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.

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Aim: The aim of the study was to determine the potentials and effects of Bacillus subtilis and lactic acid bacteria (LAB) as probiotics on broiler growth, health, and Salmonella infection. Materials and Methods: To evaluate the inoculum size applicable for broilers, 1-day-old broilers were orally fed fresh cultures of single strains and a B. subtilis KKU213/Pediococcus pentosaceus NP6 mixture at 108 and 1012 colony-forming unit (CFUs)/mL/chick. The body weight gain (BWG), Salmonella contamination level and total Bacillus and LAB abundances in the crop and intestine were measured. Subsequently, 1-day-old broilers were orally fed of KKU213, CH403, and Pediococcus acidilactici SH8 at 1010 CFUs/mL, followed by inulin. After 35 days, the BWG, Bacillus and LAB abundances in the cecum, blood parameters, and KKU213 colonization were assessed. Results: The broilers fed single strains or KKU213+NP6 exhibited a higher BWG and a higher crop LAB abundance than the controls (p<0.05). Probiotic feeding decreased the intestinal Salmonella abundance and correspondingly increased the LAB abundance. The broilers fed the mixed culture (KKU213+CH403+SH8) followed by prebiotics showed lower mortality, higher blood high-density lipoprotein levels, and lower blood uric acid levels than the controls (p<0.0004). Probiotic feeding significantly increased the Bacillus and LAB counts (p<0.05). A CE330 isolate obtained from the cecum after 35 days of KKU213 feeding was closely related to B. subtilis KKU213. Conclusion: B. subtilis KKU213 is a potent probiotic strain that can survive, colonize and reduce Salmonella infection in broilers and improve their growth and health. This strain, combined with different LAB can act synergistically in the gut and promote broiler growth.