Kefirde probiotic mikroorganizmaların RT-PCR ile tespiti ve lactobacilus türlerinin izolasyonu tanısı, probiotic özellikleri

Kefir is a dairy product rich in probiotics. In this study, aSYBR green-based real-time PCR (RT PCR) method was used for the detection of kefir probiotic strains. This assay allowed species-specific detection ofL. acidophilus, L. delbrueckii subsp.bulgaricus, L. kefiri, L. kefiranofaciens, L. fermentum, L. plantarum, L. amylovorus, L. casei, L. paracasei, Streptococcus thermophilusand Saccharomyces cerevisiae. This method was performedusing DNA isolated directlyfrom five different sources of kefir from Turkey and one milk product from Kyrgyzstan.Results of amplification and melting curve from real time PCR assay showed that L. kefiri, L. kefiranofaciens, L. casei, andL. paracaseiwere the dominant bacteria in all kefir sampleswhile L. acidophilus, L. delbrueckii subsp.bulgaricus, L. fermentum,L. plantarum, L. amylovorus, Streptococcus thermophilus, andSaccharomyces cerevisiaewere detected in a few kefir samples. Based on microscopic examination and biochemical tests, 30 of 100 bacterial isolates were confirmed to belong to the genus Lactobacillus. It was also indicated that isolated Lactobacillus species from kefir samples have potential probiotic properties. In addition, identification studies for the isolates from the kefir samples were also carried out. These isolates were identified by 16S rRNA sequencing BLAST analysis and alignment. BLAST results showed that 19 isolates had more than 97% similarity with Lactobacillus paracasei subsp. toleransstrain NBRC 15906, 5 isolates had more than 99% similarity with Lactobacillus gallinarum strain ATCC 33199, 2 isolates (k5-14 and k5-15)showed 99.21 and 98.71 % similarity of Lactobacillus zeae respectively,and 2 isolates (K4-6a and K6-3a) displayed more than 99.22 % similarity withLactobacillus helveticus strain NBRC 15019.In addition, 2 more isolates were also identified, K6-14 which was identical toLactobacillus rhamnosus strain NBRC 3425with 99.45% similarityandK2-3whichshowed98.27 %similarity toLactobacillus intestinalis strain TH4.Sequence alignment using the Clustal omega program was useful for identifıcation of some isolates. Keywords: Kefir, Lactobacillus, real-time PCR, L.kefiri, L.kefiranofaciens, L.casei, L.paracasei, L.acidophilus, L.delbrueckii subsp.bulgaricus, L.fermentum, L.plantarum, L.amylovorus, Streptococcus thermophilus, L.gallinarum, L.zeae, L.helveticus, L. rhamnosus, L.intestinalis, Saccharomyces cerevisiae,biochemical tests,16 S rRNA sequencing analysis.