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Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04 Page 11 of 20

Figure 5. Gut microbiota composition of in vitro fermentations (A) and (B) to produce fermentation supernatants and their effect on
colonic biopsies during the Ussing chamber experiment (C) and (D). (A) Common genera (mean relative abundance of at least 1% and
mean prevalence of 50% in all samples) detected in the control (Control; only inoculum) and dried chicory root cube (Cubes) in vitro
fermentation at baseline (T = 0), 6 h, 24 h, and 48 h; (B) Principal response curve assessing the combined response of all genera over
time [relative abundance (%)] and thereby revealing genera differentiating between dried chicory root cube and control fermentation.
Genera driving the difference are depicted on the right scale with taxa equally present in both dried chicory root cubes and control
having small weights and taxa deviating most having larger weights; (C) Change in transepithelial resistance after 90 min as a
percentage of baseline (%TER) in control or stimulated colonic biopsies using either the stressor SDC, the fermentation supernatant, or
their combination (fermentation supernatant + stressor); (D) Change in FITC-dextran concentration between 0 and 90 min expressed
as factor of the baseline concentration (fold-Δ). Information on all time points of all four donors can be found in
Supplementary Figure 12. TER: Transepithelial resistance; %TER: TER at 90 min expressed as a percentage of each biopsies respective
baseline; SDC: sodium deoxycholate; FITC: fluorescein isothiocyanate-dextran; fold-Δ FITC-dextran: relative change in FITC-dextran
concentration between 0 to 90 min expressed as factor of each biopsies respective baseline (respective level expressed as factor of its
baseline).

up to three times higher than for the control fermentation [Supplementary Table 7]. Butyrate production
was largest between 6 to 24 h with 8.04 ± 1.71 mM. We observed lactate to be produced at 6 h (5.83 ±
1.70 mM) during fermentation of dried chicory root cubes, but this metabolite was completely consumed at
24 h. Formate followed the same dynamics as lactate, while the BCFA iso-butyrate levels peaked in dried
chicory root cubes at 24 h before decreasing again at 48 h [Supplementary Table 7]. The fermentation
outcomes based on four different donors were similar to the previous experiment with a single donor but
followed a faster kinetic related to the higher concentration of fecal inoculum used.

Changes in gut barrier integrity of colonic biopsies following the stimulation with fermentation supernatants
Fermentation supernatants at 48 h were used to test their effect on gut barrier integrity of human colonic
biopsies in an ex vivo Ussing chamber model. Human colonic biopsies were stressed with SDC, a secondary
bile acid known to increase paracellular gut permeability and thereby affect gut barrier integrity, simulating
low-grade inflammation [50-53] . Based on the measured SCFA concentrations in the fermentation supernatant
at 48 h, biopsies stimulated with the fermentation supernatant (dilution to 2% v/v) were on average exposed
to 0.20 to 0.34 mM butyrate (mean ± SE: 0.2 ± 0.03 mM), 0.17 to 0.34 mM propionate (0.24 ± 0.04 mM), and
0.74 to 0.86 mM acetate (0.77 ± 0.04 mM). In all biopsies, TER, as a measure of overall gut integrity,