Development of rapid and highly specific TaqMan probe-based real-time PCR assay for the identification and enumeration of Lactobacillus kefiri in kefir milk

Lactobacillus kefiri is one of the key functional lactic acid bacteria in kefir milk. We designed a novel real-time PCR primer/probe set, LK_508 targeting the recA gene, for the rapid identification and enumeration of L. kefiri. In inclusivity and exclusivity test using standard strains and kefir isolates, only the 9 tested L. kefiri strains were positive, with the remaining 38 closely related microorganisms testing negative, thus indicating 100% sensitivity and specificity of the assay. The population of L. kefiri was 3.77, 4.30, 4.79, and 5.63 log cfu mL−1 of kefir milk fermented at 25 °C with 5% grain-milk ratio for 12, 24, 36, and 48 h, respectively. The newly developed qPCR assay could be applied to investigate the quantitative relationship of kefir microbiota in fermentation process.