Showing posts with label kefir grains. Show all posts
Showing posts with label kefir grains. Show all posts
Monday, September 10, 2007
Tuesday, January 30, 2007
Lactic acid bacteria and yeasts in kefir grains and kefir made from them
E Simova1, D Beshkova1, A Angelov2, Ts Hristozova1, G Frengova1 and Z Spasov3
1
Laboratory of Applied Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, 26 Maritza Boulevard,
4002 Plovdiv, Bulgaria; 2Higher Institute of Food and Flavour Industries, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria;
3
ELBY Center of Research, Development and Production, 44 - A Lagera Boulevard, 1612 Sofia, Bulgaria
In an investigation of the changes in the microflora along the pathway: kefir grains ( A ) ! kefir made from kefir grains
( B ) !kefir made from kefir as inoculum ( C ) , the following species of lactic acid bacteria ( 83 – 90% ) of the microbial
count in the grains ) were identified: Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus
delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp. pseudoplantarum and
Lactobacillus brevis. Yeasts ( 10 – 17% ) identified were Kluyveromyces marxianus var. lactis, Saccharomyces
cerevisiae, Candida inconspicua and Candida maris. In the microbial population of kefir grains and kefir made from
them the homofermentative lactic streptococci ( 52 – 65% and 79 – 86%, respectively ) predominated. Within the group
of lactobacilli, the homofermentative thermophilic species L. delbrueckii subsp. bulgaricus and L. helveticus ( 70 –
!!
87% of the isolated bacilli ) predominated. Along the pathway A! B! C, the streptococcal proportion in the total kefir
microflora increased by 26 – 30% whereas the lactobacilli decreased by 13 – 23%. K. marxianus var. lactis was
permanently present in kefir grains and kefirs, whereas the dominant lactose - negative yeast in the total yeast flora of
the kefir grains dramatically decreased in kefir C.
Journal of Industrial Microbiology & Biotechnology ( 2002 ) 28, 1 – 6 DOI: 10.1038 / sj / jim / 7000186
Keywords: kefir grains; kefir; lactic acid bacteria; yeasts; identification; composition
Introduction Materials and methods
The wholesome properties of fermented milks ( kefir, kumis ) and Kefir grains and conditions of cultivation
their nutritional value have motivated a considerable interest in The kefir grains studied were from the collection of the Center of
such ‘‘novel’’ milk foods [ 10,20,22 – 24 ] . Kefir has been defined Research, Development and Production ( LB Bulgaricum Trading,
as the yogurt of the 21st century [ 9 ] . Kefir differs from other Bulgaria ) . The lyophilized kefir grains were activated for 1 month
fermented milks in its starter, which exists in the form of ‘‘grains.’’ in the following way: Sterilized skim milk ( 0.1% milk fat, 1 - l
The grains have a specified structure and behave as biologically flasks, 1158C, 15 min ) was cooled to 18 – 208C, and inoculated
vital organisms. They grow, propagate and pass their properties on using kefir grains ( the weight ratio of kefir grains to milk was 1:10 )
to the following generations of new grains. The microflora of kefir and incubated at 18 – 208C for 24 h with three to five stirrings. Kefir
grains is remarkably stable, retaining its activity for years if grains were separated from the fermented milk by filtration through
preserved and incubated under appropriate cultural and physio- a sieve and were washed with cooled sterile water before being used
logical conditions. Kefir grains have a complex microbiological
in the next culture passage. Activated kefir grains were propagated
composition. Lactobacilli, lactic streptococci, yeasts and acetic as follows: From the grown and fresh kefir grains six grains were
acid bacteria have been shown to be present in them selected. Each was cut and cultivated separately in sterile skim milk
[ 23,25,27,28,32,37,38 ] . The kefir - specific yeasts play a key role under the conditions given. After daily transfers of the particles of
in the formation of flavour and aroma [ 8 ] . They are represented by the six grains into sterile milk for a period of 2 weeks, the new kefir
the species Kluyveromyces marxianus, Torulaspora delbrueckii, grains were cut again. This procedure of transferring and cutting
Saccharomyces cerevisiae, Candida kefir, Saccharomyces unispo- lasted ( about 2 months ) until a sufficient amount of new kefir
rus, Pichia fermentans and Yarrowia lypolytica [ 1,18,28 ] . For the grains was obtained from the initial six. The six populations of kefir
formation of a kefir starter culture, which would produce a milk grains thus obtained were used in the present study. Five grains
beverage with characteristics similar to those of traditional kefir, were taken out of each population; each was cut and the pieces were
more information needs to be acquired about the specific cultures
thoroughly washed with sterile water and suspended in a sterile
isolated from kefir grains. solution of 0.9% NaCl. By plating the diluted homogenates of every
The aim of this study was to describe changes in the microbial one of the grains on appropriate cultural media, strains of lactic acid
population in kefir grains while they underwent two subsequent bacteria and yeasts were isolated [ 12 – 15 ] .
fermentation cycles: kefir grains!kefir!second batch of kefir.
Isolation and identification of lactic acid bacteria from
Correspondence: E Simova, Laboratory of Applied Microbiology, Institute of
kefir grains
Microbiology, Bulgarian Academy of Sciences, 26 Maritza Boulevard, 4002 Plovdiv,
For isolation of the lactobacilli from kefir grains, 0.1 - ml portions of
Bulgaria
the respective dilutions of homogenates were plated on MRS agar
Received 2 August 2000; accepted 15 July 2001
Lactic acid bacteria and yeasts in kefir grains
E Simova et al
2
[ 2 ] were applied. The yeasts were also identified on the basis of
( Fluka RdH, Buchs, Switzerland ) and on Rogosa agar with cheese
fermentation tests ID 32C ( bioMerieux ) using API LAB Plus
whey ( Fluka RdH ) [17]. The plates were incubated at 308C and
software.
378C for 3 – 7 days in aerobic and anaerobic (10% CO2 )
atmospheres. For isolation of lactic streptococci the diluted
homogenates were plated on M-17 medium (Fluka RdH), and Preparation of kefir, isolation and identification of lactic
azide agar medium (Difco, Detroit, USA) was used aerobically.
acid bacteria and yeasts
The plates were incubated at 288C and 378C for 4 – 8 days.
Using kefir grains A from the six populations obtained, six samples
After microscopic examination and on the basis of their
of traditional kefir ( kefir B ) were prepared as follows: Milk ( 3%
morphology, the colonies were divided into groups and trans-
milk fat ) , pasteurised at 928C for 15 min, homogenized at 12.5 –
ferred in triplicate in sterile skim milk and on plates. Some of
17.5 MPa and cooled to 18 – 208C, was inoculated with 3% kefir
the lactobacilli, isolated directly from the kefir grains, appeared
grains. Incubation was at 18 – 208C for 18 h ( pH = 4.5 – 4.6 )
to have very limited fermentative capability until further passage
followed by coagulum cooling to 8 – 108C and kefir ripening for
through laboratory media or sterile skim milk. By microscopic
24 h. After separation of the kefir grains from the six kefir B
analysis of each culture following incubation, confirming the
samples, strains of lactic acid bacteria and yeasts were isolated and
purity of the isolates and their activation, the cultures were
identified on the basis of taxonomic characteristics and biochemical
characterized taxonomically according to the criteria described in
tests as already described.
