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Kefir

작성자 식품공학부 작성일 2013/05/16 조회수 416
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    Kefir??? 무엇이지요?  Lactobacillus와 Kluyveromyces의 mixed culture입니다....

    다음을 보시면 도움이 됩니다.
    J. Gen. Appl. Microbiol., 52, 375–379 (2006)
    Short Communication

    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.
    Acknowledgments
    This study was supported by a grant (20050301–034–474–
    006) from BioGreen 21 Program, Rural Development Administration,
    Republic of Korea.
    다음글 : 무슨 버섯 식품공학부 2013/05/16
    현재글 : Kefir 식품공학부 2013/05/16
    이전글 : Busy Bee Learning Center 식품공학부 2013/05/16