Unraveling Lactobacillus helveticus species inhabiting cultivable fraction of natural whey starter: a genotype and phenotype-based study

The lactic acid bacterium (LAB) Lactobacillus helveticus is a key player in thermophilic dairy fermentation. LAB also inhabit the human gut, which has often been exploited as a reservoir of potential novel probiotics. We analyzed the cultivable fraction of 12 natural whey starters (NWS) sampled in 2 different sampling times through DNA barcoding and genotypic approach. Twenty-five candidates were evaluated for tolerance to bile acids (BA), low pH, and lysozyme as probiotic traits. Pentaplex-PCR and 16S-ARDRA analyses were used as DNA barcoding techniques. (GTG)5 REP-PCR was carried out to estimate the intra-species diversity (Simpson’s index) and dereplicate the clones library. BA tolerance and pH tolerance were assessed by cultivating strains in the presence bile salt mixture (0.05%, 0.3%, 0.6%, and 1%), and at pH of 3.5, 4.5, and 5.4, respectively. Lysozyme (0.1 mg/mL) was used to test lysozyme tolerance. Inter-species diversity was >0.9 Simpson’s index for NWS enriched in L. helveticus (type-H), while was slightly lower in NWS enriched in L. delbrueckii (type-H). Huge phenotypic diversity was found within a pool of 25 genotypically unique strains. Two candidates showed the best growth response to all three stresses tested.