Characterization of Bacteriocins Produced by Lactobacillus brevis NM 24 and L.fermentum NM 332 Isolated from Green Olives in Iran

Abstract

The aim of the study was to isolate and characterize bacteriocin producing Lactic Acid Bacteria (LAB) from local olive samples and to exploit their potential as biopreservative. The two lactobacillus strains namely L.brevis NM 24 and L.fermentum NM 332, exhibiting wide spectrum of activity against closely related strain were selected and screened for their bacteriocin producing ability. Cell free supernatant fluid collected from both the isolates was sensitive to the action to proteolytic enzymes, and complete loss of their antagonistic activity was seen. Several gram positive and gram negative pathogens such as Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus were inhibited by the inhibitory action of bacteriocins in study. The bacteriocinogenic potential in these strains appeared non-inducible and no increase in their titer was observed after exposure to different concentrations of Mitomycin C and UV light.The concentrated crude bacteriocin samples subjected to ammonium sulphate precipitation resulted in an increased activity and high protein yield. During ultrafiltration studies, bacteriocin NM 332 produced by L.fermentum NM 332, was able to pass through the cellulose membranes with 10 KDa and 30KDa molecular weight cut off indicating the presence of a small protein molecule. By non-denaturing gel a band of approximately 8 KDa for L.fermentum NM 332 and band corresponding to 37 KDa in L.brevis NM 24 was seen. Physicochemical characterization of the partially purified bacteriocin samples indicated heat (121°C for 60 min) and acidic pH stability (pH 2-6) of bacteriocin NM 24. Exposure to surfactants resulted in a significant increase in the bacteriocin titer of both the strains, except for Tween 20 which resulted in complete loss of activity. None of the strains showed presence of plasmids indicating chromosomal associated genes responsible for bacteriocin production. The bactericidal activity displayed by the bacteriocins in study was maintained during storage at -20 °C for more than three years.