Role of gut microbiome in obesity and type 2 diabetes (T2D) became apparent from several independent studies, indicating that gut microbiome modulators like prebiotics may improve microbiome perturbations (dysbiosis) to ameliorate metabolic derangements. We herein isolate water soluble, non-digestible polysaccharides from five plant based foods (acorn, quinoa, sunflower, pumpkin and sago seeds). Various drying procedures were applied during the process of isolating acorn polysaccharide to investigate the structure and functional properties of this new prebiotic. Three drying methods (freeze, hot air and vacuum drying) in different temperatures were comparatively studied on the physicochemical properties (solubility, water and oil-holding capacity [OHC/WHC]), bioactivity (resistance to acidic and enzymatic digestions, effect on a probiotic strain growth) and antioxidant activity of AP along with the structural changes. Results suggest that the drying methods in combinations of temperatures and time of drying process affect physicochemical properties, antioxidant activity and bioactivities of AP. Freeze dried AP exhibited the highest solubility, WHC, OHC and antioxidant activity, digestibility with simulated gastrointestinal juices and fermentable by a Lactobacillus plantarum. Whereas, hot air dried (80 °C) exhibited second highest antioxidant and functional activities like solubility, WHC, OHC and fermentation. FTIR analysis showed that the changes caused by varying drying methods of AP starch are related to its amorphous or crystallinity structure and differences in functional group. Overall, these results suggest that freeze drying and hot air drying at 80 °C can be appropriately use to obtain a functional polysaccharide from acorn, as a prebiotic (resistant starch). In the final phase of this study, During polysaccharide isolation, purification, biochemical and digestion resistance characterization, and fermentation pattern by human fecal microbiome, we select acorn- and sago- derived prebiotics (on the basis of relatively higher purity and yield and lower protein contamination) and examine their effects in comparison to inulin to assess their impact on human fecal microbiome and amelioration of high fat diet (HFD)-induced obesity/T2D in mice. Prebiotics treatments in human fecal microbiome culture system not only preserve microbial diversity but also appear to foster beneficial bacteria and short-chain fatty acids (SCFAs). Feeding of acorn- and sago-derived prebiotics ameliorates HFD-induced glucose intolerance and insulin resistance in mice, with effects comparatively superior to those seen in inulin-fed mice. Feeding of both of novel prebiotics as well as inulin increases SCFAs levels in the mouse gut. Interestingly, gut hyperpermeability and mucosal inflammatory markers were significantly reduced upon prebiotics feeding in HFD-fed mice. Hypothalamic energy signaling in terms of increased expression of pro-opiomelanocortin, was also modulated by prebiotics administration. Results demonstrate that these (and/or such) novel prebiotics can ameliorate HFD-induced defects in glucose metabolism via positive modulation of gut-microbiome-brain axis and hence could be useful in preventing/ treating diet-induced obesity/T2D. Key words : Prebiotic, polysaccharides, fibers, functional properties, drying, microbiome, metabolites, obesity, diabetes, mice.