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Abstract: Lactic acid bacteria were screened for potential probiotics for use as feed additives. We obtained 3,000 isolates from feces of: cattle, dogs, goats, and infants; milk; yogurt; cheese; fermented sausages; Kimchi; and Cheonggukjang and tested their antibacterial activity toward indicator pathogens, including Bacillus cereus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica Enteritidis. We further tested their tolerance to artificial gastric juice (1% [w/v] pepsin, pH 2.5) and bile acid (0.1% [w/v] oxgall, pH 6.8). Six isolates exhibited strong antibacterial activity against indicator pathogens. The PA40 isolate from Kimchi exhibited marked resistance to artificial gastric juice and bile acid. The antibacterial substances produced by PA40 were stable to heat, pH, and enzymes. Strain PA40 was identified as a Lactobacillus curvatus strain using chemical tests and 16S rDNA sequencing and produced 248.4 mmol/L lactic acid after 48 hr of fermentative growth. The L. curvatus PA40 strain was also highly tolerant of the artificial gastrointestinal model system. Our results indicate that L. curvatus PA40 could be used as a potential probiotic feed additive. [ABSTRACT FROM AUTHOR]

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus . It showed stable activity in the range 0-70°C and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.