Unveiling the formation mechanism of Moringa oleifera leaf flavonoids aglycones during Monascus anka fermentation by using integrated widely targeted metabolomics and proteomics analysis

This study revealed the formation mechanism of flavonoid aglycones in Moringa oleifera leaf (MOL) during Monascus anka fermentation via widely targeted metabolomics and data-independent acquisition-based proteomics. Bioactivity assays demonstrated that M. anka fermentation significantly enhanced MOL's bioactivity; total flavonoid and phenolic content, ABTS and DPPH radical scavenging capacities were increased by 226.8%, 45.59%, 1.28-, and 1.25-fold compared with the unfermented control, respectively. Widely targeted metabolomics identified 172 phenolic metabolites, of which flavonoids accounted for 70.9%. Notably, flavonoid aglycones (quercetin and kaempferol) were significantly accumulated during fermentation, being 5.05-, 2.65-, and 5.54-fold higher than the unfermented control. Proteomics analysis identified key carbohydrate-active enzymes associated with cell wall degradation. Importantly, β -glucosidases (GH1, GH3) and α -L-rhamnosidases (GH78) hydrolyzed flavonoid glycosidic linkages, facilitating free aglycone accumulation. Collectively, these findings indicated that M. anka -secreted enzymes exerted catalytic roles in cell wall degradation and flavonoid deglycosylation, providing a theoretical basis for targeted enhancement of MOL bioactivity.