Selective detection, isolation and enumeration of Bifidobacterium animalis subsp. lactis BB-12 from Iranian commercial probiotic yoghurts

Abstract

Fermented dairy products are one of the most prevalent vehicles for the delivering of probiotic bacteria to the consumer. A minimal concentration of 106 CFU/g(mL) of a product is required to exert probiotic effects. In this study, we first evaluated the selectivity of WCM 50 and WCM 100 (Wilkins-Chalgren agar supplemented with 50 mg/L and 100 mg/L mupirocin), as well as mMRS (De Man Rogosa Sharpe agar supplemented with 0.1 mg/L Clindamycin plus 10 mg/L Ciprofloxacin) media, using pure cultures of prevalent Bifidobacterial and Lactobacilli probiotic strains. For each strain, the selectivity and cell recovery rate on each medium was compared statistically with that obtained on the non-selective media. Afterwards, one tuf gene-based specific primer set was designed for the detection of Bifidbacterium animalis subsp. lactis BB-12 in commercial probiotic yoghurts. The specificity of designed primer set was evaluated by operation of PCR reactions with extracted DNAs from reference strains and commercial probiotic yoghurts. Finally, strain BB-12 was detected, enumerated and confirmed through tuf gene-based PCR, selective plate count (using WCM 100 medium) and fructose-6-phophate-phosphoketolase assay (F6PPK) respectively, during shelf life and after expiry date of commercial probiotic yoghurts. The results showed that WCM 100 was completely selective for Bifidobacteria, with the recovery about 100%. However, mMRS was not completely selective for Lactobacilli. The PCR assays confirmed the specificity of tuf gene-based primer set for strain BB-12. Although the counts of strain BB-12 had significant decrease during shelf-life, but these counts didn’t fell below CODEX standard (106 CFU/mL), until expiry date of products.

Keywords

• Probiotic Yoghurt, BB-12, Enumeration, Detection, PCR, tuf gene.