## 4 Discussion
In a competitive microbial environment, nutrient acquisition is a major contributor to the survival of any individual bacterial species, and the ability to access uncommon energy sources can provide a fitness advantage. The enhancement of metabolic conditions for probiotic CU1 could be of great interest to improve probiotic efficiency. For this reason, we next evaluated the effect of IMOSs on bacteria physiology. We have shown that high DP IMOSs are hydrolyzed in shorter oligosaccharides, including DP1 and DP2, in the extracellular medium of _B. subtilis_ CU1, suggesting that a glucosidase is released by the strain during its growth. We suggested that the slight increase in DP1 concentration between 10-12 h might have been due to the hydrolysis of DP3 and higher DP oligosaccharides. We showed that _B. subtilis_ CU1 produced extracellular \(\alpha\)-glucosidase, but not \(\beta\)-glucosidase, confirming the ability of the strain to hydrolyze high DP oligosaccharides in the culture medium. No study has previously reported the degradation mechanism of high DP IMOSs in the extracellular medium of _Bacillus_, and this ability could favor the development of the probiotic within the gut microbiota. [PERSON] et al. [45] showed that the metabolism of IMOSs by _Lactobacillus reuteri_ was attributed to \(\alpha(1\to 6)\)-specific glucanase DexB and maltose phosphorylase, but limited to short-chain oligosaccharides. This extracellular hydrolytic activity could favor bioavailability for shorter IMOSs to stimulate the beneficial members of intestinal microbiota and increase microbiota bioactivity, as previously observed with other prebiotics [46; 47]. We then compared the proteome of CU1 following growth on glucose or IMOSs. Several proteins involved in carbohydrate metabolism and transport were highlighted, including maltose-6-phosphate glucosidase (MalA) and phosphoenolpyruvate-protein phosphotransferase (PEP-PTS), which were overexpressed with IMOS when it was used as a unique carbon source. A proposed model for IMOS metabolism by _B. subtilis_ CU1 is presented in Figure 3. Our observations suggest that IMOSs are metabolized into maltose in the extracellular medium through the secretion of certain glycolytic enzymes, such as glucoamylases and \(\alpha\)-amylases [48], which cleaves \(\alpha(1\to 6)\) ramifications and \(\alpha(1\to 4)\) linkages, respectively. Maltose-6-phosphate is then accumulated into the bacteria via the PEP-PTS system. The maltose-6-phosphate was then taken up by GlyA to be hydrolyzed into glucose and glucose-6-phosphate, which can be used directly for glycolysis. GlyA is known to be induced in the presence of maltose and repressed in the presence of glucose [49], which agrees with our observations. However, glucose-6-phosphate dehydrogenase (G6 PDH), the first enzyme of the pentose-phosphate pathway [50], was identified during a previous analysis conducted by one-dimensional electrophoresis and overexpressed in the presence of IMOSs (data not shown). These observations suggest that the metabolism of IMOSs could fuel the pentose phosphate pathway and increase the production of NADPH. Moreover, we observed a decreased expression of several enzymes involved in glycolysis and the TCA cycle, as well as a reduced amount of lactic and acetic acid production with IMOS as a substrate, suggesting a downregulation of mixed acid fermentation. Among the proteins overexpressed in IMOS cultures, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopilenate ligase plays a central role in the biosynthesis of peptidoglycan, while a spore coat-associated protein N is decreased. Taken together, our results show that IMOSs can drastically change the physiology and energy metabolism in CU1. Finally, we reported that, in the presence of bile salt, IMOS utilization can provide an advantageous fitness to vegetative and spores of _B. subtilis_ CU1. These results show that, in the presence of bile, IMOSs can promote spore germination more effectively than glucose.