Ehretia macrophylla fruit is a traditionally used but underutilized functional resource that is noted for its diverse phytochemical composition, which includes flavonoids and polyphenols. However, the systematic metabolic transformations that occur in the fruit during post-harvest storage—particularly those associated with its traditional processing into dark fruit tea—remain largely unknown, limiting efforts to optimize quality and processing strategies. In the study, to address this gap, we employed a widely targeted metabolomics approach based on ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) to dynamically profile the fruit metabolome across four storage stages (T0, T4, T8, T12). A total of 1101 metabolites were putatively annotated, and day 8 (T8) emerged as a potential pronounced metabolic inflection point. From T8 onward, 30–40% of metabolites showed differential accumulation, characterized by a gradual rise in nutrition-relevant lipids, amino acids, and vitamins. In contrast, key antioxidant-related metabolites, including phenolics and flavonoids, increased transiently before T8 but declined sharply thereafter. These coordinated shifts correlated with an increased representation of lipid- and alkaloid-related metabolic pathways, whereas flavonoid-associated features showed a relative decline. Collectively, these findings provide insights into the biochemical processes potentially associated with fruit blackening and the marked decline in antioxidant capacity during traditional processing. Our study provides the first metabolic blueprint connecting traditional processing practices with phased metabolic remodeling, offering a scientific foundation for quality assessment and the development of informed post-harvest strategies for processing E. macrophylla fruit.
Pectin oligosaccharides have emerged as promising prebiotics whose biological activities are closely tied to their structural features, particularly monosaccharide composition. However, systematic experimental evidence regarding how different monosaccharide compositions collectively influence gut microbiota modulation, SCFA production, and the metabolomic responses of mixed pectin oligosaccharides remains limited. To address this gap, Pool-I (27.92% galacturonic acid, rich in neutral sugars including galactose, glucose, and arabinose) and Pool-II (98.73% galacturonic acid) were evaluated by comparison with inulin, a reference prebiotic, using an in vitro human fecal fermentation model. Microbial community structure, SCFA production, and metabolomic profiles were assessed at 12 h and 24 h via 16S rRNA gene sequencing, gas chromatography, and untargeted metabolomics, respectively. Inulin exhibited superior enrichment of Actinobacteria (predominantly Bifidobacterium) and consistently higher total SCFA production (24.07 ± 0.25 mmol/L acetic acid at 24 h vs. 20.27 ± 0.28 and 20.53 ± 0.76 mmol/L for pectin oligosaccharides), particularly propionic and butyric acids. Although Pool-I and Pool-II yielded lower overall SCFA concentrations, they significantly enriched beneficial SCFA-producing genera (including Coprococcus_3, Butyricicoccus, and Parabacteroides) at 24 h. Metabolomic analysis revealed that Pool-I uniquely upregulated twenty-six differential metabolites (VIP > 1, p < 0.05), including taurine (VIP = 2.40, p < 0.001), L-proline (VIP = 1.72, p = 0.015), and pantothenate (VIP = 1.63, p < 0.001), while reducing uric acid (VIP = 1.05, p < 0.001), whereas only two differential metabolites were identified for Pool-II. We conclude that monosaccharide composition is a key determinant of the prebiotic efficacy of pectin oligosaccharides: Pool-II selectively promotes SCFA-producing taxa, whereas Pool-I elicits broader metabolic regulation. These effects provide a theoretical foundation for developing pectin oligosaccharides as potential prebiotic candidates.
