Response surface methodology optimization of cell-free supernatant from P. pentosaceus BJQ fermentation of CeRS3 and...

This study established and optimized a high-density fermentation process for BJQ-CeRS3, a co-fermentation system of Pediococcus pentosaceus BJQ (P. pentosaceus BJQ) and type 3 resistant starch derived from canna (CeRS3), and evaluated its metabolic characteristics and lipid-lowering activity in vitro. Single-factor experiments and response surface methodology (RSM) based on a Box-Behnken design (BBD) identified the optimal conditions as 62 h of fermentation, a liquid-solid ratio of 10.6:1, and an inoculation size of 3.3%. Under these conditions, the viable count reached 11.00 ± 0.04 log₁₀ CFU/mL, in close agreement with the predicted value. Untargeted LC-MS analysis showed that the fermentation process was characterized by the enrichment of organic acids, amino acids, and lipid-derived metabolites, with citric acid, acetic acid, and L-phenylalanine among the major compounds. In an oleic acid-induced HepG2 steatosis model, BJQ-CeRS3 significantly reduced intracellular triglyceride and total cholesterol levels (TC) within the non-cytotoxic concentration range, with a stronger effect on triglycerides (TG). These results indicate that optimized fermentation enhanced both biomass accumulation and the formation of potentially bioactive metabolites. This study provides a practical workflow for fermentation optimization and functional evaluation of microbial products.