The Two-synergistic Peptide Bacteriocin Produced by Enterococcus faecium NKR-5-3 Isolated from Thai Fermented Fish (Pla-ra)

Bacteriocin produced by Enterococcus faecium NKR-5-3 isolated from Thai fermented fish was characterized and purified. According to its physical and chemical properties, E. faecium NKR-5-3 produced heat tolerant bacteriocin with broad spectrum activity against indicator strains. Treatment of bacteriocin in cell-free neutralized supernatant (CFNS) with some proteases eliminated or reduced its activity, suggesting it is proteinaceous. Furthermore, it was stable in 0 to 24% NaCl, heat tolerant at pH 2 to 10 and bactericidal toward Enterococcus faecalis ATCC 19433T. Two antibacterial peptides, named enterocin NKR-5-3A and enterocin NKR-5-3B, were purified from the culture supernatant by four steps of column chromatography. Enterocin NKR-5-3A had the same mass, MW=5,241, and N-terminal amino acid sequence as brochocin A produced by Brochothrix campestris ATCC 43754, suggesting it may be identical to brochocin A. Enterocin NKR-5-3B exhibited MW=6,320, but its N-terminal amino acid sequence could not be determined. When these two purified peptides were mixed in a 1:1 ratio, the bacteriocin activity reached a maximum, which corresponded to 64 times the total calculated bacteriocin activity in the mixture, suggesting their synergism.