title: Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain.
abstract: The antimicrobial activity and probiotic properties of Bacillus subtilis strain KKU213, isolated from local soil, were investigated. The cell-free supernatant (CFS) of a KKU213 culture containing crude bacteriocins exhibited inhibitory effects on Gram-positive bacteria, including Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, and Staphylococcus aureus. The antibacterial activity of the CFS precipitated with 40% ammonium sulfate (AS) remained even after treatment at 60 and 100 °C, at pH 4 and 10 and with proteolytic enzymes, detergents and heavy metals. When analyzed by SDS-PAGE and overlaid with the indicator strains B. cereus and S. aureus, the 40% AS precipitate exhibited inhibitory activity on proteins smaller than 10 kDa. However, proteins larger than 25 kDa and smaller than 10 kDa were still observed on a native protein gel. Purified subtilosin A was prepared by Amberlite XAD-16 bead extraction and HPLC and analyzed by Nano-LC-QTOF-MS. Its molecular mass was found to be 3.4 kDa, and it retained its antibacterial activity. These results are consistent with the detection of the anti-listerial subtilosin A gene of the sbo/alb cluster in the KKU213 strain, which is 100% identical to that of B. subtilis subsp. subtilis 168. In addition to stable and cyclic subtilosin A, a mixture of many extracellular antibacterial peptides was also detected in the KKU213 culture. The KKU213 strain produced extracellular amylase, cellulase, lipase and protease, is highly acid-resistant (pH 2) when cultured in inulin and promotes health and reduces infection of intestinally colonized broiler chickens. Therefore, we propose that bacteriocin-producing B. subtilis KKU213 could be used as a potential probiotic strain or protective culture.
authors.affiliation: Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Nalisa
authors.initials: N
authors.lastname: Khochamit
authors.affiliation: Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Surasak
authors.initials: S
authors.lastname: Siripornadulsil
authors.affiliation: Research Group for Preventive Technology in Livestock, Department of Anatomy, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Peerapol
authors.initials: P
authors.lastname: Sukon
authors.affiliation: Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand. Electronic address: swilai@kku.ac.th.
authors.forename: Wilailak
authors.initials: W
authors.lastname: Siripornadulsil
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D000898:Antibiosis; D001412:Bacillus subtilis / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D001430:Bacteriocins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D023061:Gene Order; D005838:Genotype; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009876:Operon; D010455:Peptides; D010641:Phenotype; D010802:Phylogeny; D055550:Protein Stability; D012336:RNA, Ribosomal, 16S
chemical_list: D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D010455:Peptides; D012336:RNA, Ribosomal, 16S
keywords: Bacillus subtilis; Bacteriocin; Bioactive peptide; Probiotic; Subtilosin A