not shown). The reason for the
contamination of S. aureus could have been the poor
personal hygiene of food handlers and inadequate control
of cold temperatures.
In the present survey, the mean counts for yeast
were 7.7x104 cfu/ml (Table 1). These results were similar
to those obtained by Adamavičiūtė et al. (1), Irigoyen et
al. (10), Wszolek et al. (21) and Witthuhn et al. (20).
They found yeast counts of 4.2 log cfu/ml, 5 log cfu/ml,
4.8 log cfu/ml and 4.6 cfu/ml, respectively. However, our
results were lower than those found by Gulmez et al. (8)
in kefir 9.4 log cfu/ml, and were higher than those (3 to
3.9 log cfu/ml) reported by other some authors (4, 6). In
Turkey, Turkish Food Codex established a guideline with
a minimum level of 104 cfu/ml for yeast (18). According
to our results, the counts of yeast were below the
Table 2. The distribution of the microorganism counts (cfu/ml)
Tablo 2. Mikroorganizma sayılarının dağılımı (kob/ml)
Contamination levels
Microorganisms
<102 102 103 104 105 106 107 108
Lactobacilli 1 (2)a 2 (4) 3 (6) 8 (16) 6 (12) 10 (20) 15 (30) 5 (10)
Lactococci 0 (0) 0 (0) 0 (0) 0 (0) 5 (10) 6 (12) 20 (40) 19 (38)
Enterococci 33 (66) 4 (8) 3 (6) 7 (14) 3 (6) 0 (0) 0 (0) 0 (0)
Enterobacteriaceae 45 (90) 2 (4) 2 (4) 0 (0) 1 (2) 0 (0) 0 (0) 0 (0)
S. aureus 42 (84) 6 (12) 1 (2) 1 (2) 0 (0) 0 (0) 0 (0) 0 (0)
Yeast 33 (66) 1 (2) 3 (6) 5 (10) 7 (14) 1 (2) 0 (0) 0 (0)
a:The number (%) of samples in different population groups
Table 3. Analysis results for coliform bacteria
Tablo 3. Koliform bakterilere ilişkin analiz sonuçları
MPN/ml
<0.30 0.3 0.36 0.62 1.1 1.5 1.6 3.5 9.3 29 46 >110
26 (52)a 3 (6) 4 (8) 2 (4) 2 (4) 2 (4) 2 (4) 1 (2) 1 (2) 1 (2) 1 (2) 5 (10)
a: the number of samples (%)
Figen Cetinkaya - Tulay Elal Mus
220
detectable limit (<102 cfu/ml) in 33 samples and between
102-<104 cfu/ml in 4 samples. On the basis of this
guideline, a total (74%) of 37 kefir samples were
regarded as being of unsatisfactory quality.
The contamination levels with coliform bacteria of
26 samples were <0.30 MPN/ml and 5 samples were
found to be contaminated at levels of >110 MPN/ml. For
remaining 19 samples, coliform counts were in the range
of 0.3 to 46 MPN/ml, with an average of 5.3 MPN/ml
(Table 3). This finding does not agree with that of
Wszolek et al. (21) suggesting the absence of coliform
bacteria. Another study (4) of kefir samples on retail sale
in Turkey (Ankara) carried out between December 2006
and May 2007, indicated that the samples contained
coliforms with mean contamination level of 11.58
MPN/ml, which is higher than a mean coliform count
reported here. According to Turkish Food Codex,
pathogen microorganisms should not be detected in kefir
as well as other cultured dairy products (18). In this
investigation, the incidence of E. coli was 22% (11/50) in
the samples, which is close to the incidence (25%)
reported by Dinc (4). The presence of coliforms
including E. coli in samples is often an indication of
contamination during production, which can occur from
poor hygienic conditions.
Table 4 shows the values of the main
physicochemical parameters in kefir samples. The
samples had a pH value between 3.9 and 4.7, with a
mean 4.3. This finding was similar to values observed in
kefir by other some workers (4, 6, 10, 15). However,
higher values than ours (as high as 9.5) were found by
Gulmez et al. (8). The values of titratable acidity in the
current study were a mean of 0.8% L.A, ranging from
0.7% to 1.4% L.A. These values were consistent with the
limit (min. 0.6 % L.A.) laid down in Turkish Food
Codex, and also similar to those noted by Dinc (4) and
by Fontan et al. (6), who found an average of 2.7% L.A.
and 1.32% L.A., respectively, in kefir. The fat and dry
matter values of kefir samples analysed in this work
ranged from 0.3 to 4.5% and 8.0 to16.5%, respectively.
Other published studies also reported similar results.
Irigoyen et al. (10) indicated the fat and dry matter
contents of 3.2-3.59% and 11.3-11.6%, respectively, in
kefir samples made using the 1% and 5% kefir grains.
Dinc (4) demonstrated the mean values of 2.7% and
13.3% for fat and dry matter contents, respectively.
In conclusion, the results of this work on the
microbiological quality of kefir sold in Bursa province
indicated the high levels of hygiene indicator bacteria
such as coliforms and enterococci, or the presence of
potential pathogenic bacteria such as E. coli and S.
aureus. So that, approximately 38% of the samples failed
to meet the legal requirements in terms of overall
microbiological quality. Thus, these products are of
unsatisfactory and/or unacceptable microbiological
quality and there is reason to suspect possible public
health concerns. Good hygienic practices should be
applied throughout manufacture and at retail to ensure
that contamination and pathogen growth do not occur in
kefir.