Bergey’s Manual [ 16,31 ] . Identification was carried out also on
The six kefir B samples were used as starter cultures for
the basis of biochemical tests with API 20A ( for identification
preparing samples of kefir C. Kefir C was made in the following
of anaerobic bacteria ) and with API 50CH ( test for lactobacilli
way: Milk ( 3% milk fat ) was pasteurised at 928C for 15 min,
and lactococci — API 50CHL Medium, bioMerieux, Marcy
homogenized at 12.5 – 17.5 MPa and cooled to 22 – 238C. Each of
l’Etoile, France ) using the software of the company API LAB
the six milk samples was inoculated with 3% of the sample of kefir
Plus.
B. Fermentation was carried out at 238C for 8 – 12 h to pH = 4.5 –
4.6, followed by cooling to 8 – 108C with simultaneous ripening for
Isolation and identification of yeasts from kefir grains 10 – 12 h. The microflora of kefir C samples was then isolated and
Yeasts were isolated by surface spreading on plates of malt extract identified as already described.
agar ( Fluka RdH ) and YM agar ( Fluka RdH ) [ 19 ] . After
incubation at 288C for 3 – 6 days, colonies of different morphology
were obtained. Initial cultures were prepared from these colonies.
Analytical methods
Inclined agar tubes were inoculated and after incubation ( 288C for
3 days ) an initial suspension was prepared from each culture. A In order to calculate the percentage of each characterized
solution of yeast extract – peptone – glucose ( 0.45%, 0.75%, 1.0% ) microorganism in kefir grains A, kefir B and kefir C, the
was used as medium, whose pH value was corrected to 6.4 by number of viable bacteria and yeasts expressed as colony -
forming units per ml ( CFU ml À 1 ) was estimated. Serial dilutions
adding tartaric acid. After sterilization, the glucose was added to the
medium as a sterile filtered solution. The medium was inoculated of each sample were plated in triplicate and plates were incubated
with one loop of the inclined agar culture and the sample was at 308C and 378C ( for lactic acid bacteria ) , and 288C ( for
incubated to the appearance of distinct darkening. For identification yeasts ) until growth of the colonies. The results were the mean
of the yeasts the methods of Kreger van Rij [ 19 ] and Barnett et al of six counts at three different dilutions.
Table 1 Composition of the microflora of kefir grains from six populations
Microorganisms Colony count in population no.
1 2 3 4 5 6
22 ± 2.0 ( 83 ) a 19 ± 2.6 ( 88 ) 15 ± 44 ( 90 ) 24 ± 3.6 ( 84 ) 16 ± 4.4 ( 87 ) 21 ± 2.6 ( 83 )
Lactic acid bacteria
Streptococci 14 ± 2.6 ( 53 ) 13 ± 2.0 ( 61 ) 11 ± 1.7 ( 65 ) 16 ± 2.0 ( 55 ) 10 ± 1.7 ( 54 ) 13 ± 3.0 ( 54 )
L. lactis subsp. lactis 10 12 9 14 8 10
S. thermophilus 4 1 2 2 2 3
Lactobacilli 8 ± 2.0 ( 30 ) 6 ± 2.6 ( 27 ) 4 ± 1.0 ( 24 ) 8 ± 3.0 ( 29 ) 6 ± 3.0 ( 33 ) 8 ± 2.6 ( 29 )
L. delbrueckii subsp. bulgaricus 5 2 2 4 3 4
L. helveticus 2 2 – 2 2 2
L. casei subsp. pseudoplantarum – 1 1 1 – –
L. brevis 1 1 1 – 1 2
Yeasts 5 ± 1.7 ( 17 ) 3 ± 2.0 ( 12 ) 2 ± 1.0 ( 10 ) 5 ± 1.7 ( 16 ) 2 ± 1.0 ( 13 ) 5 ± 2.0 ( 17 )
K. marxianus var. lactis 1 1 – 1 – 1
S. cerevisiae 2 – – 2 – 1
C. inconspicua 2 1 1 1 2 2
C. maris – 1 – – – 1
Unidentified – – 1 1 – –
Data represent mean values for five grains from each population and standard deviation.
a
Data in parentheses represent percentage of total microflora.
Lactic acid bacteria and yeasts in kefir grains
E Simova et al
3
Table 2 Composition of the microflora of kefir samples ( kefir B ) prepared with kefir grains from six populations
Microorganisms Colony count in population no.
1 2 3 4 5 6
24 ± 3.6 ( 94 ) a 24 ± 5.2 ( 94 ) 20 ± 6.2 ( 92 ) 24 ± 3.6 ( 92 ) 20 ± 2.6 ( 96 ) 20 ± 2.6 ( 95 )
Lactic acid bacteria
Streptococci 19 ± 2.6 ( 74 ) 20 ± 2.6 ( 78 ) 16 ± 2.0 ( 76 ) 20 ± 2.6 ( 77 ) 16 ± 4.3 ( 78 ) 16 ± 4.3 ( 76 )
L. lactis subsp. lactis 15 18 13 17 12 13
S. thermophilus 4 2 3 3 4 3
Lactobacilli 5 ± 1.7 ( 20 ) 4 ± 1.0 ( 16 ) 4 ± 2.0 ( 16 ) 4 ± 1.7 ( 15 ) 4 ± 1.0 ( 18 ) 4 ± 1.7 ( 19 )
L. delbrueckii subsp. bulgaricus 2 2 2 3 2 2
L. helveticus 2 – – 1 1 2
L. casei subsp. pseudoplantarum – 1 4 – – –
L. brevis 1 1 1 – 1 –
Yeasts 1±0 (6) 2 ± 1.0 ( 6 ) 2 ± 1.7 ( 8 ) 2 ± 1.0 ( 8 ) 1±0 (4) 1±0 (5)
K. marxianus var. lactis 1 1 – 1 – 1
S. cerevisiae – – – 1 – –
C. inconspicua – – 1 – 1 –
C. maris – 1 – – – –
Unidentified – – 1 – – –
Data represent the mean values from three kefir samples from each population and standard deviation.
a
Data in parentheses represent percentage of total microflora.
were identified: the mesophilic species Lactococcus lactis subsp.
Viable streptococci were estimated on plates of M - 17 agar,
lactis ( Streptococcus lactis ) and the thermophilic species Strepto-
viable lactobacilli count on plates of MRS agar and the total yeast
coccus thermophilus. These two species were the predominating
count was determined using YM agar.
microflora in all kefir grains ( 53 – 65% of the total microflora ) and
Lactic acid and alcohol were determined by enzymatic methods
samples of kefir ( 74 – 78%; 79 – 86% ) . S. lactis prevailed over
as described by Boehringer Mannheim [ 4 ] . CO2 content ( as
S. thermophilus ( up to 12 - fold in kefir grains and up to 14 - fold in
dissolved gas ) was analysed by the enzymatic method for dissolved
kefir samples ) . During fermentation the proportion of streptococci
CO2 [ 3,6 ] . Viscosity ( cst ) was measured on an Oswald ( Sibata,
increased and reached 79 – 86% of the total microflora in kefir C.
Japan ) cinematic viscosimeter.
The following homofermentative lactobacilli were isolated: Lacto-
bacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus and
Lactobacillus casei subsp. pseudoplantarum. The heterofermenta-
Results tive lactobacillus Lactobacillus brevis was also isolated. Lactoba-
cilli constituted about 24 – 33% of the total flora in the kefir grains.
