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Cascade cell lyses and DNA extraction for identification of genes and microorganisms in kefir grains

Published online by Cambridge University Press:  13 October 2011

Magdalena Kowalczyk ,
Piotr Kolakowski ,
Joanna M Radziwill-Bienkowska ,
Agnieszka Szmytkowska  and
Jacek Bardowski
Magdalena Kowalczyk*
Affiliation:
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
Piotr Kolakowski
Affiliation:
Danisco Biolacta, Innovation, Olsztyn, Poland
Joanna M Radziwill-Bienkowska
Affiliation:
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
Agnieszka Szmytkowska
Affiliation:
Department of Pharmaceutical Microbiology, Medical University of Warsaw, Warsaw, Poland
Jacek Bardowski
Affiliation:
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
*
*For correspondence; e-mail: mk@ibb.waw.pl
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Abstract

Kefir is a dairy product popular in many countries in Central Europe, especially in Poland and other countries of Eastern and Northern Europe. This type of fermented milk is produced by a complex population of symbiotic bacteria and yeasts. In this work, conditions for DNA extraction, involving disruption of kefir grains and a cascade of cell lysis treatments, were established. Extraction procedure of total microbial DNA was carried out directly from fresh kefir grains. Using different lysis stringency conditions, five DNA pools were obtained. Genetic diversity of DNA pools were validated by RAPD analysis, which showed differences in patterns of amplified DNA fragments, indicating diverse microbial composition of all the analysed samples. These DNA pools were used for construction of genomic DNA libraries for sequencing. As much as 50% of the analysed nucleotide sequences showed homology to sequences from bacteria belonging to the Lactobacillus genus. Several sequences were similar to sequences from bacteria representing Lactococcus, Oenococcus, Pediococcus, Streptococcus and Leuconostoc species. Among homologues of yeast proteins were those from Candida albicans, Candida glabrata, Kluyveromyces lactis and Saccharomyces cerevisiae. In addition, several sequences were found to be homologous to sequences from bacteriophages.

Keywords

Complex ecosystemskefir grainscascade cell lysisDNA isolationbiodiversity
Type
Research Article
Information
Journal of Dairy Research , Volume 79 , Issue 1 , February 2012 , pp. 26 - 32
Copyright
Copyright © Proprietors of Journal of Dairy Research 2011

