K.V. Bespomestnykh
Kemerovo State Institute of Agriculture
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Summary. This article presents the results of research to develop a PCR-test-system for indication and identification of lactic acid bacteria starter cultures used in the manufacture of dairy products. The nucleotide sequence of DNA encoding 16S rRNA synthesis of bacteria of the genus Lactobacillus, submitted to GenBank, to identify conserved regions of nucleic acid sequences and for laying the genus-specific species-specific primers. Designed new genus-specific and species-specific primers for the identification of bacteria of the genus Lactobacillus, with significant differences from other lactic acid bacteria genera and species. Theoretical specificity synthesized primers confirmed experimentally bacterial ferments, concentrates and dairy products. Revealed that virtually created primers have the highest degree of homology to the nucleotide sequence of DNA sections 16S rRNA microorganisms L. delbrieckii subsp. bulgaricus and have high specificity. They can detect a particular species of microorganism the difference with respect to other bacteria and to determine their quantitative content in the product. The optimal values of the 16S rRNA gene amplification of lactic acid bacteria (temperature, incubation time, number of cycles): 95 ºС – 200 sec – 1 cycle [62 ºС – 50 sec, 95 ºС – 20 sec] – 25 cycles of 72 ºС – 120 sec – 1 cycle. A comparative evaluation of the species specificity of PCR with the classical biochemical methods. The results of identification of species other L. bulgaricus and Lactobacillus obtained using polymerase chain reaction are comparable to the classical biochemical identification methods based on different species saccharolytic activity of lactobacilli. Experimentally confirmed the higher specificity and sensitivity of PCR-test-systems for the identification of lactic acid bacteria belonging to the bacterial starters. Studies have been conducted on a wide range of dairy products.
Keywords: polymerase chain reaction primers, bacteria of the genus Lactobacillus, milk products , quality control.