The distribution of the species of lactic acid bacteria and yeasts in
A twofold decrease in the quantity of lactobacilli in the pathway
the kefir grains A and in the samples of kefir B and kefir C is given
‘‘A!B!C’’ was noted. Of the isolated lactobacilli in all the
in Tables 1 – 3. Two species of homofermentative lactic streptococci
Table 3 Composition of the microflora of kefir samples ( kefir C ) prepared with B as inoculum
Microorganisms Colony count in population no.
1 2 3 4 5 6
17 ± 4.0 ( 94 ) a 17 ± 2.6 ( 96 ) 18 ± 4.4 ( 96 ) 19 ± 2.6 ( 95 ) 22 ± 2.6 ( 96 ) 16 ± 2.0 ( 95 )
Lactic acid bacteria
Streptococci 14 ± 2.6 ( 79 ) 15 ± 4.4 ( 84 ) 16 ± 2.0 ( 85 ) 17 ± 2.6 ( 86 ) 20 ± 2.6 ( 86 ) 13 ± 2.6 ( 79 )
L. lactis subsp. lactis 11 14 14 13 15 10
S. thermophilus 3 1 2 4 5 3
Lactobacilli 3 ± 1.0 ( 15 ) 2 ± 1.7 ( 12 ) 2 ± 1.0 ( 11 ) 2 ± 1.7 ( 9 ) 2 ± 1.0 ( 10 ) 3 ± 1.7 ( 16 )
L. delbrueckii subsp. bulgaricus 1 1 1 1 1 1
L. helveticus 1 – – 1 1 1
L. casei subsp. pseudoplantarum – 1 1 – – –
L. brevis 1 – – – – 1±0
Yeasts 1±0 (6) 1±0 (4) 1±0(4) 1±0 (5) 1±0 (4) 1±0 (5)
K. marxianus var. lactis 1 1 – 1 – 1
S. cerevisiae – – – – 1 –
C. inconspicua – – 1 – – –
C. maris – – – – – –
Unidentified – – – – – –
Data represent the mean values from three kefir samples ( kefir C ) prepared with kefir B as inoculum and standard deviation.
a
Data in parentheses represent percentage of total microflora.
Lactic acid bacteria and yeasts in kefir grains
E Simova et al
4 Table 4 Characteristics of kefir B and kefir C after 24 h storage at 48C
Kefir Ba Kefir Cb
Component
pH 4.5 ± 0.26 4.35 ± 0,20
Lactic acid ( g l À 1 ) 8.18 ± 0.61 8.20 ± 0,57
Alcohol ( % ) 0.25 ± 0.04 0.09 ± 0,04
Carbon dioxide ( g l À 1 ) 0.85 ± 0.15 0.15 ± 0,06
Mesophilic streptococci ( CFU ml À 1 ) 8 ( ± 4.58 ) .109 6 ( ± 2,64 ) .109
Thermophilic streptococci ( CFU ml À 1 ) 3 ( ± 2,64 ) .106 4 ( ± 2,00 ) .106
Homofermentative lactobacilli ( CFU ml À 1 ) 8 ( ± 2,64 ) .106 2 ( ± 1,00 ) .106
Yeasts ( CFU ml À 1 ) 2 ( ± 2,64 ) .105 1 ( ± 2,00 ) .104
Viscosity ( cst ) 1.075 ± 0,21 1.043 ± 0,28
Aroma strong, typically yeasty very light yeasty
Flavour kefir - specific, refreshingly pungent predominating lactic acid
Texture homogeneous, creamy, abundantly effervescent slightly viscous, very slight effervescence
a
Kefir B was prepared with kefir grains No. 4 and received the best organoleptic evaluation of kefirs B, prepared with the six populations.
b
Kefir C was prepared with kefir Ba as inoculum.
Data represent the mean values from three kefir B and kefir C, and their standard deviations.
L. casei subsp. pseudoplantarum was found in the populations
samples L. bulgaricus prevailed, followed by L. helveticus. During
from three kefir grains and in two samples of kefir B and kefir C.
the two consecutive fermentations L. helveticus decreased. The
The presence of this species in kefir grains has been noted in other
heterofermentative species L. brevis, present in five out of the six
studies [ 7 ] . According to some authors, the heterofermentative
populations of kefir grains, dramatically decreased, and in kefir C it
lactobacilli predominate over the homofermentative ones [ 1 ] , and
was found in two out of six samples. L. casei subsp. pseudoplan-
according to others, they are the only representatives of the
tarum was found in three out of six samples of kefir grains in kefir
lactobacilli [ 38 ] . The isolated species L. brevis decreased along the
B and kefir C.
pathway A!B!C. These results are very similar to those of
The lactose - fermenting yeast K. marxianus var. lactis was
Takizawa et al [ 33 ] , who found that 90% of the bacterial species in
found in kefir grains A, kefir B and kefir C together with non -
kefir grains were homofermentative lactobacilli. In other studies,
lactose - fermenting yeasts ( S. cerevisiae, Candida inconspicua,
however, the lactobacilli represented 65 – 80% of the microbial
Candida maris and two unidentified yeasts ) . The yeast microflora
count of the grain, with streptococci and various yeasts as the
of kefir grains was marked by the prevailing presence of lactose -
remainder [ 1,23 ] .
negative yeasts ( 60 – 100% of the total quantity of yeasts ) . Among
Yeasts in kefir grains represented 10 – 17% of the total isolated
then the species C. inconspicua predominated, followed by
microflora with lactose - negative yeast species predominating
S. cerevisiae.
( 66 – 100% ) . The inability of these yeasts to ferment lactose
In the preliminary organoleptic evaluation of kefirs B prepared
makes them dependent on lactic acid bacteria capable of
with kefir grains from the six populations, kefir B prepared with
hydrolyzing this disaccharide. The prevalence of lactose - negative
kefir grains from population No. 4 ranked first with the best
yeasts in the grains was consistent with the studies carried out by
characteristics. The data comparing the physicochemical and
other authors [ 29,36 ] . It is worth noting that the group of
microbiological composition and organoleptic properties of kefir
lactose - negative yeasts contains the two species C. inconspicua
B with population No. 4 and those of kefir C, prepared with kefir B,
and C. maris, whose presence in kefir has not been reported
are shown in Table 4.
previously. C. inconspicua predominated in the yeast microflora
of the kefir grains and drastically decreased along the pathway
A!B!C. C. maris was present in the microflora of two kefir
Discussion grains. These two species did not ferment or assimilate galactose.
C. inconspicua assimilated DL -lactic acid and was, therefore,
Lactic acid bacteria represented 83 – 90% of the microbial count of
the grains. In the microbial populations of the kefir grains, the completely dependent on the metabolism of lactic acid bacteria.
homofermentative species of lactic acid streptococcus S. lactis C. maris did not assimilate DL - lactic acid. Possibly, its presence
prevailed in kefir B and kefir C ( 40 – 55% and 60 – 80%, in kefir grains was connected not only with the presence of
respectively ) , which determined it as the main fermenting glucose and certain vitamins ( thiamine, pyridoxine ) , but also
organism. Our results differ from those studies that found a very with the assimilation of some acids produced by lactic acid
scarce presence of streptococci ( a single diplococcal cell ) in the bacteria by the transformation of pyruvic acid. The lactose -
grain - associated population [ 5,26 ] . However, a number of authors positive species K. marxianus var. lactis was identified in the
consider S. lactis as an integral part of the microflora of kefir grains isolates from four kefir grains. Its presence in kefir grains has
irrespective of its low numbers [ 11,30 ] . been reported in other studies [ 1,35,36 ] . According to some
Within the lactobacilli, the thermophilic homofermentative authors, K. marxianus var. lactis isolated from kefir grains is
species L. bulgaricus and L. helveticus ( 70 – 87% of the isolated distinguished by the peculiarity of always occurring with lactose -
lactobacilli ) predominated. There are reports on the presence of negative yeasts, which is also confirmed by our results [ 1 ] .
