Metabolic profiling of polysaccharides from Leccinum crocipodium (Letellier.) Watliag stem fermented by Bacteroides thetaiotaomicron and their immunomodulatory effects

Leccinum crocipodium (Letellier.) Watliag has attracted increasing attention for their biological activity. In this study, the active polysaccharide components (LCSP11 and LCSP22) extracted from the stem of L. crocipodium (Letellier.) Watliag were investigated to assess structural characterization of LCSP11 and LCSP22, their effects on the metabolic profile of Bacteroides thetaiotaomicron, and the immune activities of the resulting fermentation products. The results showed that LCSP11 and LCSP22 were a type of heteropolysaccharide and amorphous state with good stability, which displayed molecular aggregation in aqueous solutions. LCSP11 and LCSP22 were effectively fermented by Bacteroides thetaiotaomicron, producing a variety of microbial metabolites, including organic acids and derivatives (30.13 %), lipids and lipid-like molecules (21.33 %), and organoheterocyclic compounds (17.45 %). Multiple differential metabolites were identified in the fermentation products (F11 and F22), with significant accumulation of peptides, amino acids, nucleotides, steroids, and fatty acids, such as murabutide and L-cystine. KEGG pathway analysis identified six enriched metabolic pathways in F11 and five in F22, with the histidine metabolic pathway significantly enriched in F11. Furthermore, LCSP22 fermentation by Bacteroides thetaiotaomicron produced short-chain fatty acids, including acetic acid, propionic acid, isovaleric acid, and caproic acid. Cellular experiments suggested that these fermentation metabolites exhibited immunoactivating effects on RAW264.7 cells, significantly enhancing phagocytic capacity and promoting the secretion of nitric acid (NO) and cytokines, including TNF-α, IL-6, and IL-2. These results provide new insights into the immunomodulatory activities of polysaccharides from the stem of L. crocipodium (Letellier.) Watliag fermented by Bacteroides thetaiotaomicron and broadens the potential applications of this natural resource in food, nutrition, and biomedicine.