Fermentation Optimization of Ergosta‐4,6,8(14),22‐Tetraen‐3‐One From <i>Aspergillus oryzae</i> and Its Anti‐Inflammatory Mechanism via Multi‐Pathway Inhibition of <scp>MyD88</scp> / <scp>NF</scp> ‐ <scp>κB</scp> / <scp>MAPK</scp> / <scp>NLRP3</scp> S

<i>Aspergillus oryzae</i> is a key microorganism in the production of various traditional food products, including rice wine, soy sauce, and baijiu. In this study, we identified and quantified ergosta-4,6,8 (14), 22-tetraen-3-one (ETO), a bioactive compound derived from <i>A. oryzae</i>, using high-performance liquid chromatography (HPLC). Our results confirmed that ETO is one of the major components produced by <i>A. oryzae</i>. Bioactivity assays of the crude extract revealed that ETO exhibits significant anti-inflammatory properties. To enhance ETO production, we optimized the fermentation medium. Single-factor experiments identified glycerol and N-acetylglucosamine as the optimal carbon and nitrogen sources, respectively. An orthogonal experimental design was then employed to further refine the fermentation conditions, resulting in the determination of an optimal medium composition. The feasibility and effectiveness of this optimized fermentation process were validated, laying the groundwork for the industrial-scale production of ETO. To elucidate the anti-inflammatory mechanism underlying ETO's bioactivity, further investigations were performed in LPS-stimulated RAW264.7 macrophages. The results demonstrated that ETO significantly reduced the secretion of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, and suppressed the activation of the MyD88/NF-κB/MAPK/NLRP3 signaling pathway, indicating its potential as a potent anti-inflammatory agent. In conclusion, ETO is not only abundantly produced by <i>A. oryzae</i>, but also exhibits pronounced anti-inflammatory activity, providing a promising foundation for its future development in anti-inflammatory therapeutics.