|Kefir??? 무엇이지요? Lactobacillus와 Kluyveromyces의 mixed culture입니다....|
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J. Gen. Appl. Microbiol., 52, 375–379 (2006)
Isolation and molecular taxonomy of two predominant types of microflora in Kefir
Jin-Chul Heo1 and Sang-Han Lee1,2,*
1 Food & Bio-industry Research Institute and 2 Department of Food Science & Technology,
Kyungpook National University, Daegu 702–701, Korea
Key Words——Kefir; Kluyveromyces marxianus; Lactobacillus kefiri; phylogenetic analysis; rDNA sequencing
Kefir is a traditional fermented beverage in the Caucasian Mountains that can be prepared with fresh
milk. It is known that Kefir is composed by a symbiotic association of lactic acid bacteria and yeasts
living together into gelatinous and irregular materials secreted by them. This association sometimes
is mistaken for mushrooms like Camella assamica and Cordyceps sinensis. Kefir is similar to products
that exhibit some anti-bacterial, anti-mycotic, anti-tumor, and anti-inflammatory activity. Although
Kefir and its related products (Tibetan mushrooms) are very similar in structure, microbial content,
cultivation procedures and fermentation products, Kefir is usually reported to lead to health benefits
of a probiotic nature (Cevikbas et al., 1994;Diniz et al., 2003).
Microbial strains belonging to the genera, Lactobacillus, and Streptococcus are known to exist in
fermented products such as yoghurt, cheese, and kimchi, but there is no specific data on those
involved in the fermentation of Kefir (Baruzzi et al., 2000; Simova et al., 2002). Therefore, the
present study was undertaken to isolate and identify the predominant microflora involved in Kefir fermentation.
In this work, we describe the phylogenetic characteristics of the two major microbes isolated from
Kefir. The Kefir sample was obtained from Dominic Anfiteatro, a Kefir producer from Australia. The
Kefir was washed twice from the white and gelatinous lump. The lump was added to commercial
milk (5–10 fold of volume) every 3 days at room temperature in order to maintain the seed culture.
The lump was split by vigorous mixing and suspended in phosphate-buffered saline. Each vial of cell
clump (containing a final concentration of 50% glycerol) was stored at 196°C in a liquid nitrogen tank.
Three types of media were used for the isolation of microbes. The predominant strains A and B were
isolated from colonies cultured on PDA, PCA, and MRS medium (Difco, Detroit, MI, USA). To
investigate their morphological and physiological characteristics, strains A and B were mainly
cultivated aerobically at 30°C on MRS medium. The cells for DNA extraction were produced from
liquid MRS medium. The strains were cultivated aerobically at 30°C on a horizontal shaker at 150
rpm. For fatty acids methyl ester (FAME) analysis, strains A and B were cultivated at 30°C for 3 days
on MRS agar. The morphology of cells was examined using a scanning electron microscope (Hitachi
S-2500, Tokyo, Japan) as described by the manufacturer’s manual. For the isolation of DNA,
chromosomal DNA was isolated and purified according to the method previously described (Tamaoka
and Komagata, 1984; Yoon et al., 1997), with the exception that ribonuclease T1 was used together
with ribonuclease A. The 16S rDNA of strain A was amplified by PCR using two universal primers as described previously (Yoon and Park, 2000). The PCR product was purified by using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). The purified 16S rDNA was sequenced using an ABI PRISM BigDye Terminator Cycle sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) as recommended by the manufacturer. The purified sequencing reaction mixtures were automatically electrophoresed using an Applied Biosystems model 310 automatic DNA sequencer. The 16S rDNA sequences of strains A were aligned with 16S rRNA gene sequences of Lactobacillus sp., and the representatives of some related genera by using CLUSTAL W software (Collins et al., 1991;
Thompson et al., 1994). Gaps at the 5 and 3 ends of the alignment were omitted from further
analyses. Evolutionary distance matrices were calculated by using the algorithm with the DNADIST
program within the PHYLIP package (Felsenstein, 1993). A phylogenetic tree was constructed by using
the neighbor-joining method (Saitou and Nei, 1987) as implemented within the NEIGHBOR program of the same package.
The stability of the relationships was assessed by bootstrap analysis of 1,000 data sets using the programs
SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE of the PHYLIP package. The 26S rDNA of strain B was amplified by PCR using two universal primers as described previously (Boisselier-Dubayle et al., 2002; Vanderpoorten et al., 2002) with slight modification; the PCR product was purified by using a QIAquick
PCR purification kit (Qiagen). The purified 26S rDNA was sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) as recommended by the manufacturer.
