## 3 Results
### Fermentation of Fractan-Type Carbohydrates by BB-12 and Complex Microbiota (Experiment 3)  
To characterize the symbiotic potential of the fructan-type carbohydrates in combination with BB-12 in presence of a complex microbiota, short-term colonic incubations were performed using the fecal inocula of the three selected donors. Upon performing 16 S-targeted Illumina sequencing coupled with flowcytometry on the samples collected after 48 h of incubation to obtain absolute numbers, an OTU5 related to _Bifidobacterium animals_, i.e., species to which BB-12 belongs to, was detected in all reactors to which BB-12 was dosed (72 independent reactors), while not being detected in any of the reactors to  
Figure 3: Metabolic activity of BB-12 in presence of sterile fecal suspension of six donors (‘BB-12’) and of the fecal microbiota of the six same donors as such (‘FS’) upon dosing starch as carbon source. Bars represents the average changes (\(\pm\)SEM) in pH (**A**), gas production (**B**), acetate ((**C**); mM), lactate ((**D**); mM), propionate ((**E**); mM), butyrate ((**F**); mM) and bCFA ((**G**); mM) levels between 0 and 6 h (light grey), 6 h and 24 h (dark grey) and 24 h and 48 h (stripes) as well as changes in _Bifidobacterium_ levels ((**H**); expressed as the ratio of their absolute abundance as measured via qPCR at the end versus the beginning of the incubation (48 h/0 h)) (\(n=6\)). BB-12 = _Bifidobacterium animals_ subsp. _lactis_; SEM = standard error of means. which BB-12 was not dosed (again 72 independent reactors; data not shown). Therefore, although 16S-targeted Illumina sequencing cannot be considered as a species or strain-specific detection method in general, detection of OTU5 was considered to reflect the abundance of BB-12 for this very specific experimental setup. This allowed to conclude that upon its administration to the complex microbiota, mild enrichment of BB-12 (as reflected by OTU5) was observed in the probiotic incubations (blank) of donors C (Figure 4A), D (Figure 4B) and F (Figure 4C). The selective growth of BB-12 was strongly boosted upon its co-administration with each of the seven fructan-type carbohydrates, as seen by the significant enrichment compared to the blank incubations for all fructans and for all donors tested. When averaged over the three donors (Figure 4D), the strongest growth of BB-12 was observed for OF1, followed by OF2 and OF/IN1, OF/IN2, while the mildest growth of BB-12 was observed for IN1. While addition of BB-12 as such did not alter the pH, acetate, lactate, butyrate or bCFA levels compared to the blank (Table 2), minor, yet significant increases in gas and propionate production were observed, mainly due to increases for donor C (Table S1). In contrast, marked and significant metabolic changes were observed upon dosing the fructan-type carbohydrates, including reduction of pH values, increased gas production, stimulation of acetate and lactate levels and decreased bCFA production (Table 2). Similar effects were observed upon supplementation of OF1, OF2 and OF/IN1, which were characterized by a more pronounced increase of lactate levels. Treatment with OF/IN2, IN1, IN2 and IN3 on the other hand significantly increased propionate and butyrate levels, coinciding with  
Figure 4: Selective growth of OTU5 (related to _Bifidobacterium animals_ and upon data analysis reflecting the levels of BB-12) in presence of a complex microbiota upon dosing BB-12 and fructans with short (OF1, OF2), intermediate (OF/IN1, OF/IN2) and high degree of polymerization (IN1, IN2 and IN3) versus a blank incubation. Bars represent the average absolute levels (cells/mL) of OTU5 after 48 h of colonic incubation for donors C (**A**), D (**B**) and F (**C**) together with the averages over the three donors (**D**) (\(n=3\) for each donor). Results are expressed as average \(\pm\) SD for the individual donors and average \(\pm\) SEM for the average over the donors. Conditions that have no letter in common are statistically significantly different from one another (\(p<0.05\)).