Isolation and Molecular Identification of Lactic Acid Bacteria from Raw Milks of Central Alborz Heights, by High Resolution Melting Real Time PCR & 16SrDNA PCR Sequencing

Industrial importance of Lactic Acid Bacteria (LAB) is widely known. LAB are the greatest former of Probiotics and have numerous applications in health and industry. The aim of this study was to identify LAB from the raw milks of virgin heights of Alborz and introduce local species with industrial potential to be replaced for imported species. Also survey of present LAB diversity, preservation of local LAB in a microbial bank and looking for new species were the other goals of the study. Raw milk samples were collected from 14 selected areas of Alborz Heights and transported to the laboratory under standard terms. LAB colonies were isolated by macroscopic and microscopic observations and differential tests. DNAs of the 64 isolates were extracted and grouped by High Resolution Melting-Real Time PCR using 16SrDNA universal primers. Finally 1 sample was chosen from each group and sent for sequencing after performing PCR. Alignment of the resulted sequences with NCBI GeneBank identified 6 genuses, 9 species and 2 potentially new species. Streptococcus vestibularis, Enterococcus faecalis, Lactococcus lactis, Streptococcus infantarius, Leuconostoc argentinum, Lactobacillus brevis, Pediococcus pentosaceus, Lactobacillus rhamnosus, Lactobacillus plantarum and two bacteria from genuses Pediococcus and Enterococcus were identified by 16SrDNA analysis. The results show an amazing diversity of LAB in raw milks of Central Alborz. Most of the isolates have industrial potential and can be replaced for imported strains. Also due to 16SrDNA analysis, there is a chance for presence of two new species. The important point is that application of High Resolution Melting-Real Time PCR in this study resulted in a great decrease in time and expenses which proves the importance of using molecular techniques in microbiology and their combination with classic methods.