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Detection of probiotic microorganisms using RT-PCR, and isolation, identification, probiotic properties of lactobacillus species from kefir

Hassan, Manal Sami El Sayed
Kefir is a dairy product rich in probiotics. In this study, a SYBR green-based realtime PCR (RT PCR) method was used for the detection of kefir probiotic strains. This assay allowed species-specific detection of L. acidophilus, L. delbrueckii subsp.bulgaricus, L. kefiri, L. kefiranofaciens, L. fermentum, L. plantarum, L. amylovorus, L. casei, L. paracasei, Streptococcus thermophilus and Saccharomyces cerevisiae. This method was performed using DNA isolated directly from 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, and L. paracasei were the dominant bacteria in all kefir samples while L. acidophilus, L. delbrueckii subsp. bulgaricus, L. fermentum, L. plantarum, L. amylovorus, Streptococcus thermophilus, and Saccharomyces cerevisiae were 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. tolerans strain 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 with Lactobacillus helveticus strain NBRC 15019. In addition, 2 more isolates were also identified, K6-14 which was identical to Lactobacillus rhamnosus strain NBRC 3425 with 99.45% similarity and K2-3 which showed 98.27 % similarity to Lactobacillus intestinalis strain TH4. Sequence alignment using the Clustal omega program was useful for identifıcation of some isolates