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Geliş tarihi: 14.09.2011 / Kabul tarihi: 10.01.2012
Address for correspondence:
Doç. Dr. Figen Çetinkaya
Uludağ Üniversitesi, Veteriner Fakültesi,
Besin Hijyeni ve Teknolojisi Anabilim Dalı,
16059 Görükle Kampüsü, Bursa.
Phone: +90 224 2941212, Fax: +90 224 2941202,
E-mail: figcetinkaya@yahoo.com
... The species and the amount of various microorganisms show significant variation based either on its region of origin or the methods of production, fermentation conditions and the substrates used (Pintoda et al., 1996;Beshkova et al., 2003;Cetinkaya and Elal-Mus, 2012;Altay et al., 2013). Fermentation conditions such as time and temperature are important parameters that influence the microbiota of kefir grain and also kefir produced by this grain Altay et al., 2013). ...
... In previous studies, the obtained total dry matter contents ranged between 8.88-16.73% (Ertekin and Guzel-Seydim, 2010;Uslu, 2010;Cetinkaya and Elal-Mus, 2012). The results obtained in the present study were also in the range of those values. ...
... Kefir and kefir grains can be affected from the ratio of kefir grains to milk, the origin and method of cultivation (Beshkova et al., 2003;Fröhlich-Wyder, 2003;Mistry, 2004;Witthuhn et al., 2005;Cetinkaya and Elal-Mus, 2012). Koroleva (1991) described the microorganisms composition of properly prepared kefir and according to this description kefir's microbiota should contain homofermentative mesophilic lactic acid streptococci (10 8 -10 9 CFU/ mL), thermophilic lactobacilli (10 5 CFU/mL), heterofermentative lactic acid streptococci (10 7 -10 8 CFU/mL), yeasts (10 5 -10 6 CFU/mL), acetic acid bacteria (10 5 -10 6 CFU/mL). ...
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... Lactic acid bacteria colony was cultured on De Man, Rogosa and Sharpe (MRS) (Cetinkaya and Elal, 2012), and pour-plating method was used to enumerate them. Samples were homogenised with maximum recovery diluent (MRD) solution in the stomacher bag for 2 min and 10 -7 serial dilutions were made. ...
... Acidified Potato Dextrose Agar (PDA) was used to culture yeasts (Cetinkaya and Elal, 2012), and spread-plating method was used to enumerate them. Around 15 -20 mL of sterilised agar was poured into an empty plate, and stored in the refrigerator (4°C). ...
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... They stated that 35% and 10% of samples were contaminated with E. coli and S. aureus. Cetinkaya and Elal (2012) [24] conducted a study to investigate the microbial contamination of 50 samples of kefir. They reported 2.4 × 10 2 cfu/ml contamination of S. aureus. ...
... The microbiological quality and physicochemical properties of kefir are influenced by the microbial property of used kefir grains, the grains to milk ratio, incubation temperature and time, storage temperature and sanitation conditions [29]. Kefir has been reported to exhibit antimicrobial activity which is attributable to lactic acid, bacteriocins, hydrogen peroxide, acetaldehyde, volatile acids, diacetyl, and/or carbon dioxide produced by the bifidobacteria and the LAB present in the drink [30]. ...
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... Banana kefir had the lowest value (4.53 log10 cfu/ml) at the end of the incubation, while at the end of the storage period, apricot kefir's level dropped to below its value at the beginning of the incubation (3.04 log10 cfu/ml). Öner et al. (2010) reported a yeast count of 5.34 log10 cfu/ml at the end of the storage period, while Çetinkaya and Elal-Mus (2012) reported a count of <1.0x10 2 -1.10x10 6 . Uslu (2010) reported an average yeast count of 6.70 cfu/ml in plain kefir and 6.44 log10 cfu/ml in fruit kefir. ...
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In this study, a total of 21 coccoid forms of lactic acid bacteria (lactococci) were isolated from Turkish kefir samples. As a result of the identification tests, 21 lactococci isolates were identified as Lactococcus cremoris (11 strains), Lactococcus lactis (4 strains), Streptococcus thermophilus (3 strains) and Streptococcus durans (3 strains). The amount of produced lactic acid, hydrogen peroxide, proteolytic activity, diacetyl and acetaldehyde productions of the lactococci were determined. Different amounts of lactic acid were produced by strains studies; however, lactic acid levels were 2.3–9.9 mg/ml. While Lac. lactis Z13S, Str. durans Z7S, Z8S, Z15S, Lac. cremoris Z9S, Z16S, Z17S, Z19S, Z20S and Z21S strains were not shown hydrogen peroxide, Lac. lactis Z1S and Z2S strains had a maximum hydrogen peroxide (0.17 μg/ml). Lac. lactis Z2S, Z3S, Str. thermophilus Z5S, Z12S, Str. durans Z7S, Z8S, Lac. cremoris Z14S and Z16S strains were not show proteolytic activity, Lac. cremoris Z20S strain produced the maximum amount (0.09 mg/ml) of proteolytic activity. Acetaldehyde concentration produced in Lactobacillus strains ranged between 0.18 and 3.96 μg/ml. Antimicrobial effects of the lactococci on Escherichia coli NRLL B-704, Staphylococcus aureus 4-63, Pseudomonas aeruginosa ATCC 29212 were also determined by an agar diffusion method. All of the strains were able to inhibit S. aureus, while Lac. lactis Z1S, Z2S and Lac. cremoris Z6S strains were able to inhibit E. coli and P. aeruginosa. Also, Str. thermophilus Z5S strain were able to inhibit P. aeruginosa.