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Journal of Culture Collections, Volume 3, 2000-2002, pp. 48-52
IDENTIFICATION OF LACTIC ACID BACTERIA ISOLATED FROM
KATYK, GOAT’S MILK AND CHEESE Lilia Tserovska*, Stanka Stefanova and Tanya Yordanova Code Number: cc02007 Summary Eighteen lactic
acid bacterial strains were isolated from home made katyk, goat’s milk
and cheese. Nine of them belonged to lactic acid cocci and others were
referred to genus Lactobacillus. Five strains were identified to species
by determination of morphological, cultural, physiological and biochemical
characteristics - Pediococcus acidilactici, Pediococcus pentosaceus, Lactobacillus
delbrueckii ssp. bulgaricus, Lactobacillus helveticus and Lactobacillus
plantarum. Lactic acid bacteria are widely distributed in the nature.
In this group are included representatives of the genus Lactobacillus,
Lactococcus, Pediococcus and Leuconostoc. They could be isolated
from soils, waters, plants, silages, waste products, and also from the intestinal
tract of animals and humans. The lactic acid fermentation, which these bacteria
perform has long been known and applied by the humans for making different
foodstuffs. It plays an essential role in the production of all dairy products
and is involved in the production of many other foods and drinks – sausages,
pickles, boza etc. Since decades by these processes the application of well-studied
starter cultures was established. They should possess stable fermentation
characteristics and should be resistant to bacteriophages [5]. In our country
at domestic conditions dairy products are prepared from cow, but also from
sheepish, goat’s and buffalo-cow’s milk – such as cheese, curds, katyk (specific
national dairy product), yellow cheese, etc. By these products the species
composition of lactic acid bacteria is more various and inconstant when compared
with those of the trade products. The “wild” strains, in biotechnological
aspect are perspective as bacteriocin producers [6, 10] and probiotics [3,
4]. They have a potential use for the establishing of new, the so-called “functional
foods” [3, 9]. The aim of the
recent study was the isolation and taxonomic determination of lactic acid
bacteria from home made katyk and products of goat’s milk in order for their
including in original Bulgarian collection of lactic acid strains. Isolation of lactic acid bacteria. The lactic acid
bacteria were isolated form home made katyk of cow’s milk in the Sofia
region (2 samples), yogurt of goat’s milk (2) and goat’s cheese (2) of Tabashko
village, Troyan region. The isolation was performed by the routine microbiological
procedure and inoculation on a solid medium. Selective media for lactic acid
bacteria were used – MRS-agar and M-17 agar. The cultivation was performed
without shaking at the appropriate temperature (32 and 40°C) for five
days. From katyk were isolated only termophilic strains and they were cultivated
at temperature 42-45°C. Once single colonies were obtained they were
inoculated in MRS broth or M-17 broth. After growing the cultures were diluted
and tested for purity on the suitable agar medium. Identification of the bacterial strains. The determination
of the strains was performed according to their morphological, cultural,
physiological and biochemical characteristics by the procedures described
in the Bergey’s Manual [1]. For performing the biochemical tests a medium
with the following com-position was used (g/l): bovine extract – 10.0; neopeptone – 10.0;
yeast extract – 5.0; Tween 80 –1 ml; K2HPO4 – 2.0;
CH3COONa x 3H2O – 5.0; diamonium citrate – 2.0; MgSO4 – 0.2;
MnSO4 – 0.05; bromcresol purple – 0.17. The carbon source was
added to the sterile basal medium as filter sterilized solution to a final
concentration of 1 %. Carbohydrates utilization was assessed at the 24th,
48th hour and on the 7th day of the strain growth at
the corresponding temperature. For identification of the lactic acid bacteria API 50 CH
tests (bioMerieux) were also used. From the tested samples eighteen lactic acid bacterial cultures
were isolated. After the original characterization nine of them were determined
as representatives of the lactic acid cocci, and the rest were referred to
genus Lactobacillus. The strains K1, K2, Ch1, Ch2 and Ch3 were
determined to species. The strains K1 and K2 isolated from katyk were cocci – Gram-positive,
nonspore-forming, nonmotile, catalase-negative, facultative an-aerobes. Their
morphological, cultural and physiological characteristics are shown in Table
1, and the biochemical – in Table 2. As a
result of the investigations the strain K1 was determined as Pediococcus
acidilactici and the strain K2 as Pediococcus pentosaceus.
Both strains curdled the milk very well, which is not typical for the pediococci.
This characteristic could be explained with adaptive alterations as a result
of growth in the tested substrate. The strains were deposited in the National
Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) with accession
numbers 3437 and 3545, respectively, and were kept in a freeze-dried state.
Representatives of these two species from the genus Pediococcus find
biotechnological application by the processing and conservation of meat and
vegetable foods and play an essential role as components of trade starter
cultures by sausage production [8]. Table 1. Morphological, cultural and physiological characteristics of the isolated strains.
Legend: positive reaction (+), negative reaction (-), not determined (ND). Table 2. Biochemical characteristics of the tested strains – acid production by utilization of carbon sources.
Legend: positive reaction (+), negative reaction (-), not determined (ND). The other three lactic acid cultures were referred to genus Lactobacillus.
They are Gram-positive, nonspore-forming rods, catalase-negative, microaerophilic,
with fermentative metabolism. For their identification to species the morphological,
cultural, physiological and biochemical characteristics were tested. The
data are shown in Table 1 and Table
2. The strain Ch1 could be determined as a representative of
the obligate homofermentative species Lactobacillus delbrueckii. The
all three subspecies of the species (subspp. bulgaricus, delbrueckii and lactis)
are characterized with a narrow fermentation profile. The studied strain
was able to ferment glucose, fructose, lactose, galactose and maltose. It
is the closest to Lactobacillus delbrueckii ssp. bulgaricus although
the dissimilation of the last two carbohydrates is not characteristic for
the type strain. As a result of the taxonomic studies the tested strain Ch2
was determined as Lactobacillus helveticus. Its morphological, cultural
and biochemical characteristics were identical with those of the type strain.
This species belongs to the group of the termophilic lactobacilli, obligate
homofermentative type and is included in starter cultures by the production
of the cheese Gruyere, Gorgonzola, Mozarella [2]. The phenotype analysis of the strain Ch3 indicated its relativity
to Lactobacillus plantarum. This strain belongs to the group of the
mesophilic lactobacilli, which are commonly met in the later phase of the
maturing of the cheese, together with Lb. casei, Lb. brevis and Lb.
buchneri [2]. Olarte noted that the presence of Lb. plantarum by
the production of the cheese Cameros from goat’s milk decreased the number
of the enterobacteria and fecal coliforms in the final product [7]. The two strains from genus Pediococcus were isolated
from katyk – Pediococcus acidilactici and Pediococcus pentosaceus.
From goat’s yogurt was isolated Lactobacillus delbrueckii ssp. bulgaricus and
from home made goat’s cheese Lactobacillus helveticus and Lactobacillus
plantarum. These strains showed some non-essential phenotypic differences
from the type strain due to the specificity of the substrates and the growth
conditions. The “wild” strains have potential as producers of growth inhibition
factors, which play important role in the interaction within the mixed cultures
and the investigations in this direction will continue. References
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