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Bacillus subtilis CU1 is a probiotic strain with beneficial effects on immune health in elderly subjects and diarrhea. Commercialized under spore form, new strategies to improve the germination, fitness and beneficial effects of the probiotic once in the gut have to be explored. For this purpose, functional food ingredients, such as isomaltooligosaccharides (IMOSs), could improve the fitness of Bacillus probiotics. IMOSs are composed of α(1 → 6)- and α(1 → 4)-linked oligosaccharides and are partially indigestible. Dietary IMOSs stimulate beneficial members of intestinal microbiota, but the effect of a combination of IMOSs with probiotics, such as B. subtilis CU1, is unknown. In this study, we evaluate the potential effect of IMOSs in B. subtilis CU1 and identify the metabolic pathways involved. The biochemical analysis of the commercial IMOSs highlights a degree of polymerization (DP) comprised between 1 and 29. The metabolism of IMOSs in CU1 was attributed to an α-glucosidase, secreted in the extracellular compartment one hundred times more than with glucose, and which seems to hydrolyze high DP IMOSs into shorter oligosaccharides (DP1, DP2 and DP3) in the culture medium. Proteomic analysis of CU1 after growth on IMOSs showed a reshaping of B. subtilis CU1 metabolism and functions, associated with a decreased production of lactic acid and acetic acid by two times. Moreover, we show for the first time that IMOSs could improve the germination of a Bacillus probiotic in the presence of bile salts in vitro, with an 8 h reduced lag-time when compared to a glucose substrate. Moreover, bacterial concentration (CFU/mL) was increased by about 1 log in IMOS liquid cultures after 48 h when compared to glucose. In conclusion, the use of IMOSs in association with probiotic B. subtilis CU1 in a synbiotic product could improve the fitness and benefits of the probiotic. [ABSTRACT FROM AUTHOR]