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This study aimed to investigate in vitro effects of the selected pre-biotics alone, and in combination with two potential probiotic Lactobacillus strains on the microbial composition of Apis ceranagut microbiota and acid production. Four prebiotics, inulin, fructo-oligosaccharides, xylo-oligosaccharides, and isomalto-oligosac¬charides were chosen, and glucose served as the carbon source. Supplementation of this four prebiotics increased numbers of Bifidobacterium and lactic acid bacteria while decreasing the pH value of in vitro fermentation broth inoculated with A. cerana gut microbiota compared to glucose. Then, two potential probiot¬ics derived from A. cerana gut at different dosages, Lactobacillus helveticus KM7 and Limosilactobacillus reuteri LP4 were added with isomalto-oligosaccharides in fermentation broth inoculated with A. cerana gut microbiota, respectively. The most pronounced impact was observed with isomalto-oligosaccharides. Compared to isomalto-oligosaccharides alone, the combination of isomalto-oligo¬saccharides with both lactobacilli strains induced the growth of Bifi¬dobacterium, LAB, and total bacteria and reduced the proliferation of Enterococcus and fungi. Consistent with these results, the altered metabolic activity was observed as lowered pH in in vitro culture of gut microbiota supplemented with isomalto-oligosaccharides and lactobacilli strains. The symbiotic impact varied with the typesand concentration of Lactobacillus strains and fermentation time. The more effective ability was observed with IMO combined with L. helveticus KM7. These results suggested that isomalto-oligosac¬charides could be a potential prebiotic and symbiotic with certain lactobacilli strains on A. cerana gut microbiota.