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Pectin recovered from mango peel biomass can be used as a potential source for pectic oligosaccharide hydrolysate with excellent probiotic growth-enhancing performance and prebiotic potentials. Consequently, the objectives of the current study were to optimise the enzyme hydrolysis treatment of mango peel pectin (MPP) and to evaluate the pectic oligosaccharide effects of Lactobacillus reuteri DSM 17938 and Bifidobacterium animalis TISTR 2195. Mango of “chok anan” variety was chosen due to its excessive volume of biomass in processing and high pectin content. The optimal treatment for mango peel pectic oligosaccharide (MPOS) valorisation was 24 h of fermentation with 0.3% (v/v) pectinase. This condition provided small oligosaccharides with the molecular weight of 643 Da that demonstrated the highest score of prebiotic activity for both of B. animalis TISTR 2195 (7.76) and L. reuteri DSM 17938 (6.87). The major sugar compositions of the oligosaccharide were fructose (24.41% (w/w)) and glucose (19.52% (w/w)). For the simulation of prebiotic fermentation, B. animalis TISTR 2195 showed higher proliferation in 4% (w/v) of MPOS supplemented (8.92 log CFU/mL) than that of L. reuteri (8.53 CFU/mL) at 72 h of the fermentation time. The main short chain fatty acids (SCFAs) derived from MPOS were acetic acid and propionic acid. The highest value of total SCFA was achieved from the 4% (w/v) MPOS supplementation for both of B. animalis (68.57 mM) and L. reuteri (69.15 mM). The result of this study therefore conclusively advises that MPOS is a novel pectic oligosaccharide resource providing the opportunity for the sustainable development approach through utilising by-products from the fruit industry.