## 4 Discussion  
Environmental probiotic _Bacillus_ species display the potential to support human and animal microbiota. Bacterial spore formers of the _Bacillus_ genus are being used as probiotic supplements for use in animals and humans. Indeed, endospore formers are interesting because their spores are resistant to acidic pH and stable for long periods of time in probiotic preparations [31]. However, new strategies to improve the germination, fitness and bioactivity of _Bacillus_ probiotics, once in the gut, have to be explored [32]. Among these strategies, the combination with dietary fibers, such as prebiotics, has retained the attention of the research and industry communities. In this work, we showed the ability of several _Bacillus_ probiotic strains to grow on commercial prebiotics. Among them, _B. subtilis_ CU1 was grown on a commercial prebiotic substrate isomaltooligosaccharide (IMOS, Nanjing Zelang, China). _B. subtilis_ CU1 is used in a probiotic preparation for oral bacteriotherapy of gastrointestinal disorders, microbiota dysbiosis and to enhance immunity [13; 14; 33]. However, little is known regarding the possible association of _B. subtilis_ CU1 with certain prebiotics, such as IMOS. Structural spectrometric analysis of the commercial IMOS purchased for this study showed a composition with glucose units with \(\alpha(1\to 4)\) and \(\alpha(1\to 6)\) linkages, and a degree of polymerization (DP) estimated between 1 and 29. Depending on the manufacturer, com  
Figure 4: IMOS provides an advantageous fitness to _B. subtilis_ CU1 spores in the presence of bile salts. Graphs (**A,B**) represent the growth curves for _B. subtilis_ CU1 obtained in minimal medium with glucose or IMOS as carbon source in the presence or absence of 0.5% bile salts and inoculated with vegetative bacteria (**A**) or spores (**B**), respectively. Growth curves have been obtained by OD\({}_{959\text{nm}}\) measurement. (**C**) Bacterial numeration in CFU/mL in the culture medium inoculated with spores after 48 h of culture in the presence of bile (**D**) DP1, DP2 and DP3 determination by LC during the growth of _B. subtilis_ CU1 with 0.5% bile, with IMOS as carbon substrate. The data presented are from one representative experiment among three. Data are presented as means \(\pm\) SD (Mann–Whitney test; \({}^{*}\)_p_\(<\) 0.05), excepted for (**D**). merical preparations of IMOSs could be quite different and influence their digestibility [34]. As a functional oligosaccharidesacharide, IMOS has been demonstrated to improve gut microbiota and promote the proliferation of beneficial bacteria, especially _Bifidobacteria_[35]. Moreover, the beneficial effects of IMOSs on animal and human health have been previously described [36; 37], but little is known about the effect of a diet supplementation with IMOSs on the metabolism and fitness of probiotic _Bacilii_, especially in humans. It is known that _Bacillus subtilis_ strains can be used to attenuate dysbiosis in vitro and in animals [38; 39; 40; 41]. Moreover, [PERSON] et al. [42] reported that _Bacillus_ and IMOSs complement each other in improving gut microbiota populations and intestinal disorders. More recently, [PERSON] et al. [43; 44] reported that dietary IMOSs and _Bacillus_ supplementation regulated the concentration of serum reproductive hormones and the duration of farrowing and post-weaning estrus by changing the gut microbiota of sows. If few papers have reported the beneficial effects of the IMOS and _Bacillus_ combination, to our knowledge, there is no study evaluating if IMOSs could support _Bacillus_ metabolism and physiology once in the gut and how IMOS could improve _Bacillus_ probiotic effects. Commercial probiotic _Bacilii_, including _B. subtilis_ CU1, are commercialized under spore form. This means that the bacteria reach the intestinal tract as a spore, and are able to germinate if the conditions are favorable [7; 8].