Efficient Production Strategy of a Novel Postbiotic Produced by Bacillus subtilis and Its Antioxidant and Anti-Inflammatory Effects

Microbially synthesized postbiotics have unique properties and advantages; however, systematic studies on the efficient production and biological functions of postbiotics from <i>Bacillus subtilis</i> are limited, which greatly restricts their applications. In this study, we obtained a novel crude exopolysaccharide (EPS) postbiotic from <i>Bacillus subtilis</i> H4. We systematically optimized the EPS production strategy using single-factor analysis, Plackett-Burman design, the path of steepest ascent method, and response surface methodology. The optimized EPS yield was significantly improved, with a maximum yield of 15.01 g/L under the addition of 4.12% soy peptone, 8.99% sucrose, and 0.06% MnSO₄. We found that EPS is a neutral, heterogeneous polysaccharide with a pyranose ring, with a molecular weight of 44,304.913 kDa and a melting point of 218 °C. It consists of glucose, galactose, arabinose, glucosamine, and mannose at a molar ratio of 58.85:19.81:14.75:10.89:6.58. EPS exhibits strong antioxidant capacities, scavenging ABTS and DPPH radicals with IC50 values of 1 and 6 mg/mL, respectively. Moreover, it shows notable anti-inflammatory properties, dramatically inhibiting the lipopolysaccharide (LPS)-induced elevation of nitric oxide (NO) levels and over-activation of the TLR4-NF-κB signaling pathway. These findings highlight the potential of EPS as a multifunctional bioactive compound, offering great promise for its application in the food, clinical, and feed industries.