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

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 theLactobacillusgenus. Several sequences were similar to sequences from bacteria representingLactococcus, Oenococcus, Pediococcus, StreptococcusandLeuconostocspecies. Among homologues of yeast proteins were those fromCandida albicans, Candida glabrata, Kluyveromyces lactisandSaccharomyces cerevisiae. In addition, several sequences were found to be homologous to sequences from bacteriophages.