In Vitro Characterization of Probiotic Strains Bacillus subtilis and Enterococcus durans and Their Effect on Broiler...

In vitro experiments were conducted to characterize the effect of bile salt supplementation and pH on the proliferation of Bacillus subtilis CE330 and Enterococcus durans CH33 probiotics and in vivo experiments on production performance, cecal Salmonella enterica serovar Enteritidis (S. Enteritidis) load, and the immune response of broilers. A one-way ANOVA was used to examine the effect of bile and pH on probiotic species proliferation. B. subtilis. CE330 was more tolerant to high bile concentrations and pH levels compared to E. durans CH33. Bile concentrations between 3.0 and 4.0% and a pH range between 2 and 4 decreased (p < 0.05) the proliferation of E. durans CH33. In vitro, cell-free supernatants (CFSs) of B. subtilis CE330 and E. durans CH33 at a ratio of 1:1 significantly (p < 0.05) reduced S. Enteritidis proliferation, with the highest inhibition observed at a 5:1 ratio of E. durans CH33 CFS. The cultures of B. subtilis CE330 and E. durans CH33 with 4% bile salt for 72 h had a higher proline concentration of 56.95 (13.1-fold) and 20.09 (2.5-fold) µmol/g of fresh weight, respectively. A total of 144 one-day-old male Cobb broiler chicks were randomly allocated to four treatment groups-basal diet, basal diet + challenge, probiotics (B. subtilis CE330 and E. durans CH33, 0.5 g/kg feed), and probiotics + challenge in six replications. On day 14, birds in the challenge treatment were orally challenged with 1 × 10⁸ CFU of S. Enteritidis. A two-way ANOVA was used to examine the effects of probiotic supplementation and Salmonella challenge on dependent variables after 10 d post-Salmonella infection. Probiotic supplementation did not alter the body weight gain, the feed conversion ratio, the intestinal histomorphology (p > 0.05), or IL-1β and IL-10 gene expression (p > 0.05) at 10 dpi. However, probiotic supplementation decreased the Salmonella load by 38% compared to the control group. In conclusion, B. subtilis CE330 and E. durans CH33 reduced cecal S. Enteritidis load by 38%, thereby demonstrating their potential as probiotic interventions to enhance food safety and serve as alternatives to antibiotics in poultry. Hence, when developing multi-strain probiotic formulations, it is essential to emphasize the biocompatibility of various strains within the host system.