Co-Culturing Bacillus Strains for Enhanced Antimicrobial Activity

<b>Background/Objectives</b>: <i>Bacillus</i> probiotic mixtures have been used as a novel source of antimicrobial production. However, there is a need to emphasize the potential of co-cultured <i>Bacillus</i> strains and to understand the underlying mechanisms involved in their combination formulation. <b>Methods</b>: <i>Bacillus</i> strains BPR-11, BPR-16, and BPR-17, were cultured either as mono or multi-cultures. The bacterial growth and density were evaluated by measuring their optical density. The chemical profiles of their bioactive extracts were analysed by LC-MS, and their antimicrobial activity were tested against selected pathogens. <b>Results</b>: Our results reveal that co-cultured strains significantly increased bacterial growth, with an optical density level of, 2.67 which was significantly higher than the monocultures of BPR-11 (2.24), BPR-16 (2.32), and BPR-17 (2.22). Furthermore, the ethyl acetate extracts from these bacterial cultures showed that the combined co-culture (F1) exhibited the highest antimicrobial activity, with MICs of 25 µg/mL against <i>C. perfringens</i>, <i>E. coli</i>, and <i>S. aureus</i>, and 50 µg/mL against <i>P. aeruginosa</i> and <i>S. enterica</i>. In contrast, the monocultures BPR-16 and BPR-17 showed moderate activity with MICs of 50 µg/mL against <i>C. perfringens</i>, <i>E. coli</i>, and <i>S. aureus</i>, and 100 µg/mL against <i>P. aeruginosa</i> and <i>S. enterica</i>. BPR-11 had the lowest antimicrobial activity, with MICs of 100 µg/mL against <i>C. perfringens</i>, <i>E. coli</i>, and <i>S. aureus</i>, and no activity against <i>P. aeruginosa</i> and <i>S. enterica</i>. Metabolite profiling showed that the extract from the co-culture had a marked increase in the production of antimicrobial metabolites, including C₁₃-C₁₆ surfactin C. Lastly, the metabolism study of surfactin C analogues suggested that they were highly stable (99%) when incubated with cytochromes P450 over 120 min. <b>Conclusions</b>: Together, these findings highlight the potential for multi-strain co-culturing to develop new antimicrobials and provide valuable insights into the synergistic effects for antimicrobial production.