Characterization of probiotic Lactobacillus spp. isolates from commercial fermented milks

The aim of this project was to study the identity of probiotic lactobacilli in fermented milk products from the United Kingdom/European markets during their survival during shelf-life. This in vitro study was also aimed at undertaking studies on some of the physiological probiotic criteria, such as resistance to stomach/intestine conditions and also possible functional properties of the isolates, such as antimicrobial activities, antibiotic resistance/susceptibility and antibiotic resistance genes, biofilm formation and production of conjugated linoleic acid (CLA). Primarily, a comparative study was carried out on selectivity of MRS-Clindamycin, MRS-Sorbitol and MRS-IM Maltose, to select the right medium for enumeration of probiotic Lactobacillus. Based on selectivity of medium for recovery of the targeted lactobacilli and also simplicity of preparation, MRS-Clindamycin was chosen as the best medium for enumeration of probiotic Lactobacillus in fermented milks. The results of enumeration of lactobacilli showed that 22 out of a total 36 tested products contained more than 106 colony forming units/g at the end of their shelf-life, which comply with the recommended minimum therapeutic level for probiotics. Rep-PCR using primer GTG-5 was applied for initial discrimination of isolated strains, and isolates, which presented different band profile, were placed in different groups. The isolated Lactobacillus spp. were identified mainly as Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus paracasei by analysis of partial sequences of the 16S ribosomal RNA and rpoA genes. In order to characterize the isolates for probiotic properties, this study was focused on six Lactobacillus isolates along with two commercial Lactobacillus cultures from Chr. Hansen (Lactobacillus acidophilus La5 and Lactobacillus casei C431) and three Lactobacillus type strains (Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei and Lactobacillus acidophilus) which were purchased from NCIMB. The stomach and intestine conditions were mimiced using a batch culture fermentation system, and the combined effects of pH, enzymes and bile salts on survival of tested isolates was tested. The tested isolates were able to survive at low pH environment and also high concentrations of bile salts of the upper digestive tract. The potential of tested isolates for biofilm formation was determined in different conditions of nutritional and physiological stresses. The capability of tested isolates to produce biofilm in nutrient rich medium was recorded. However, the growth limitation, such as nutrient shortage in diluted media and also using inulin rather than glucose in synthetic medium, did not induce biofilm formation. Antimicrobial activities of tested bacteria against indicator bacteria namely Escherichia coli NCTC 12900, Salmonella enterica serovar Typhimurium DT124 and Salmonella enterica serovar Enteritidis PT4 and Lactobacillus delbruckii subsp. bulgaricus were studied. The production of organic acids and bacteriocin was considered as key mechanisms for antimicrobial activity of tested strains. Screening the isolates competence for production of CLA demonstrated that this feature is species dependent and also entirely related to the level of initial linoleic acid in the medium. Eleven tested isolates were also assessed for their antibiotic resistance profile by determination of minimum inhibitory concentration (MIC). The acquired resistance to cefoxitin, ceftriaxone, chloramphenicol, erythromycin, gentamycin, kanamycin, lincomycin, streptomycin, tylosin tartarate, tetracycline and vancomycin was observed in all tested isolates. Also their genetic background of antibiotic resistance genes was studied by PCR reactions and none of the tested isolates showed positive bands for investigated resistance genes.