Metabolomic Characterization of Baby Spinach Phenolics Transformation During Gastrointestinal Digestion and Microbiome-Mediated Metabolism

Gastrointestinal digestion and colonic fermentation determine phenolic transformation and reciprocal microbiome modulation, influencing the generation of gut-derived metabolites associated with epithelial integrity, inflammatory regulation, and metabolic homeostasis. Baby spinach phenolics possess antioxidant and microbiome-modulating potential; however, their functional efficacy is constrained by storage-induced degradation and limited gastrointestinal bioaccessibility. This study investigated phenolic transformation in fresh and stored baby spinach (4 °C and 25 °C) during simulated gastrointestinal digestion and subsequent colonic fermentation. Standardized in vitro digestion revealed limited phenolic bioaccessibility (10–15%), with storage at 25 °C accelerating oxidative degradation and reducing antioxidant capacity. Storage at 25 °C reduced TPC from approximately 465 to 265 µg GAE/g and decreased antioxidant activity by nearly 30%, whereas refrigerated storage (4 °C) better preserved phenolic stability and antioxidant capacity throughout the storage period. LC–MS/MS–based untargeted metabolomics characterized digestion-driven structural remodeling and identified diverse colonic metabolites generated during human fecal fermentation. Despite storage-induced alterations in precursor phenolics, 16S rRNA sequencing demonstrated microbiome relative microbial stability, with fermentation time exerting a stronger influence on community assembly than storage conditions. Microbial metabolism produced shared downstream metabolites, particularly phenylpropionic and flavonoid-derived intermediates. These results suggest that storage modifies phenolic availability during digestion, while gut microbial metabolism sustains the production of functionally relevant metabolites.