L. helveticus as a prevailing member of the lactic acid microflora in The composition of the yeast flora in kefir B and kefir C was
kefir grains [ 35 ] . The mesophilic homofermentative species highly dependent on the starter used. During fermentation and
Lactic acid bacteria and yeasts in kefir grains
E Simova et al
5
4 Boehringer Mannheim GmbH Biochemica. 1983. In: Methods of
ripening, alterations occurred only in the group of lactose -negative
enzymatic food analysis using test combinations. Boehringer
yeasts. In kefir B samples, the lactose - negative species ( five out
Mannheim GmbH Biochemica, Mannheim, Germany, pp. 17 – 18,
of nine isolated yeast species ) predominated slightly. In all kefir C 33 – 34.
samples, where K. marxianus var. lactis was present, other yeast 5 Duitschaever C, N Kemp and A Smith. 1988. Microscopic studies of
the microflora of kefir grains and kefir made by different methods.
species were not identified. In two of the samples where
Milchwissenschaft 43: 479 – 481.
K. marxianus var. lactis was not found, the species C. inconspicua
6 Forrester R, L Wataji, D Silverman and K Pierre. 1976. Enzymatic
and S. cerevisiae were identified. The unidentified yeast present in method for determination of CO2 in serum. Clin Chem 22: 243 – 245.
kefir B disappeared in kefir C. The quantity of the lactose - 7 Galli A, E Fiori, L Franzetti, M Pagani and G Ottogalli. 1995.
fermenting yeast K. marxianus var. lactis remained the same in the Composizione microbiologica e chimica dei granuli di Kefir ‘‘di
frutta.’’ Ann Microbiol Enzimol 45: 85 – 95.
kefir grains, traditional kefir B and kefir C. The total yeast
8 Glaeser H, E Hangst and K Ziegler. 1986. Biochemische charakter-
quantity decreased because of the decrease of lactose - negative
isierung der in molkereikefir und kefirkulturen vorkommende hefen.
yeast in the course of the process: kefir grains A!kefir B!kefir Dtsch Molk - Ztg 16: 483 – 490.
C. A number of authors maintain that lactose - fermentative yeast 9 Gorski D. 1994. Kefir: 21st century yogurt? Dairy Foods 95: 49.
10 Honer C. 1993. Now kefir. Dairy Field 176: 91.
should be defined as one of the main components of the ‘‘specific
11 Hosono A, T Tanabe and H Otani. 1990. Binding properties of lactic
cultures,’’ whereas the lactose - negative yeasts should be desig-
acid bacteria isolated from kefir milk with mutagenic amino acid
nated as recontamination yeast [ 8,21,34 ] . Participation of pyrolyzates. Milchwissenschaft 45: 647 – 651.
K. marxianus var. lactis in the microflora of the kefir grains 12 IDF Standard 94 B. 1990. Milk and milk products - enumeration of
ensures metabolism of lactose through alcohol fermentation and yeast and moulds — colony count technique at 258C, Brussels,
Belgium.
the formation of the typical yeasty flavour and aroma. According
13 IDF Standard 146. 1991. Yogurt identification of characteristic
to some authors, kefir must contain an approximate minimum of
microorganisms ( Lactobacillus delbrueckii subsp. bulgaricus and
1Â105 lactose - fermenting yeasts to obtain the characteristic Streptococcus salivarius subsp. thermophilus ) , Brussels, Belgium.
flavour and aroma [ 8 ] . That statement was supported by four 14 IDF Standard 163. 1992. General standard of identity for fermented
milks, Brussels, Belgium.
samples of kefir C ( out of six ) and two samples of kefir B ( out of
15 IDF Standard 117 B. 1997. Yogurt - enumeration of characteristic
six ) , in which only K. marxianus var. lactis was present ( Tables 2
microorganisms — colony count technique at 378C, Brussels,
and 3 ) . On the other hand, special emphasis should be laid upon Belgium.
the role of lactose - negative yeast in the formation of the yeasty 16 Kandler O and N Weiss. 1986. Regular, nonsporing Gram - positive
flavour of kefir. Although K. marxianus var. lactis was similar in rods. In: Sneath P, N Mair, M Sharpe and J Holt ( Eds ) , Bergey’s
Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore,
kefir B and kefir C, the stronger and typical yeasty aroma and the
MD, vol. 2, section 14, pp. 1208 – 1234.
refreshing, pungent taste of traditional kefir B may have been due
17 Kojima S, S Takizawa, S Tamura, S Fujinaga, Y Benno and T Nakase.
to the lactose - negative yeast that was present. The remaining 1993. An improved medium for the isolation of lactobacilli from kefir
samples of kefir B ( with populations Nos. 2, 3, 5 ) and kefir C, grains. Biosci Biotechnol Biochem 57: 119 – 120.
18 Koroleva N. 1988. Starters for fermented milks. Section 4: Kefir and
which contained lactose - negative yeasts, were noticed to possess
kumis starters. Bull IDF 227: 35 – 40.
the typical yeasty flavour and aroma that were absent in the kefirs
19 Kreger van Rij NJW. 1984. The Yeast: A Taxonomic Study. 3rd ed.
with K. marxianus var. lactis only. Kefir B had the flavour and Elsevier, Amsterdam.
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significantly stronger carbonated taste than kefir C ( Table 4 ) . 21 Krusch U. 1884. Mikrobiologische charakterisierung von kefir.
Bakteriologie, Keimzahlen, Hauptgarprodukte. Dtsch Molk - Ztg 105:
There were significant differences in alcohol and CO2 concen-
1332 – 1336.
trations ( three and six times higher in kefir B ) , which have an
22 Kubo M, T Odani, S Nakamura, S Tokumaru and H Matsuda. 1992.
important role in the formation of kefir flavour and aroma. Pharmacological study on kefir — a fermented milk product: I.
It can be concluded that the composition of lactic acid Antitumour activity. J Pharm Soc Jpn 112: 489 – 495.
23 Libudzisz Z and A Piatkiewicz. 1990. Kefir production in Poland.
microflora in the samples of kefir grains and kefir was quite
Dairy Ind Int 55: 31 – 33.
homogeneous. Along the pathway A!B!C, streptococci
24 Marshall V. 1993. Starter cultures for milk fermentation and their
increased their proportion in the total kefir microflora by 26 – characteristics. J Soc Dairy Technol 46: 49 – 56.
30%, whereas lactobacilli decreased by 13 – 23%. The composi- 25 Mitsue T, K Tachibana and Y Fujio. 1999. Efficient kefiran production
tion of the yeast flora undergoes a complete change at the by a mixed culture of Lactobacillus kefiranofaciens KF - 75 and yeast
strains. Seibutsu - Kogaku Kaishi 77: 99 – 103.
expense of the lactose - negative yeasts. K. marxianis var. lactis
26 Neve H. 1992. analysis of kefir grain starter cultures by scanning
was permanently present in kefir grains and kefirs, whereas the
electron microscopy. Milchwissenschaft 47: 275 – 278.
dominant lactose - negative yeasts in the total yeast flora of kefir 27 Pintado M, J Lopez da Silva, P Fernandes, F Malcato and T Hogg.
grains decreased drastically in kefir C. Lactic acid bacteria and 1996. Microbiological and rheological studies on Portuguese kefir
grains. Int J Food Sci Technol 31: 15 – 26.
yeasts isolated from kefir grains can be used to form kefir
28 Rehm H and M Lehner. 1990. Characteristische Eigenschaften von
starters.
osterreichischem Kefir. Ernahrung 14: 571 – 574.
