Molecular methods for identification ofLactobacillus delbrueckiisubsp.bulgaricusandStreptococcus thermophilususing methionine biosynthesis and 16S rRNA genes

Abstract

Yoghurt and starter culture producers are still searching strains ofLactobacillus delbrueckiisubsp.bulgaricusandStreptococcus thermophilusto produce healthier yogurt with longer shelf life, better texture, taste and quality. However, selective identification ofLb.delbrueckiisubsp.bulgaricusandStrep. thermophilusfrom a mixed population using microbiological and biochemical methods is difficult, time consuming and may not be accurate. In this study, a quick, sensitive and accurate method is proposed to identify bothLb. delbrueckiisubsp.bulgaricusandStrep. thermophilususing PCR. The method is comprised of two parts. In the first part, methionine biosynthesis genes, known to be present in both species were partially amplified by designed primers (cysmet2F and cysmet2R). Partial amplification of the methionine biosynthesis gene which gives 700 bp fragment resulted in selective identification ofLb. bulgaricusandStrep. thermophilus. All 16Lb. bulgaricusand 6Strep. thermophilusisolates assessed by this method gave the expected amplification. On the other hand, further analysis of other closely related species with the same primers have indicated that the same product was also amplified in two more lactobacilli namely,Lb. delbrueckiisubsp.lactisandLb. helveticusspecies. Thus, in the second part of the method, further differentiation ofLb. delbrueckiisubsp.bulgaricusandStrep. thermophilusfrom each other and these species was achieved using restriction analysis of 16S rRNA gene withEcoRI.