Characterization of Partially Purified Bacteriocin Like Substance (BLIS) Produced by Probiotic Lactobacillus Strains

There is an increasing interest in search for antimicrobial peptides (bacteriocins and bacteriocin-like compounds) produced by lactic acid bacteria (LAB) because of their potential to be used as antimicrobial agents for improving the safety of food products. The main objective of study was to evaluate the antibacterial potential of locally isolated Lactic Acid bacteria (LAB) and determine their bacteriocin producing ability in in-vitro conditions. The antibacterial activity of 77 isolated LAB strains was tested against a number of pathogens by well-diffusion method. The isolates demonstrating antimicrobial potential were selected and tested for the production of bacteriocin or bacteriocin like substance. The bacteriocin produced by two of the isolates were partially purified and characterized. The results indicated the neutralized supernatant fluid of two of the isolates identified as L. brevis LB32 and L. pentosus LP05, were active against the growth of Listeria monocytogenes, Salmonella enteritidis, Shigella dysenteriae, Staphylococcus aureus and Streptococcus pneumoniae. Additionally, L. brevis LB32 was able to inhibit the growth of Salmonella typhi and Klebsiella pneumoniae, while, S. pnuemoniae and L. monocytogenes appeared to be the most sensitive strain as apparent by highest zone of inhibition against these pathogens, respectively. The antimicrobial activity in the supernatant fluids of the mentioned strains remained unaffected after treating with enzymes catalase, lipase and lysozyme, while were strongly sensitive to the action of proteolytic enzymes, suggesting the presence of bacteriocin like inhibitory substance (BLIS) in the two isolates. The inhibitory substance produced by the two isolates appeared heat resistant and tolerated 100˚C and 121˚C for 55 minutes and 20 minutes, respectively. Partial purification of the concentrated culture supernatant fluids of L. brevis LB32 and L. pentosus LP05 by ammonium sulphate (80%) and DEAE cellulose columns resulted in an enhanced activity (AU/mL) and yield. Using different pore size ultra filter membranes and SDS-PAGE analysis, the approximate molecular weight of the BLIS produced by L. brevis LB32 and, L. pentosus LP05 appeared to be approximately 4.5 and 6 KDa, respectively. In contrast to L. brevis LB32, L. pentosus LP05 harbored an 18Kb plasmid DNA which appeared to be carrying the bacteriocin gene as evident by plasmid curing experiments. All the mutants retained their host immunity and were resistant to the bacteriocin produced by the parent strain. In conclusion, the antibacterial activity possessed by these isolates might be used for the control of unwanted pathogens mainly in dairy products, and could be investigated further for using in fermented dairy products.