29 Rosi J. 1978. The kefir microorganisms: acetic acid bacteria. Sci Tec
Latt - Casearia 29: 221 – 227.
30 Rossi J and M Gobbetti. 1991. Use of a multi - component starter
References for continuous manufacture of kefir. Ann Microbiol Enzimol
1 Angulo L, E Lopez and C Lema. 1993. Microflora present in kefir 41:223 – 226.
grains of the Galician region ( North - West of Spain ) . J Dairy Res 60: 31 Schleifer K. 1986. Gram - positive cocci. In: Sneath P, N Mair, M
263 – 267. Sharpe and J Holt ( Eds ) , Bergey’s Manual of Systematic
2 Barnett J, R Payne and D Yarrow. 1990. Yeasts: characterisation and Bacteriology. Williams & Wilkins, Baltimore, MD. vol. 2, section 12,
identification. Cambridge Univ Press, Cambridge, UK. pp. 1043 – 1071.
3 Barrere C, M Ducasse, C Vigne, M Vidal and A Tallec. 1978. Dosage 32 Takizawa S, S Kojima, S Tamura, S Fujinaga, Y Benno and T Nakase.
du CO2 dans les vins. Vignes et Vins 273: 31 – 38. 1994. Lactobacillus kefirgranum sp. nov. and Lactobacillus parakefir
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6 sp. nov., two new species from kefir grains. Int J Syst Bacteriol 44: characterisation of lactic acid bacteria and yeasts isolated from kefir
435 – 439. grains in Taiwan. Aust J Dairy Technol 54: 14 – 18.
33 Takizawa S, S Kojima, S Tamura, S Fujinaga, Y Benno and T Nakase. 36 Wyder M, H Spillman and Z Puhan. 1997. Investigation of the yeast
1998. The composition of the Lactobacillus flora in kefir grains. Syst flora in dairy products: a case study of kefir. Food Technol Biotechnol
Appl Microbiol 21: 121 – 127. 35: 299 – 304.
34 Weis W and G Burgbacher. 1986. 100 Jahre Kefir in Deutschland - Nach 37 Yokoi H, T Watanabe, Y Fujii, T Toba and S Adachi. 1990. Isolation
wie vor ein aktuelles Thema. Untersuchung von ‘‘Kefir’’ aus and characterization of polysaccharide - producing bacteria from kefir
Molkereien und Handel sowie dessen Problematik. Dtsch Milch- grains. J Dairy Sci 73: 1684 – 1689.
wirtschaft 37: 81 – 90. 38 Yoshida T and K Toyoshima. 1994. Lactic acid bacteria and yeast from
35 Wen L, C HsiaoLing and L JeRuei. 1999. Identification and kefir. J Jpn Soc Nutr Food Sci 47: 55 – 59.
1
Laboratory of Applied Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences, 26 Maritza Boulevard,
4002 Plovdiv, Bulgaria; 2Higher Institute of Food and Flavour Industries, 26 Maritza Boulevard, 4002 Plovdiv, Bulgaria;
3
ELBY Center of Research, Development and Production, 44 - A Lagera Boulevard, 1612 Sofia, Bulgaria
In an investigation of the changes in the microflora along the pathway: kefir grains ( A ) ! kefir made from kefir grains
( B ) !kefir made from kefir as inoculum ( C ) , the following species of lactic acid bacteria ( 83 – 90% ) of the microbial
count in the grains ) were identified: Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus
delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus casei subsp. pseudoplantarum and
Lactobacillus brevis. Yeasts ( 10 – 17% ) identified were Kluyveromyces marxianus var. lactis, Saccharomyces
cerevisiae, Candida inconspicua and Candida maris. In the microbial population of kefir grains and kefir made from
them the homofermentative lactic streptococci ( 52 – 65% and 79 – 86%, respectively ) predominated. Within the group
of lactobacilli, the homofermentative thermophilic species L. delbrueckii subsp. bulgaricus and L. helveticus ( 70 –
!!
87% of the isolated bacilli ) predominated. Along the pathway A! B! C, the streptococcal proportion in the total kefir
microflora increased by 26 – 30% whereas the lactobacilli decreased by 13 – 23%. K. marxianus var. lactis was
permanently present in kefir grains and kefirs, whereas the dominant lactose - negative yeast in the total yeast flora of
the kefir grains dramatically decreased in kefir C.
Journal of Industrial Microbiology & Biotechnology ( 2002 ) 28, 1 – 6 DOI: 10.1038 / sj / jim / 7000186
Keywords: kefir grains; kefir; lactic acid bacteria; yeasts; identification; composition
Introduction Materials and methods
The wholesome properties of fermented milks ( kefir, kumis ) and Kefir grains and conditions of cultivation
their nutritional value have motivated a considerable interest in The kefir grains studied were from the collection of the Center of
such ‘‘novel’’ milk foods [ 10,20,22 – 24 ] . Kefir has been defined Research, Development and Production ( LB Bulgaricum Trading,
as the yogurt of the 21st century [ 9 ] . Kefir differs from other Bulgaria ) . The lyophilized kefir grains were activated for 1 month
fermented milks in its starter, which exists in the form of ‘‘grains.’’ in the following way: Sterilized skim milk ( 0.1% milk fat, 1 - l
The grains have a specified structure and behave as biologically flasks, 1158C, 15 min ) was cooled to 18 – 208C, and inoculated
vital organisms. They grow, propagate and pass their properties on using kefir grains ( the weight ratio of kefir grains to milk was 1:10 )
to the following generations of new grains. The microflora of kefir and incubated at 18 – 208C for 24 h with three to five stirrings. Kefir
grains is remarkably stable, retaining its activity for years if grains were separated from the fermented milk by filtration through
preserved and incubated under appropriate cultural and physio- a sieve and were washed with cooled sterile water before being used
logical conditions. Kefir grains have a complex microbiological
in the next culture passage. Activated kefir grains were propagated
composition. Lactobacilli, lactic streptococci, yeasts and acetic as follows: From the grown and fresh kefir grains six grains were
acid bacteria have been shown to be present in them selected. Each was cut and cultivated separately in sterile skim milk
[ 23,25,27,28,32,37,38 ] . The kefir - specific yeasts play a key role under the conditions given. After daily transfers of the particles of
in the formation of flavour and aroma [ 8 ] . They are represented by the six grains into sterile milk for a period of 2 weeks, the new kefir
the species Kluyveromyces marxianus, Torulaspora delbrueckii, grains were cut again. This procedure of transferring and cutting
Saccharomyces cerevisiae, Candida kefir, Saccharomyces unispo- lasted ( about 2 months ) until a sufficient amount of new kefir
rus, Pichia fermentans and Yarrowia lypolytica [ 1,18,28 ] . For the grains was obtained from the initial six. The six populations of kefir
formation of a kefir starter culture, which would produce a milk grains thus obtained were used in the present study. Five grains
beverage with characteristics similar to those of traditional kefir, were taken out of each population; each was cut and the pieces were
more information needs to be acquired about the specific cultures
thoroughly washed with sterile water and suspended in a sterile
isolated from kefir grains. solution of 0.9% NaCl. By plating the diluted homogenates of every
The aim of this study was to describe changes in the microbial one of the grains on appropriate cultural media, strains of lactic acid
population in kefir grains while they underwent two subsequent bacteria and yeasts were isolated [ 12 – 15 ] .
fermentation cycles: kefir grains!kefir!second batch of kefir.
