A mixture of xylo-/manno-oligosaccharides (XMOS) was produced from Gleditsia sinensis peel (GSP) and seed endosperm (GPP) using glutamic acid (GA) catalysis. An enzymatic hydrolysis-HPLC method was established to quantify xylo-oligosaccharides (XOS) and manno-oligosaccharides (MOS) in XMOS. Rheological analysis revealed that changing the GSP-to-GPP ratio lowered initial shear stress from over 2000 Pa to below 200 Pa, affecting XOS and MOS yields. Under optimal conditions (0.3 M GA, GSP:GPP of 1.5:1), yields of 69.66% XOS and 57.49% MOS were achieved with 0.256 g/L furfural and undetectable 5-HMF. Optimized XMOS promoted the growth of Bifidobacterium adolescentisto 0.91 × 108 mL–1, exceeding XOS, MOS, and fructooligosaccharide by 93.1%, 6.6-fold, and 46.3%, respectively. XMOS fermentation produced 19.34 g/L of short-chain fatty acids, with butyric and propionic acids significantly enriched, each reaching 6.52 g/L and 5.16 g/L. This study establishes a green route for converting biomass into mixed oligosaccharides with tunable composition and distinct fermentability.
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