Dietary Probiotic Bacillus subtilis AAHM-BS2360 and Its Postbiotic Metabolites Enhance Growth, Immunity, and Resistance to Edwardsiellosis in Pangasianodon hypophthalmus

Edwardsiellosis, caused by <i>Edwardsiella tarda</i>, poses a significant threat to the aquaculture industry, particularly in pangasius farming. This study investigates the effects of probiotic <i>Bacillus subtilis</i> AAHM-BS2360 and its postbiotic metabolites on growth performance, immune responses, antioxidative activity, and disease resistance against <i>E. tarda</i> infection. A total of 240 healthy pangasius (37.0 ± 4.9 g) were divided into four treatment groups with four replicate tanks each, as follows: (1) the Control group, which received feed top-dressed with 100 mL of 0.85% NaCl/kg diet; (2) the Probiotic group, which received feed supplemented with 100 mL of <i>B. subtilis</i> AAHM-BS2360 cells at the concentration of 1 × 10¹² CFU/kg diet; (3) the Postbiotic group, which received feed supplemented with <i>B. subtilis</i> AAHM-BS2360 cell-free supernatant 100 mL/kg diet; and (4) the Pro + Post group, which received a combination of <i>B. subtilis</i> AAHM-BS2360 cells and cell-free supernatant. After 30 days of feeding treatment, biochemical serum analysis revealed a significant increase in the AST/ALT ratio in the Postbiotic group. The Probiotic and Postbiotic treatments increased lysozyme activity in mucus, indicating an innate immune response to pathogens. The Pro + Post group exhibited the highest levels of catalase (CAT) in serum and upregulated antioxidant-related genes. All treatment groups receiving <i>B. subtilis</i> AAHM-BS2360, metabolites, and their combinations showed significant upregulation of immune-related genes, like <i>lygl1</i>, <i>tgfb</i>, <i>b2ml</i>, and <i>tnf.</i> The expression of proinflammatory genes (<i>litaf</i>, <i>ifngr1l</i>, <i>c3</i>, <i>il13</i>, and <i>il1b</i>) increased, with the most pronounced effects observed in the Pro + Post group. The Probiotic group showed significant upregulation of the growth-related gene <i>igf1</i>. Meanwhile, the Pro + Post group showed significantly higher values in SGR and ADG parameters, with values of 3.29 ± 0.98%/day and 1.42 ± 0.52 g/day respectively (<i>p</i> < 0.05). Survival rates were significantly higher in the Pro + Post (87.5%), Postbiotic (84.37%), and Probiotic (81.25%) groups when challenged with <i>E. tarda</i>. Dietary supplementation with <i>B. subtilis</i> AAHM-BS2360, its metabolites, and their combination enhanced immune response, reduced oxidative stress, and improved growth performance in pangasius, highlighting its potential as a functional feed additive for sustainable aquaculture.