The Antimicrobial Effects of Bacillus subtilis B-1 isolated from a Fish Culture Pond

Bacillus subtilis B-1, isolated from fish culture pond water, produced antimicrobial substances that inhibited an array of pathogenic bacteria, including Streptococcus agalactiae ABRCS-1, Aeromonas hydrophila ABRCA-1, Staphylococcus aureus ATCC 12600, Listeria innocua ATCC33090, Micrococcus luteus IFO 12708, Bacillus circulans JCM 2504 and Bacillus coagulans JCM 2257. Particularly, L. innocua ATCC 33090 showed the highest sensitivity to B. subtilis B-1 out of all indicator strains tested. The cell-free neutralized supernatant from B. subtilis B-1 was stable when tested at 100 °C for 50 min across a range of pH values (2-10) and had a maximum activity of 320 AU∙mL^-1. When tested after 24 h at 4 °C the stability results were the same.However, the maximum activities decreased to 80 AU∙mL^-1 at pH values of 4, 5 and 6.The activity of B-1 also decreased when subjected to a temperature of 100 °C for 60 min. The antimicrobial substances in B. subtilis B-1 supernatants were sensitive to the proteolysis by α–chymotrypsin, trypsin, proteinase K and a serine type protease from Bacillus sp. They were stable in the presence of sodium chloride (NaCl) up to a solution of 15%. The high stability of antimicrobial substances in B. subtilis B-1 makes it suitable for the inhibition of food-borne spoilage bacteria and pathogenic bacteria in aquaculture. Purifying the antimicrobial substances by amberlite adsorption and reverse-phase chromatography resulted in a single active peak, which was designated B1-1. The molecular weight of this fraction by mass spectrometry was 3,398.05 m/z.