IDENTIFI CATION OF BACTERIOCIN GENES IN PROBIOTIC STRAINS OF LACTIC ACID BACTERIA Lactobacillus Acidophilus D-75 And Lactobacillus Acidophilus D-76

Aim: to detect the bacteriocin genes of probiotic strains Lactobacillus acidophilus D-75 and Lactobacillus acidophilus D-76 (components of Vitaflor dietary supplement). Materials and Methods: The antagonistic activity of L. acidophilus D-75 and L. acidophilus D-76 strains was estimated by the deferred antagonism method. The identification of bacteriocin genes was performed by PCR using helveticin J gene primers. Amplified fragments were sequenced using ABI PRISM® 310 Genetic Analyzer and were analyzed using NCBI/BLASTX. Results: The test bacteria exhibit pronounced antagonistic activity (AA) against Escherichia coli O75 and Salmonella Enteritidis 209. In the presence of Actoflor-S (metabiotic dietary supplement) the antagonistic activity of tested probiotic strains was 2-2.5-fold increased indicating its inducible nature. The identical sequences of 537 bp homologous to gene fragment of helveticin of L. helveticus DPC4571 (lhv_1632 gene) were detected in DNA of both strains. Sequencing of these fragments showed difference in three nucleotides compared to the reference DNA of DPC4571 strain (A instead of G at position 46, C instead of T at position 249 and A instead of T at position 537), but all these replacements do not lead to changes in the amino acid sequence of a bacteriocin. For L. acidophilus D-76 another bacteriocin gene fragment of 283 bp was identified (in addition to 537 bp fragment). The latter had 95% homology with the helveticin J gene of L. helveticus R0052 (R0052_09025 gene). In NCBI/BLASTX database the sequences homologous to the helveticin gene of L. helveticus DPC4571 were found in 11 microorganisms related to L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. helveticus and L. kitasatonis. Conclusion: Thus, our study shows that there are at least two bacteriocins in L. acidophilus D-76 and one bacteriocin in L. acidophilus D-75. However, the true potential of these probiotic bacteriocins for human health has yet to be realized.

Actoflor-S, antagonistic activity, bacteriocin gene, helveticin, Lactobacillus, probiotics, sequencing, Vitaflor

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