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Page 10 of 20 Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04

only differed statistically significantly for dried chicory root powder and not between cubes and inulin at
any time point [Supplementary Table 4]. Finally, ammonium increased mainly between 0 and 24 h and was
highest for control and least for inulin, concomitant with the largest decrease in pH in inulin, while dried
chicory root cubes and powder did not differ. In summary, dried chicory root cube fermentation yielded
similar total SCFAs but higher final butyrate levels compared to chicory root powder or isolated inulin with
less gas produced.

Effect of fermentation supernatant produced from in vitro fermentation of dried chicory root on gut
permeability
We continued to assess the effect of fermentation supernatants produced during the fermentation of dried
chicory root on gut permeability in an ex vivo model using human colonic biopsies from four donors. Dried
chicory root cubes were chosen as they were found to yield the highest butyrate levels in vitro.

Gut microbiota changes underlying the fermentation supernatants
To produce the fermentation supernatant, dried chicory root cubes were again fermented in a fecal in vitro
batch fermentation model and compared to a negative control (control; only inoculum), but at higher
inoculum concentration. Again, we observed an increase in Escherichia-Shigella spp., which especially
dominated the control fermentation [Figure 5A]. We observed a similar time-dependent modulation of the
overall microbiota composition with changes in relative abundance levels of individual taxa mostly peaking
at 6 h and leveling off at 24 h [Supplementary Table 5, Supplementary Figures 9 and 10]. In contrast to the
single-donor in vitro fermentation, donors for this experiment were not chosen based on a low
bifidobacteria count, and all four donors had considerable levels of Bifidobacterium spp. present in their
feces (mean relative abundance across all conditions of 4.3% ± 1.5%; Figure 5A and Supplementary Figure
11). Following dried chicory root cube fermentation, we observed the highest changes at the genus level in
Bifidobacterium spp. (6 h: P = 0.018, q = 0.216; 24 h: P = 0.081, q = 0.452; 48 h: P = 0.029, q = 0.152).
Bifidobacterium spp. levels increased rapidly up to a third of the whole microbiota community and were
statistically significantly higher (up to 10-fold) from control levels at 6 and 48 h (Supplementary Table 6 and
for individual microbiota profiles, Supplementary Figure 11). Constructing a principal response curve
[Figure 5B], which assessed the combined response over time of gut bacterial genera to the product using
redundancy analysis on the first principal component, confirmed that relative levels in Bifidobacterium spp.
clearly differentiated the microbial community of dried chicory root cubes from the control. Besides the
changes in Bifidobacterium spp. levels, changes in Coprococcus spp. levels differed, too, but reached the
highest levels in the control. We also observed several taxa decreasing in relative abundance in both
conditions, but notably, butyrate-producing genera decreased only in the control to statistically significantly
lower levels [Supplementary Table 5]. Butyricicoccus spp. levels fluctuated but were statistically significantly
higher at 24 h in the dried chicory root cube fermentations and virtually absent in the control at 48 h
[Supplementary Table 6]. Again, we observed that Roseburia spp. re-increased at 48 h for dried chicory root
cubes but not control, while bacteria from the Eubacterium hallii/Anaerobutyricum group increased only
[49]
for cubes at 6 and 24 h but then decreased at 48 h, reaching lower levels than the control [Supplementary
Tables 5 and 6]. The dynamics of these gut microbiota changes were in line with those observed for the
single donor low in bifidobacteria while demonstrating the difference in gut microbiota modulation when
Bifidobacterium spp. are present.

Changes in pH, gas production and fermentation metabolites underlying the fermentation supernatants
We determined pH, gas, and production of SCFAs to understand the changes underlying the metabolites
present in the fermentation supernatants [Supplementary Table 7]. The pH dropped again rapidly within
6 h, while gas production leveled off already between 6 and 24 h. Dried chicory root cubes yielded, on
average, 12.56 ± 1.50 mM butyrate, 12.12 ± 1.97 mM propionate, and 38.60 ± 2.03 mM acetate, which were