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Abstract

Edible Boletus mushrooms hold considerable development potential due to their exceptional nutritional and biological profiles. This study characterized two novel galactans, NBP and BRP, extracted from Neoboletus brunneissimus and Butyriboletus roseoflavus , respectively. Structural analysis revealed that both NBP and BRP possess a backbone composed of α-1,6-linked galactopyranosyl residues substituted at O -2, with structural diversity arising from variations in the side-chain substituents. Although both polysaccharides exhibit low viscosities, BRP forms a shear-stable elastic gel network, contrasting with the predominantly linear structure of NBP. In vitro fermentation demonstrated that both galactans markedly promoted the proliferation of beneficial probiotics, optimized gut microbiota composition, and enriched butyrate-producing bacteria including Faecalibacterium prausnitzii . Furthermore, they stimulated the production of lactic acid and short-chain fatty acids (SCFAs), leading to a reduction in fermentation pH and thereby modulating microbial ecology and host energy metabolism. Metagenomic annotation revealed that galactan degradation was driven by glycoside hydrolases (GHs) from Bacteroidaceae, and molecular docking analyses indicated that these GHs exhibit distinct binding preferences for specific structural regions of the polysaccharides. These results explain the basis for the microbiota-dependent improvement of gut health by Boletus galactans, providing a theoretical foundation for their development as precision prebiotics.