Isolation and identification of lactic acid bacteria from
Correspondence: E Simova, Laboratory of Applied Microbiology, Institute of
kefir grains
Microbiology, Bulgarian Academy of Sciences, 26 Maritza Boulevard, 4002 Plovdiv,
For isolation of the lactobacilli from kefir grains, 0.1 - ml portions of
Bulgaria
the respective dilutions of homogenates were plated on MRS agar
Received 2 August 2000; accepted 15 July 2001
Lactic acid bacteria and yeasts in kefir grains
E Simova et al
2
[ 2 ] were applied. The yeasts were also identified on the basis of
( Fluka RdH, Buchs, Switzerland ) and on Rogosa agar with cheese
fermentation tests ID 32C ( bioMerieux ) using API LAB Plus
whey ( Fluka RdH ) [17]. The plates were incubated at 308C and
software.
378C for 3 – 7 days in aerobic and anaerobic (10% CO2 )
atmospheres. For isolation of lactic streptococci the diluted
homogenates were plated on M-17 medium (Fluka RdH), and Preparation of kefir, isolation and identification of lactic
azide agar medium (Difco, Detroit, USA) was used aerobically.
acid bacteria and yeasts
The plates were incubated at 288C and 378C for 4 – 8 days.
Using kefir grains A from the six populations obtained, six samples
After microscopic examination and on the basis of their
of traditional kefir ( kefir B ) were prepared as follows: Milk ( 3%
morphology, the colonies were divided into groups and trans-
milk fat ) , pasteurised at 928C for 15 min, homogenized at 12.5 –
ferred in triplicate in sterile skim milk and on plates. Some of
17.5 MPa and cooled to 18 – 208C, was inoculated with 3% kefir
the lactobacilli, isolated directly from the kefir grains, appeared
grains. Incubation was at 18 – 208C for 18 h ( pH = 4.5 – 4.6 )
to have very limited fermentative capability until further passage
followed by coagulum cooling to 8 – 108C and kefir ripening for
through laboratory media or sterile skim milk. By microscopic
24 h. After separation of the kefir grains from the six kefir B
analysis of each culture following incubation, confirming the
samples, strains of lactic acid bacteria and yeasts were isolated and
purity of the isolates and their activation, the cultures were
identified on the basis of taxonomic characteristics and biochemical
characterized taxonomically according to the criteria described in
tests as already described.
Bergey’s Manual [ 16,31 ] . Identification was carried out also on
The six kefir B samples were used as starter cultures for
the basis of biochemical tests with API 20A ( for identification
preparing samples of kefir C. Kefir C was made in the following
of anaerobic bacteria ) and with API 50CH ( test for lactobacilli
way: Milk ( 3% milk fat ) was pasteurised at 928C for 15 min,
and lactococci — API 50CHL Medium, bioMerieux, Marcy
homogenized at 12.5 – 17.5 MPa and cooled to 22 – 238C. Each of
l’Etoile, France ) using the software of the company API LAB
the six milk samples was inoculated with 3% of the sample of kefir
Plus.
B. Fermentation was carried out at 238C for 8 – 12 h to pH = 4.5 –
4.6, followed by cooling to 8 – 108C with simultaneous ripening for
Isolation and identification of yeasts from kefir grains 10 – 12 h. The microflora of kefir C samples was then isolated and
Yeasts were isolated by surface spreading on plates of malt extract identified as already described.
agar ( Fluka RdH ) and YM agar ( Fluka RdH ) [ 19 ] . After
incubation at 288C for 3 – 6 days, colonies of different morphology
were obtained. Initial cultures were prepared from these colonies.
Analytical methods
Inclined agar tubes were inoculated and after incubation ( 288C for
3 days ) an initial suspension was prepared from each culture. A In order to calculate the percentage of each characterized
solution of yeast extract – peptone – glucose ( 0.45%, 0.75%, 1.0% ) microorganism in kefir grains A, kefir B and kefir C, the
was used as medium, whose pH value was corrected to 6.4 by number of viable bacteria and yeasts expressed as colony -
forming units per ml ( CFU ml À 1 ) was estimated. Serial dilutions
adding tartaric acid. After sterilization, the glucose was added to the
medium as a sterile filtered solution. The medium was inoculated of each sample were plated in triplicate and plates were incubated
with one loop of the inclined agar culture and the sample was at 308C and 378C ( for lactic acid bacteria ) , and 288C ( for
incubated to the appearance of distinct darkening. For identification yeasts ) until growth of the colonies. The results were the mean
of the yeasts the methods of Kreger van Rij [ 19 ] and Barnett et al of six counts at three different dilutions.
Table 1 Composition of the microflora of kefir grains from six populations
Microorganisms Colony count in population no.
1 2 3 4 5 6
22 ± 2.0 ( 83 ) a 19 ± 2.6 ( 88 ) 15 ± 44 ( 90 ) 24 ± 3.6 ( 84 ) 16 ± 4.4 ( 87 ) 21 ± 2.6 ( 83 )
Lactic acid bacteria
Streptococci 14 ± 2.6 ( 53 ) 13 ± 2.0 ( 61 ) 11 ± 1.7 ( 65 ) 16 ± 2.0 ( 55 ) 10 ± 1.7 ( 54 ) 13 ± 3.0 ( 54 )
L. lactis subsp. lactis 10 12 9 14 8 10
S. thermophilus 4 1 2 2 2 3
Lactobacilli 8 ± 2.0 ( 30 ) 6 ± 2.6 ( 27 ) 4 ± 1.0 ( 24 ) 8 ± 3.0 ( 29 ) 6 ± 3.0 ( 33 ) 8 ± 2.6 ( 29 )
L. delbrueckii subsp. bulgaricus 5 2 2 4 3 4
L. helveticus 2 2 – 2 2 2
L. casei subsp. pseudoplantarum – 1 1 1 – –
L. brevis 1 1 1 – 1 2
Yeasts 5 ± 1.7 ( 17 ) 3 ± 2.0 ( 12 ) 2 ± 1.0 ( 10 ) 5 ± 1.7 ( 16 ) 2 ± 1.0 ( 13 ) 5 ± 2.0 ( 17 )
K. marxianus var. lactis 1 1 – 1 – 1
S. cerevisiae 2 – – 2 – 1
C. inconspicua 2 1 1 1 2 2
C. maris – 1 – – – 1
Unidentified – – 1 1 – –
Data represent mean values for five grains from each population and standard deviation.
a
Data in parentheses represent percentage of total microflora.
Lactic acid bacteria and yeasts in kefir grains
E Simova et al
3
Table 2 Composition of the microflora of kefir samples ( kefir B ) prepared with kefir grains from six populations
Microorganisms Colony count in population no.