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References

Altschul, SF, Madden, TL, Schaffer, AA, Zhang, J, Zhang, Z, Miller, W & Lipman, DJ 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 5 3389–3402CrossRefGoogle Scholar
Andrighetto, C, Psomas, E, Tzanetakis, N, Suzzi, G & Lombardi, A 2000 Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products. Letters in Applied Microbiology 30 5–9CrossRefGoogle ScholarPubMed
Beh, AL, Fleet, GH, Prakitchaiwattana, C & Heard, GM 2006 Evaluation of molecular methods for the analysis of yeasts in foods and beverages. Advances in Experimental Medicine and Biology 571 69–106CrossRefGoogle ScholarPubMed
Chen, HC, Wang, SY & Chen, MJ 2008 Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods. Food Microbiology 25 492–501CrossRefGoogle ScholarPubMed
De Antoni, G, Zago, M, Vasek, O, Giraffa, G, Carminati, D, Marcó, MB, Reinheimer, J & Suárez, V 2010 Lactobacillus plantarum bacteriophages isolated from Kefir grains: phenotypic and molecular characterization. Journal of Dairy Research 77 7–12CrossRefGoogle ScholarPubMed
de Barros Lopes, M, Soden, A, Henschke, PA & Langridge, P 1996 PCR differentiation of commercial yeast strains using intron splice site primers. Applied and Environmental Microbiology 62 4514–4520CrossRefGoogle ScholarPubMed
Fliss, I, Emond, E, Simard, RE & Pandian, S 1991 A rapid and efficient method of lysis of Listeria and other gram-positive bacteria using mutanolysin. Biotechniques 11 453, 456–457Google ScholarPubMed
Garbers, I-M, Britz, TJ & Witthuhn, RC 2004 PCR-based denaturing gradient gel electrophoretic typification and identification of the microbial consortium present in kefir grains. World Journal of Microbiology and Biotechnology 20 687–693CrossRefGoogle Scholar
Garrote, GL, Abraham, AG & De Antoni, GL 1997 Preservation of kefir grains, a comparative study. Lebensmittel-Wissenschaft und-Technologie 30 77–84CrossRefGoogle Scholar
Garrote, GL, Abraham, AG & De Antoni, GL 2010 Microbial interactions in Kefir: a natural probiotic drink. In Biotechnology of Lactic Acid Bacteria: Novel Applications, ch 18 (Eds Mozzi, F, Raya, RR & Vignolo, GM). Oxford, UK: Wiley-BlackwellGoogle Scholar
Gill, SR, Pop, M, Deboy, RT, Eckburg, PB, Turnbaugh, PJ, Samuel, BS, Gordon, JI, Relman, DA, Fraser-Liggett, CM & Nelson, KE 2006 Metagenomic analysis of the human distal gut microbiome. Science 312 1355–1359CrossRefGoogle ScholarPubMed
Hutchinson, CA III 2007 DNA sequencing: bench to bedside and beyond. Nucleic Acid Research 35 6227–6237CrossRefGoogle Scholar
Lopitz-Otsoa, F, Rementeria, A, Elguezabal, N & Garaizar, J 2006 Kefir: a symbiotic yeasts-bacteria community with alleged healthy capabilities. Revista Iberoamericana de Micologia 23 67–74CrossRefGoogle ScholarPubMed
Oklinski, MK 2006 The influence of the medium composition and selected parameters of production for the activity of kefir grains. Master's Thesis. Olsztyn, Poland: The University of Warmia and MazuryGoogle Scholar
Pintado, ME, Lopes Da Silva, JA, Fernandes, PB, Malcata, FX & Hogg, TA 1996 Microbiological and rheological studies on Portuguese kefir grains. International Journal of Food Science and Technology 31 15–26CrossRefGoogle Scholar
Prakitchaiwattana, CJ, Fleet, GH & Heard, GM 2004 Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes. FEMS Yeast Research 4 865–877CrossRefGoogle ScholarPubMed
Rossetti, L & Giraffa, G 2005 Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases. Journal of Microbiological Methods 63 135–144CrossRefGoogle ScholarPubMed
Sambrook, J, Fritsch, EF & Maniatis, T 1989 Molecular Cloning: a Laboratory Manual, 2nd edition. Cold Spring Harbor, New York, USA: CSHL PressGoogle Scholar
Sanger, F, Nicklen, S & Coulson, AR 1977 DNA sequencing with chain terminating inhibitors. In Proceedings of the National Academy of Sciences of the United States of America 74 5463–5467CrossRefGoogle ScholarPubMed
Schloss, PD & Handelsman, J 2005 Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Applied and Environmental Microbiology 71 1501–1506CrossRefGoogle ScholarPubMed
Sherman, F, Fink, GR & Hicks, JB 1986 Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor, New York, USA: CSHL PressGoogle Scholar
Tailliez, P, Tremblay, J, Ehrlich, SD & Chopin, A 1998 Molecular diversity and relationship within Lactococcus lactis, as revealed by randomly amplified polymorphic DNA (RAPD). Systematic and Applied Microbiology 21 530–538CrossRefGoogle ScholarPubMed
Tilsala-Timisjarvi, A & Alatossava, T 2004 Rapid DNA preparation from milk and dairy process samples for the detection of bacteria by PCR. Food Microbiology 21 365–368CrossRefGoogle Scholar
Tyson, GW, Chapman, J, Hugenholtz, P, Allen, EE, Ram, RJ, Richardson, PM, Solovyev, VV, Rubin, EM, Rokhsar, DS & Banfield, JF 2004 Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428 37–43CrossRefGoogle ScholarPubMed
Venter, JC, Remington, K, Heidelberg, JF, Halpern, AL, Rusch, D, Eisen, JA, Wu, D, Paulsen, I, Nelson, KE, Nelson, W, Fonts, DE, Levy, S, Knap, AH, Lomas, MW, Nealson, K, White, O, Peterson, J, Hoffmon, J, Parsons, R, Baden-Tillson, H, Pfannkoch, C, Rogers, YH & Smith, HO 2004 Environmental genome shotgun sequencing of the Sargasso Sea. Science 304 66–74CrossRefGoogle ScholarPubMed
Wang, YY, Li, HR, Jia, SF, Wu, ZJ & Guo, BH 2006 Analysis of bacterial diversity of kefir grains by denaturing gradient gel electrophoresis and 16S rDNA sequencing. Wei Sheng Wu Xue Bao 46 310–313 [Article in Chinese]Google ScholarPubMed
Witthuhn, RC, Schoeman, T & Britz, TJ 2005a Characterisation of the microbial population at different stages of Kefir production and Kefir grain mass cultivation. International Dairy Journal 15 383–389CrossRefGoogle Scholar
Witthuhn, RC, Schoeman, T, Cilliers, A & Britz, TJ 2005b Impact of preservation and different packaging conditions on the microbial community and activity of Kefir grains. Food Microbiology 22 337–344CrossRefGoogle Scholar
Zhou, J, Liu, X, Jiang, H & Dong, M 2009 Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis. Food Microbiology 26 770–775CrossRefGoogle ScholarPubMed