| Summary: | Herein, a bactericide-secreting <i>Bacillus</i> strain, potentially useful as a biocontrol agent, was isolated from the commercial Yanjing Natto food. Following the biochemical and physiological evaluation, the molecular identification was performed using 16S rDNA sequencing of polymerase chain reaction-amplified DNA that confirmed the natto isolate as <i>Bacillus subtilis</i> natto (<i>B. subtilis</i> natto). The biocontrol (microbial inhibitory) capability of <i>B. subtilis</i> natto was investigated against <i>Staphylococcus aureus</i>, <i>Escherichia coli</i>, <i>Salmonella typhimurium</i>, and yeast (<i>Yarrowia lipolytica</i>) and recorded. The antimicrobial activity of <i>B. subtilis</i> natto was further enhanced by optimizing the growth medium for optimal bactericides secretion. Under optimized conditions, <i>B. subtilis</i> natto exhibited much higher inhibitory activity against <i>S. aureus</i> with a zone of inhibition diameter up to 27 mm. After 48 h incubation, the optimally yielded <i>B. subtilis</i> natto broth was used to extract and purify the responsible bactericides by silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography. Structural identification of purified bactericides (designated as NT-5, NT-6, and NT-7) from <i>B. subtilis</i> natto was performed by <sup>13</sup>C-nuclear magnetic resonance (NMR) and mass spectral analyses. The NMR comparison also revealed that NT-5, NT-6, and NT-7 had identical structures, except for the fatty chain. In summary, the present study suggests the improved biocontrol and/or microbial inhibitory potential of newly isolated bactericides secreting <i>B. subtilis</i> natto.
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