MOLECULAR IDENTIFICATION AND TYPING OF LACTOBACILLI ISOLATED FROM KEFIR GRAINS

HOLLMANN, AXEL; DELFEDERICO, LUCRECIA; MARTINEZ, MARIANO; IGLESIAS, GABRIEL; DE ANTONI, GRACIELA; SEMORILE, LILIANA

BARILOCHE, ARGENTINA

Congreso; XXXIX Reunión Anual SAIB-2003; 2003

Kefir is a fermented milk prepared from kefir grains. The grains are clusters of microorganisms held together by a matrix of polysaccharides and proteins that include primarily lactic acid bacteria, yeasts and acetic acid bacteria. The aims of this work were the molecular identification and typing of 17 heterofermentative lactobacilli isolated from kefir grains (CIDCA, Cs. Exactas, UNLP). The identification of two isolates was made by sequencing of the PCR product of 16S rDNA gene. In all the isolates, this gene was analyzed by ARDRA (Amplified Ribosomal DNA Restriction Analysis), using the restriction enzymes Hae III, Dde I, Alu I and Eco RI. The genotyping was performed by analysis of RAPD-PCR (Random Amplified Polymorphic DNA) patterns using primers M13 (GAGGGTGGCGGTTCT), Coc (AGCAGCGTGG), ERIC-2 (AAGTAAGTGACTGGGGTGAGCG) and 1254 (CCGCAGCCAA). The analysis of 16S rDNA subunit sequences showed that both isolates belong to the species Lactobacillus kefir. The patterns obtained by ARDRA were identical in all the isolates and in the reference strain Lb. kefir JCM 5818, whereas a homofermentative lactobacillus showed a different pattern. Lb. brevis JCM 1059 showed a different ARDRA pattern with the enzymes DdeI and Alu I. The RAPD-PCR patterns obtained with the four primers used allowed the differentiation of lactobacilli isolates. The resulting unrooted phylogenetic tree grouped the 17 isolates in six clusters.