Optimization of Total Flavonoid Production from Chenpi via Solid-state Fermentation with <italic>Bacillus subtilis</italic> and Evaluation of Antioxidant Activity

In this study, we investigated solid-state fermentation (SSF) as a bioprocessing strategy to improve the total flavonoid yield and systematically evaluate the quality enhancement of Chenpi. Bacillus subtilis was selected as the optimal fermentation strain based on total flavonoid yield. The SSF process was systematically optimized through single-factor experiments and response surface methodology using a Box–Behnken design. Subsequently, the total flavonoid content, levels of four major flavonoid compounds (hesperidin, hesperetin, nobiletin, and tangeretin), and in vitro antioxidant activity, as evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays, were compared between fermented and non-fermented Chenpi. Results showed that the optimized fermentation conditions were as follows: Inoculation volume of 20%, liquid-to-solid ratio of 0.8:1 mL/g, temperature of 34 ℃, and fermentation time of 25 h. Under these conditions, the total flavonoid yield reached 2.22%. Compared with the non-fermented control, the total flavonoid content in fermented Chenpi increased significantly by 21.08% (22.17±1.01 vs 18.31±1.02 mg RE/g, PPPPP50 values for DPPH and ABTS+ radicals from 1.17 and 1.15 mg/mL to 0.83 and 0.69 mg/mL, respectively. In summary, SSF with B. subtilis is an effective method for simultaneously improving total flavonoid yield, enhancing individual flavonoid contents, and boosting antioxidant activity in Chenpi. This method provides a technical basis and shows great application potential for developing high-value Chenpi-based products.