Microbial fermentation alters the phytochemical profile of Ginkgo biloba leaves

• Four microorganisms were screened and used for Ginkgo biloba leaves fermentation. • Microbial fermentation increased total polyphenols, flavonoids, and polysaccharides compared to unfermented GBL. • Untargeted metabolomics found 116 differentially expressed secondary metabolites, mainly in plant secondary metabolite and flavonoid biosynthesis pathways. • Crocin was hydrolyzed to crocetin during fermentation, enhancing bioactive compounds. This study employed microbial fermentation to alter phytochemical profiles in Ginkgo biloba leaves (GBL). Four microorganisms— Lactobacillus plantarum SCTM-1, Saccharomyces cerevisiae GLLB-3, Aspergillus niger ZYDC-1, and Aspergillus cristasukae LNAC-1—were used individually and in a consortium (YXSL: L. plantarum + S. cerevisiae ). All the microbial fermentation changes the GBL metabolite profiles, including total polyphenols, flavonoids, and polysaccharides. Especially, compared to unfermented GBL, YXSL increased total polyphenols, flavonoids, and polysaccharides by 76.0%, 170.6%, and 20.9%, respectively. Metabolomic analysis identified 116 differentially expressed secondary metabolites, mainly in plant secondary metabolite, phenylpropanoid, and flavonoid biosynthesis pathways. YXSL specifically enhanced flavonoid production via microbial biotransformation, with crocetin content increasing 104.5% through hydrolysis of crocin. This work highlights microbial potential to boost GBL’s bioactive components and provides a basis for improving phytochemical yields via fermentation.