Enhancing the Antioxidant and Anti-inflammatory Potentials of Mulberry-derived Postbiotics through Submerged Fermentation with B. subtilis H4 and B. amyloliquefaciens LFB112

Mulberry-derived postbiotics (MDP) have been scientifically proven to possess beneficial properties for human health, making them potential nutraceuticals. This study aims to enhance the antioxidant and anti-inflammatory effect of MDP via submerged fermentation with Bacillus subtilis H4 (H4) and Bacillus amyloliquefaciens LFB112 (LFB112) bacterial strains. Our results demonstrated compelling evidence that the combination of two strains with the optimized time (24 hours), inoculum size (8%), and substrate size (10%) significantly increased the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities. When compared to the non-fermented mulberry (NFM), the MDP with the above-optimized conditions showed substantially higher antioxidant activities such as DPPH, 2,2’-Azino-bis (3-ethylebenzothiazoline-6-sulfonic acid) (ABTS), hydroxyl scavenging activities, and reducing power capabilities. Moreover, the MDP showed a significant decrease in the lipopolysaccharides (LPS)-induced reactive oxygen species (ROS) generated in RAW264.7 cells. The antibacterial assays such as minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) showed that MDP possesses antibacterial activity against various pathogenic bacterial strains with the lowest MIC recorded as 32.5 mg/ml against S. pullorum CVCC519, while the highest MIC observed was 40 mg/ml against P. aeruginosa. The lowest and highest MBCs observed were 40 and 55 mg/ml against S. aureus ATCC43300 and S. pullorum CVCC519 respectively. Furthermore, the MDP showed good ant-inflammatory activities by significantly reducing the nitric oxide (NO) concentration (p<0.0001) and mRNA expression of the proinflammatory cytokines in response to the LPS-induced inflammation in the RAW264.7 macrophage cell line. To sum up, our findings indicate that MDP as a novel postbiotics can be utilized both as a functional food and a safe feed additive, possibly benefiting gut and metabolic health.