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BACKGROUND Novel functional polysaccharides from fungi are important nutraceuticals for food applications. A purified exopolysaccharide (MEP 2) was extracted and purified from the fermentation liquor of Morchella esculenta. The aim of this study was to investigate the digestion profile, antioxidant abilities and effect on the microbiota composition in diabetic mice of MEP 2. RESULTS MEP 2 is stable during in vitro saliva digestion but partially degraded during gastric digestion. The digest enzymes exert negligible effect on the chemical structure of MEP 2. Molecular weight and atomic force microscope (AFM) images suggest that both smaller chains and larger aggregations were produced. Scanning electron microscope (SEM) images reveal that the surface morphology was much altered after intestinal digestion. After digestion, the antioxidant ability increases as revealed by the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Both MEP 2 and its digested components show strong α-amylase and moderate α-glucosidase inhibition activities, leading us to further investigate its ability to modulate the diabetic symptoms. MEP 2 treatment ameliorate the inflammatory cell infiltration and increase the size of pancreas inlets. Serum concentration of HbA1c was significantly reduced. Blood glucose level during the oral glucose tolerance test (OGTT) was also slightly lower. MEP 2 increases the diversity of gut microbiota and modulate the abundance of several important bacteria including Alcaligenaceae, Caulobacteraceae, Prevotella, Brevundimonas, Demequina and several Lachnospiraceae species. CONCLUSION MEP 2 was partially degraded during the in vitro digestion. Its potential antidiabetic bioactivities may associate with its α-amylase inhibition and gut microbiome modulation ability. This article is protected by copyright. All rights reserved.