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.

Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.

Kefir is an acidic, mildly alcoholic dairy beverage produced by the fermentation of milk with a grain-like starter culture (Koroleva, 1988). These grains usually contain a relatively stable and specific balance of microbes that exist in a complex symbiotic relationship (Obermann & Libudzisz, 1998; Witthuhn et al. 2004). The different groups of microbes present in the grains are active at different stages of the fermentation (Koroleva, 1982). The lactococci, including Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lc. lactis subsp. diacetilactis provide rapid acid development during the first hours of the fermentation (Litopoulou-Tzanetaki & Tzanetakis, 2000). As the acidity of the milk increases it provides favourable conditions for the growth of the lactobacilli (Rea et al. 1996). The yeasts, acetic acid bacteria and the aroma-producing microbes, mainly leuconostocs, have a much slower growth rate than the lactic acid producers, resulting in the slow production of the aroma compounds and the gradual increase in the concentration of these substances in the later stages of the fermentation (Koroleva, 1982).