Assessment of the microbial diversity of Brazilian kefir grains by DGGE and pyrosequencing

Consumption of kefir has spread all over the world due its beneficial health effects. Kefir is produced by fermentation of milk with a kefir grain as the starter culture. Kefir grains are composed of an inert polysaccharide/protein matrix in which a complex microbial community coexists in a symbiotic relationship. The aim of this work was to combine two culture-independent methods for evaluating the microbial diversity and community structure of three distinct kefir grains collected in Brazil, namely DGGE and barcode pyrosequencing. For DGGE analysis, total DNA from kefir grains was used as a template to amplify the 16S rRNA genes from bacteria and the 26S rDNA from fungi. DGGE bands were excised, re-amplified, sequenced and identified by BLAST analysis against the GenBank database. For pyrosequencing, the universal primers Y1 and Y2 with 454-adaptors, followed by a 10-bp sample-specific barcode were used for amplification and sequencing. Raw sequences were processed through the Ribosomal Database Project (RDP) pyrosequencing pipeline. The fingerprints of the bacterial community in three kefir grains contained up to five bands, among them three bands in common. For yeast community the fingerprints were similar for all samples, having a additional band for grain AV. The individual bands excised were identified as Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactococcus lactis and Saccharomyces cerevisae. A total of 14314 partial 16S rRNA sequences were obtained from the three kefir grains samples in pyrosequencing, and grouped into 3 Phyla: Actinobacteria, Proteobacteria and Firmicutes. The Firmicutes were the most abundant and dominated by Bacilli belonging to the order Lactobacillales. Among these, members of the genus Lactobacillus were predominant OTUs in all samples (96%), followed by OTUs of the genera Lactococcus and Leuconostoc. The culture-independent techniques used in this study were useful to give complementary data for analysis of the microbiota present in the ecosystem of Brazilian kefir.