1 2 3 4 5 6
24 ± 3.6 ( 94 ) a 24 ± 5.2 ( 94 ) 20 ± 6.2 ( 92 ) 24 ± 3.6 ( 92 ) 20 ± 2.6 ( 96 ) 20 ± 2.6 ( 95 )
Lactic acid bacteria
Streptococci 19 ± 2.6 ( 74 ) 20 ± 2.6 ( 78 ) 16 ± 2.0 ( 76 ) 20 ± 2.6 ( 77 ) 16 ± 4.3 ( 78 ) 16 ± 4.3 ( 76 )
L. lactis subsp. lactis 15 18 13 17 12 13
S. thermophilus 4 2 3 3 4 3
Lactobacilli 5 ± 1.7 ( 20 ) 4 ± 1.0 ( 16 ) 4 ± 2.0 ( 16 ) 4 ± 1.7 ( 15 ) 4 ± 1.0 ( 18 ) 4 ± 1.7 ( 19 )
L. delbrueckii subsp. bulgaricus 2 2 2 3 2 2
L. helveticus 2 – – 1 1 2
L. casei subsp. pseudoplantarum – 1 4 – – –
L. brevis 1 1 1 – 1 –
Yeasts 1±0 (6) 2 ± 1.0 ( 6 ) 2 ± 1.7 ( 8 ) 2 ± 1.0 ( 8 ) 1±0 (4) 1±0 (5)
K. marxianus var. lactis 1 1 – 1 – 1
S. cerevisiae – – – 1 – –
C. inconspicua – – 1 – 1 –
C. maris – 1 – – – –
Unidentified – – 1 – – –
Data represent the mean values from three kefir samples from each population and standard deviation.
a
Data in parentheses represent percentage of total microflora.
were identified: the mesophilic species Lactococcus lactis subsp.
Viable streptococci were estimated on plates of M - 17 agar,
lactis ( Streptococcus lactis ) and the thermophilic species Strepto-
viable lactobacilli count on plates of MRS agar and the total yeast
coccus thermophilus. These two species were the predominating
count was determined using YM agar.
microflora in all kefir grains ( 53 – 65% of the total microflora ) and
Lactic acid and alcohol were determined by enzymatic methods
samples of kefir ( 74 – 78%; 79 – 86% ) . S. lactis prevailed over
as described by Boehringer Mannheim [ 4 ] . CO2 content ( as
S. thermophilus ( up to 12 - fold in kefir grains and up to 14 - fold in
dissolved gas ) was analysed by the enzymatic method for dissolved
kefir samples ) . During fermentation the proportion of streptococci
CO2 [ 3,6 ] . Viscosity ( cst ) was measured on an Oswald ( Sibata,
increased and reached 79 – 86% of the total microflora in kefir C.
Japan ) cinematic viscosimeter.
The following homofermentative lactobacilli were isolated: Lacto-
bacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus and
Lactobacillus casei subsp. pseudoplantarum. The heterofermenta-
Results tive lactobacillus Lactobacillus brevis was also isolated. Lactoba-
cilli constituted about 24 – 33% of the total flora in the kefir grains.
The distribution of the species of lactic acid bacteria and yeasts in
A twofold decrease in the quantity of lactobacilli in the pathway
the kefir grains A and in the samples of kefir B and kefir C is given
‘‘A!B!C’’ was noted. Of the isolated lactobacilli in all the
in Tables 1 – 3. Two species of homofermentative lactic streptococci
Table 3 Composition of the microflora of kefir samples ( kefir C ) prepared with B as inoculum
Microorganisms Colony count in population no.
1 2 3 4 5 6
17 ± 4.0 ( 94 ) a 17 ± 2.6 ( 96 ) 18 ± 4.4 ( 96 ) 19 ± 2.6 ( 95 ) 22 ± 2.6 ( 96 ) 16 ± 2.0 ( 95 )
Lactic acid bacteria
Streptococci 14 ± 2.6 ( 79 ) 15 ± 4.4 ( 84 ) 16 ± 2.0 ( 85 ) 17 ± 2.6 ( 86 ) 20 ± 2.6 ( 86 ) 13 ± 2.6 ( 79 )
L. lactis subsp. lactis 11 14 14 13 15 10
S. thermophilus 3 1 2 4 5 3
Lactobacilli 3 ± 1.0 ( 15 ) 2 ± 1.7 ( 12 ) 2 ± 1.0 ( 11 ) 2 ± 1.7 ( 9 ) 2 ± 1.0 ( 10 ) 3 ± 1.7 ( 16 )
L. delbrueckii subsp. bulgaricus 1 1 1 1 1 1
L. helveticus 1 – – 1 1 1
L. casei subsp. pseudoplantarum – 1 1 – – –
L. brevis 1 – – – – 1±0
Yeasts 1±0 (6) 1±0 (4) 1±0(4) 1±0 (5) 1±0 (4) 1±0 (5)
K. marxianus var. lactis 1 1 – 1 – 1
S. cerevisiae – – – – 1 –
C. inconspicua – – 1 – – –
C. maris – – – – – –
Unidentified – – – – – –
Data represent the mean values from three kefir samples ( kefir C ) prepared with kefir B as inoculum and standard deviation.
a
Data in parentheses represent percentage of total microflora.
Lactic acid bacteria and yeasts in kefir grains
E Simova et al
4 Table 4 Characteristics of kefir B and kefir C after 24 h storage at 48C
Kefir Ba Kefir Cb
Component
pH 4.5 ± 0.26 4.35 ± 0,20
Lactic acid ( g l À 1 ) 8.18 ± 0.61 8.20 ± 0,57
Alcohol ( % ) 0.25 ± 0.04 0.09 ± 0,04
Carbon dioxide ( g l À 1 ) 0.85 ± 0.15 0.15 ± 0,06
Mesophilic streptococci ( CFU ml À 1 ) 8 ( ± 4.58 ) .109 6 ( ± 2,64 ) .109
Thermophilic streptococci ( CFU ml À 1 ) 3 ( ± 2,64 ) .106 4 ( ± 2,00 ) .106
Homofermentative lactobacilli ( CFU ml À 1 ) 8 ( ± 2,64 ) .106 2 ( ± 1,00 ) .106
Yeasts ( CFU ml À 1 ) 2 ( ± 2,64 ) .105 1 ( ± 2,00 ) .104
Viscosity ( cst ) 1.075 ± 0,21 1.043 ± 0,28
Aroma strong, typically yeasty very light yeasty
Flavour kefir - specific, refreshingly pungent predominating lactic acid
Texture homogeneous, creamy, abundantly effervescent slightly viscous, very slight effervescence
a
Kefir B was prepared with kefir grains No. 4 and received the best organoleptic evaluation of kefirs B, prepared with the six populations.
b
Kefir C was prepared with kefir Ba as inoculum.
Data represent the mean values from three kefir B and kefir C, and their standard deviations.