To isolate the major microbes, we crushed the white gelatinous and irregular cell clump and washed the
cells twice with phosphate-buffered saline. The cells were split by vigorous mixing and suspended in phosphate-buffered saline. By serial dilution, the cells were seeded on the PCA, PDA, and MRS media. After 3–7 days, the colonies were observed with a phase contrast microscope. We were able to select two types of cells (Figs. 1A–B): strain A, which formed small-sized colonies; and strain B, which formed large-sized glassy colonies. Strain A was Gram-positive, non-spore-forming, and non-motile. The cells of this strain were short slender rods measuring 0.6–0.8 by 1.5–3.0 mm on MRS medium at 30°C, and occurred singly, in pairs, or occasionally in short chains. After incubation on MRS agar for 3 days, the colonies appeared white, circular to slightly irregular, convex, smooth, and opaque. Strain A had catalase-negative and oxidase-negative activities. Casein was hydrolyzed by this strain but gelatin, starch, and urea were not hydrolyzed, arginine was not deaminated and indole was not produced. Strain A produced both L()-lactic acid and D()-lactic acid.
Strain A fermented gluconate and did not produce gas from glucose, indicating that this microorganism is facultatively heterofermentative (Vandamme et al., 1996). Strain A grew well in aerobic and strict anaerobic con-ditions on liquid and solid MRS media at 10 and 40°C, but not at 45°C. The optimal temperature for growth of this strain was approximately 30°C. Strain B was a yeast strain that is rod-shaped and opaque. The cells were globulose, ellipsoidal and cylindrical (Hammes et al., 1992; Kandler and Weiss, 1986). Strain B formed a sediment and a ring in glucose-yeast extract broth and assimilated sucrose, raffinose, inulin, and ethanol. The spores ranged in shape from spheroidal to ellipsoidal to reniform. Sporulation occurred after 2–5 days at 17–25°C on 1% malt extract agar (Llorente et al., 2000). We randomly picked up 30 colonies each of strain A and B from the plates, and then carried out the above physiological test for the taxonomic determination of the colonies. The results showed that strain A (30 colonies) and B (30 colonies) are related to Lactobacillus sp. and Kluyveromyces sp., respectively, with the only two types of bacteria. These results indicate that the major microflora of the strains A and B are Lactobacillus sp. and Kluyveromyces sp., respectively, suggesting that the two strains are formed by a symbiotic association each other.
Next, we intentionally selected 10 colonies from
each strain and sequenced their rDNA for further molecular
taxonomy. First, the 16S rDNA of strain A was
directly sequenced following PCR amplification. The
almost complete 16S rDNA sequence determined was
1,530 bp long (Accession No. AY363303), and was
found to correspond to the region between positions
28 and 1558 by comparison with the 16S rDNA of
Escherichia coli. To determine a possible phylogenetic
classification of strain A, the 16S rDNA sequence was
subjected to similarity searches with public sequence
databases. The results showed that the nucleotide sequence
similarity of the two strains is highly conserved,
and revealed that all 10 colonies of strain A
and B are members of the genus Lactobacillus, and
Kluyveromyces sp. respectively (Figs. 2–3). This relationship
became clear from the phylogenetic analysis
and nucleotide sequence similarity value. The phylogenetic
tree shows that strain A forms an evolutionary
lineage within a radiation of a cluster comprising Lactobacillus
species and is phylogenetically most closely
related to Lact. kefiri NRIC 1693T (Fig. 2). Levels of
16S rDNA similarity between strain A and Lact. kefiri
NRIC 1693T and between strain A and Lact. buchneri
DSM 20057T were 100% and 98.9%, respectively.
In contrast, the partial 26S rDNA of strain B was sequenced
using an ABI PRISM BigDye Terminator
Cycle Sequencing Ready Reaction Kit (Applied
Biosystems) as recommended by the manufacturer.
The partial 26S rDNA sequence determined was 545
bp (Accession No. AY363304). To determine the possible
phylogenetic classification of strain B, the 16S
rDNA sequence was subjected to similarity searches
with public sequence databases. The results revealed
that all 6 colonies of strain B are members of the
genus Kluyveromyces. The phylogenetic tree shows
that strain B forms an evolutionary lineage within a radiation
of a cluster comprising Kluyveromyces species
and is phylogenetically most closely related to K.
marxianus (Fig. 3). Levels of partial 26S rDNA similarity
between strain B and K. marxianus NRRL Y-8281T,
K. lactis var. lactis NRRL Y-8279T, and K. lactis var.
drosophilarum NRRL Y-8278T were 100%, 99.8%,
99.8%, respectively (Wayne et al., 1987).
In summary, we isolated and identified two major microflora
from Kefir, a traditional Tibetan beverage. The
two predominant microbial isolates were related to
Lactobacillus kefiri and Kluyveromyces marxianus.
The mixed culture of the two strains showed the classical
microbial growth of common commercial products
such as yoghurt (data not shown). Further in vivo studies
into effects such as body weight change, constipation,
and cholesterol inhibition will shed additional light
on the usefulness of the beverage.
This study was supported by a grant (20050301–034–474–
006) from BioGreen 21 Program, Rural Development Administration,
Republic of Korea.