European Journal of Biomedical and Pharmaceutical Sciences

MOLECULAR IDENTIFICATION OF Lactobacillus spp., ISOLATED FROM TRADITIONAL BULGARIAN DAIRY PRODUCTS

Veronica Nemska, Nelly Georgieva1 and Svetla Danova*

ABSTRACT

Genus Lactobacillus constitutes a heterogeneous group of lactic acid bacteria (LAB) that currently has more than 180 species and subspecies, most of which play an important role in food fermentations. The increasing interest in these species is due to their ability to improve quality of dairy products, to ensure their safety and to promote consumers` health. The aim of this study was to perform a molecular identification of 45 newly-isolated LAB strains from traditional Bulgarian dairy products: artisanal curd, katak, yoghurt, white-brined and yellow cheese. They were characterized as Lactobacillus spp. The phenotypic and physiological similarity, observed from the studied dairy lactobacilli, created some difficulties in their identification. This required a combination of classical and genomic methods and new adapted protocols for dairy LAB. Therefore, the DNA from all LAB was extracted and several methods based on polymerase chain reaction (PCR) method were further applied: (i) Multiplex PCR with specific primers for the differentiation of closely related species from the group of Lactobacillus plantarum; (ii) 16S rDNA analyses and (iii) RAPD PCR - a high discriminative genotypic method for distinguishing strain-specific features of representatives of L. plantarum group. Twenty-three of the investigated strains were identified as L. plantarum. The non-identified lactobacilli were subjected to 16S rDNA PCR, the gold standard in bacterial taxonomy. According to the BLAST analysis of the obtained sequences, they have been identified as: Lactobacillus fermentum (3 strains), Lactobacillus rhamnosus (2 strains), Lactobacillus delbrueckii spp. bulgaricus (1 strain) and Lactobacillus paracasei (1 strain). A group of 4 strains with very low similarity to the presented Lactobacillus spp. in the GenBank – NCBI data was found, which could be new LAB species. Further characterizations, however are needed and they are still in progress.

Keywords: Lactobacillus spp., Identification, PCR, Multiplex PCR, RAPD PCR.