L. casei subsp. pseudoplantarum was found in the populations
samples L. bulgaricus prevailed, followed by L. helveticus. During
from three kefir grains and in two samples of kefir B and kefir C.
the two consecutive fermentations L. helveticus decreased. The
The presence of this species in kefir grains has been noted in other
heterofermentative species L. brevis, present in five out of the six
studies [ 7 ] . According to some authors, the heterofermentative
populations of kefir grains, dramatically decreased, and in kefir C it
lactobacilli predominate over the homofermentative ones [ 1 ] , and
was found in two out of six samples. L. casei subsp. pseudoplan-
according to others, they are the only representatives of the
tarum was found in three out of six samples of kefir grains in kefir
lactobacilli [ 38 ] . The isolated species L. brevis decreased along the
B and kefir C.
pathway A!B!C. These results are very similar to those of
The lactose - fermenting yeast K. marxianus var. lactis was
Takizawa et al [ 33 ] , who found that 90% of the bacterial species in
found in kefir grains A, kefir B and kefir C together with non -
kefir grains were homofermentative lactobacilli. In other studies,
lactose - fermenting yeasts ( S. cerevisiae, Candida inconspicua,
however, the lactobacilli represented 65 – 80% of the microbial
Candida maris and two unidentified yeasts ) . The yeast microflora
count of the grain, with streptococci and various yeasts as the
of kefir grains was marked by the prevailing presence of lactose -
remainder [ 1,23 ] .
negative yeasts ( 60 – 100% of the total quantity of yeasts ) . Among
Yeasts in kefir grains represented 10 – 17% of the total isolated
then the species C. inconspicua predominated, followed by
microflora with lactose - negative yeast species predominating
S. cerevisiae.
( 66 – 100% ) . The inability of these yeasts to ferment lactose
In the preliminary organoleptic evaluation of kefirs B prepared
makes them dependent on lactic acid bacteria capable of
with kefir grains from the six populations, kefir B prepared with
hydrolyzing this disaccharide. The prevalence of lactose - negative
kefir grains from population No. 4 ranked first with the best
yeasts in the grains was consistent with the studies carried out by
characteristics. The data comparing the physicochemical and
other authors [ 29,36 ] . It is worth noting that the group of
microbiological composition and organoleptic properties of kefir
lactose - negative yeasts contains the two species C. inconspicua
B with population No. 4 and those of kefir C, prepared with kefir B,
and C. maris, whose presence in kefir has not been reported
are shown in Table 4.
previously. C. inconspicua predominated in the yeast microflora
of the kefir grains and drastically decreased along the pathway
A!B!C. C. maris was present in the microflora of two kefir
Discussion grains. These two species did not ferment or assimilate galactose.
C. inconspicua assimilated DL -lactic acid and was, therefore,
Lactic acid bacteria represented 83 – 90% of the microbial count of
the grains. In the microbial populations of the kefir grains, the completely dependent on the metabolism of lactic acid bacteria.
homofermentative species of lactic acid streptococcus S. lactis C. maris did not assimilate DL - lactic acid. Possibly, its presence
prevailed in kefir B and kefir C ( 40 – 55% and 60 – 80%, in kefir grains was connected not only with the presence of
respectively ) , which determined it as the main fermenting glucose and certain vitamins ( thiamine, pyridoxine ) , but also
organism. Our results differ from those studies that found a very with the assimilation of some acids produced by lactic acid
scarce presence of streptococci ( a single diplococcal cell ) in the bacteria by the transformation of pyruvic acid. The lactose -
grain - associated population [ 5,26 ] . However, a number of authors positive species K. marxianus var. lactis was identified in the
consider S. lactis as an integral part of the microflora of kefir grains isolates from four kefir grains. Its presence in kefir grains has
irrespective of its low numbers [ 11,30 ] . been reported in other studies [ 1,35,36 ] . According to some
Within the lactobacilli, the thermophilic homofermentative authors, K. marxianus var. lactis isolated from kefir grains is
species L. bulgaricus and L. helveticus ( 70 – 87% of the isolated distinguished by the peculiarity of always occurring with lactose -
lactobacilli ) predominated. There are reports on the presence of negative yeasts, which is also confirmed by our results [ 1 ] .
L. helveticus as a prevailing member of the lactic acid microflora in The composition of the yeast flora in kefir B and kefir C was
kefir grains [ 35 ] . The mesophilic homofermentative species highly dependent on the starter used. During fermentation and
Lactic acid bacteria and yeasts in kefir grains
E Simova et al
5
4 Boehringer Mannheim GmbH Biochemica. 1983. In: Methods of
ripening, alterations occurred only in the group of lactose -negative
enzymatic food analysis using test combinations. Boehringer
yeasts. In kefir B samples, the lactose - negative species ( five out
Mannheim GmbH Biochemica, Mannheim, Germany, pp. 17 – 18,
of nine isolated yeast species ) predominated slightly. In all kefir C 33 – 34.
samples, where K. marxianus var. lactis was present, other yeast 5 Duitschaever C, N Kemp and A Smith. 1988. Microscopic studies of
the microflora of kefir grains and kefir made by different methods.
species were not identified. In two of the samples where
Milchwissenschaft 43: 479 – 481.
K. marxianus var. lactis was not found, the species C. inconspicua
6 Forrester R, L Wataji, D Silverman and K Pierre. 1976. Enzymatic
and S. cerevisiae were identified. The unidentified yeast present in method for determination of CO2 in serum. Clin Chem 22: 243 – 245.
kefir B disappeared in kefir C. The quantity of the lactose - 7 Galli A, E Fiori, L Franzetti, M Pagani and G Ottogalli. 1995.
fermenting yeast K. marxianus var. lactis remained the same in the Composizione microbiologica e chimica dei granuli di Kefir ‘‘di
frutta.’’ Ann Microbiol Enzimol 45: 85 – 95.
kefir grains, traditional kefir B and kefir C. The total yeast
8 Glaeser H, E Hangst and K Ziegler. 1986. Biochemische charakter-
quantity decreased because of the decrease of lactose - negative
isierung der in molkereikefir und kefirkulturen vorkommende hefen.
yeast in the course of the process: kefir grains A!kefir B!kefir Dtsch Molk - Ztg 16: 483 – 490.
C. A number of authors maintain that lactose - fermentative yeast 9 Gorski D. 1994. Kefir: 21st century yogurt? Dairy Foods 95: 49.
10 Honer C. 1993. Now kefir. Dairy Field 176: 91.
should be defined as one of the main components of the ‘‘specific
11 Hosono A, T Tanabe and H Otani. 1990. Binding properties of lactic
cultures,’’ whereas the lactose - negative yeasts should be desig-
acid bacteria isolated from kefir milk with mutagenic amino acid
nated as recontamination yeast [ 8,21,34 ] . Participation of pyrolyzates. Milchwissenschaft 45: 647 – 651.
K. marxianus var. lactis in the microflora of the kefir grains 12 IDF Standard 94 B. 1990. Milk and milk products - enumeration of
ensures metabolism of lactose through alcohol fermentation and yeast and moulds — colony count technique at 258C, Brussels,
Belgium.
the formation of the typical yeasty flavour and aroma. According
13 IDF Standard 146. 1991. Yogurt identification of characteristic
to some authors, kefir must contain an approximate minimum of
microorganisms ( Lactobacillus delbrueckii subsp. bulgaricus and
1Â105 lactose - fermenting yeasts to obtain the characteristic Streptococcus salivarius subsp. thermophilus ) , Brussels, Belgium.
flavour and aroma [ 8 ] . That statement was supported by four 14 IDF Standard 163. 1992. General standard of identity for fermented
milks, Brussels, Belgium.
samples of kefir C ( out of six ) and two samples of kefir B ( out of
15 IDF Standard 117 B. 1997. Yogurt - enumeration of characteristic
six ) , in which only K. marxianus var. lactis was present ( Tables 2
microorganisms — colony count technique at 378C, Brussels,
and 3 ) . On the other hand, special emphasis should be laid upon Belgium.
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17 Kojima S, S Takizawa, S Tamura, S Fujinaga, Y Benno and T Nakase.
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which contained lactose - negative yeasts, were noticed to possess
kumis starters. Bull IDF 227: 35 – 40.
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19 Kreger van Rij NJW. 1984. The Yeast: A Taxonomic Study. 3rd ed.
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22 Kubo M, T Odani, S Nakamura, S Tokumaru and H Matsuda. 1992.
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