Pile-fermentation and golden-flower fermentation reshape tea polysaccharides in Tibetan dark tea: structural...

Pile-fermentation and golden-flower fermentation are key processing steps in Tibetan dark tea, yet their combined effects on tea polysaccharides remain poorly understood. To distinguish the individual and combined contributions of pile-fermentation and golden-flower fermentation, four polysaccharide-enriched fractions were prepared in a 2 × 2 design: LTPS (neither process), LFTPS (golden-flower fermentation only), HTPS (pile-fermentation only), and HFTPS (both processes applied sequentially). All fractions were identified as uronic acid-rich acidic heteropolysaccharides with similar monosaccharide species but distinct molar ratios. Processing markedly altered the molecular-weight distribution, and HFTPS showed the lowest apparent molecular weight and the highest absolute zeta potential. UV-Vis, FT-IR, XRD, and SEM analyses indicated that the main polysaccharide backbone remained largely preserved, whereas microstructural and physicochemical properties were substantially reshaped by processing. Among the four fractions, HFTPS exhibited the strongest antioxidant capacity and the highest inhibitory activities against α-glucosidase, cholesterol esterase, and lipoxygenase. In a high-glucose-induced Caenorhabditis elegans model, tea polysaccharides reduced lipid accumulation, improved locomotion, prolonged lifespan, and enhanced heat- and oxidative-stress resistance. These phenotypic improvements were accompanied by biochemical changes and transcriptional responses consistent with enhanced fatty acid oxidation and reduced lipogenesis. Exploratory correlation analysis further suggested that lower apparent molecular weight, higher polyphenol content, and more pronounced acidic and composite characteristics were generally associated with improved bioactivities. Overall, the fraction obtained after pile-fermentation followed by golden-flower fermentation showed the most favourable integrated structural and functional profile among the four fractions, supporting the potential use of Tibetan dark tea polysaccharides in functional food applications.