Characterization of the Bacteriocin J4 Produced by Bacillus amyloliquefaciens J4 Isolated from Korean Traditional Fermented Soybean Paste

Abstract

In order to isolate the antibacterial bacteriocin-producing bacteria against Listeria monocytogenes, one of noxious and prevalent food-poisoning bacterium, 117 strains of indigenous ferment bacteria were isolated from Korean traditional fermented soybean paste (Doenjang) in Korea. Among the isolated strains, 4 isolates inhibited the growth of L. Monocytogenes. Strain J4, showing the strongest inhibition activity against L. Monocytogenes, was identified as a Bacillus amyloliquefaciens by 16S rDNA sequencing. The strain had a broad spectrum of antibacterial activity against various food-borne pathogens such as Micrococcus luteus, Vibrio parahaemolyticus, and Staphylococcus aureus besides L. monocytogenes. The bacteriocin J4 was purified by 60% ammonium sulfate precipitation, carboxymethyl (CM)-Sephadex column chromatography, and Sephadex G-100 gel filtration. The purified bacteriocin J4 was estimated to be 39 kDa molecular weight by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Bacterocin J4 had antimicrobial activity in a wide pH range (2.0-12.0) and was heat-stable at 80oC for 20 min, but the activity of bacterocin was inactivated by heating at 100oC for 15 min. The antimicrobial activity of the bacteriocin J4 was completely abolished by treatment with proteolytic enzymes such as protease (type I and IX), papain, trypsin, proteinase K, and pepsin, but not when treated with α-amylase, α-glucosidase, and β-glucosidase. The encoding gene of the bacteriocin J4 was included in chromosomal DNA, not plasmid. The bacteriocin J4 had a bacteriolytic mechanism, resulting in cell wall degradation of L. Monocytogenes.