β-glucosidase-mediated hydrolysis of Ophiocordyceps polysaccharides enhances antioxidant and immunomodulatory activities and ameliorates intestinal barrier function to maintain gut homeostasis

Polysaccharides derived from Ophiocordyceps (Chinese Cordyceps) exhibit notable bioactivities, yet their high molecular weight may limit their fermentability by the gut microbiota, thereby modulating the production of bioactive metabolites. This study aimed to prepare low-molecular-weight Ophiocordyceps polysaccharides (OPs) through enzymatic modification and to elucidate the associated structural alterations and mechanisms underlying intestinal barrier regulation. Hydrolysis with β-glucosidase yielded the enzymatically modified product OPs-80E. Structural characterization demonstrated that hydrolysis significantly reduced the average molecular weight from 8.64 × 10 5 Da to 2.99 × 10 4 Da and altered the monosaccharide composition profile. In vitro assays showed that OPs-80E exhibited greater antioxidant capacity and immunomodulatory activity than native OPs-80, as evidenced by enhanced RAW264.7 cell proliferation, phagocytosis, and cytokine secretion. In a cyclophosphamide-induced immunosuppressed mouse model, OPs-80E was more effective than OPs-80 in reversing intestinal histopathological damage, significantly upregulating the expression of tight junction proteins (ZO-1 and Claudin-1), and remodeling the gut microbiota architecture. 16S rRNA gene sequencing analysis indicated that OPs-80E significantly enriched butyrate-producing taxa, such as Lachnospiraceae, while suppressing potential pathogens. Correlation network analysis further revealed that OPs-80E established a symbiotic network centered on the Prevotellaceae Ga6A1 group and Clade Ia , facilitating synergistic “polysaccharide degradation and butyrate production” metabolic cross-feeding to maintain intestinal barrier integrity. Collectively, enzymatic hydrolysis represents an effective strategy for enhancing the bioactivity of Chinese Cordyceps polysaccharides. OPs-80E exerts intestinal protective effects by modulating the “microbiota and barrier” axis, highlighting its broad application prospects in functional foods and pharmaceutical development.