Partial purification and characterization of a bacteriocin produced by Bacillus subtilis NCIMB 3610 that exhibits antimicrobial activity against fish pathogens

The bacteriocin produced by Bacillus subtilis NCIMB 3610 was tested for its antimicrobial action against Enterococcus faecalis, Lactococcus lactis, Staphylococcus aureus, Escherichia coli, Bacillus sphaericus and the fish pathogens Vibrio anguillarum 408 O1 and Photobacterium damselae subsp.piscicida. Inhibition of growth was observed for all bacteria tested with the exception of B. sphaericus which appeared resistant to the bacteriocin treatment. The greatest sensitivity was observed for Enterococcus, with an inhibition of 85% compared to control and it was selected as indicator strain. Antimicrobial activity was greater for the cell free supernatant collected after 72 hrs and the bacteriocin appeared sensitive to lipase, chymotrypsin and pepsin but resistant to amylase and proteinase K. The bacteriocin was heat resistant and higher bactericidal activity was observed at pH 6-8.5. Purification was performed by ammonium sulphate precipitation of the culture cell free supernatant, ion exchange chromatography (MacroPrep 25) and solid phase extraction (SPE, C18). SDS electrophoresis revealed that the apparent molecular weight of the bacteriocin was about 16 kDa. Bactericidal activity was retained throughout the purification procedure and appeared significantly increased (p <0.001) for the final SPE product following solvent evaporation, amounting to 98% of control for Enterococcus. The strong antibacterial action of the purified bacteriocin against the two fish pathogens examined, renders B. subtilis NCIMB 3610 a potential candidate for further evaluation as a probiotic to be used for the prophylactic treatment of fish against bacterial infections.