Optimization of culture conditions for enhanced antibacterial metabolite production by Bacillus velezensis TL and evaluation of its efficacy against necrotic enteritis in broilers

Bacillus velezensis has been widely utilized in agriculture and livestock; however, the efficacy of the metabolites of B. velezensis in the prevention and control of animal disease has not been reported. This study aimed to increase the production of antibacterial metabolites of B. velezensis TL by optimizing the culture conditions and evaluating their ability to mitigate necrotic enteritis caused by Clostridium perfringens. In the Landy medium, cellobiose was screened as the carbon source among glucose, mannose, arabinose, and maltodextrin, which significantly enhanced the anti-C. perfringens activity of the fermentation supernatant. When twelve amino acids, shikimic acid and two amino acid mixtures were added to cellobiose-supplemented Landy medium, the antibacterial activity was not enhanced. Therefore, the optimized media included 10 g/L cellobiose, 5 g/L L-glutamate, 0.002 g/L L-phenylalanine, 0.5 g/L MgSO₄·7H₂O, 0.5 g/L KCl, 1.0 g/L KH₂PO₄, 0.15 mg/L FeSO₄, 5.0 mg/L MnSO₄, and 0.16 mg/L CuSO₄. The optimal fermentation time is 24 h, and the working volume is 20%. Under these conditions, the antibacterial activity of the fermentation supernatant also increased against E. coli, S. enteritidis, S. aureus and S. suis. Combined genomic and transcriptomic analyses revealed that B. velezensis TL contains seven antibacterial biosynthesis-related gene clusters that are upregulated in the optimized medium. In vivo trials demonstrated that dietary supplementation with the metabolites of B. velezensis TL significantly reduced intestinal damage, improved the duodenal villus height‒crypt depth ratio (P< 0.05), and decreased the C. perfringens load in the duodenum from 4.6 ± 0.2 to 3.4 ± 0.6 log₁₀ (copies/g) and in the jejunum from 6.3 ± 0.7 to 5.2 ± 0.3 log₁₀ (copies/g) compared with those in the group challenged with C. perfringens ML2 (P< 0.05). These results suggest that B. velezensis TL-fermented metabolites may serve as effective postbiotics for the prevention of necrotic enteritis in broilers.