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Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04 Page 15 of 20

[64]
enemas has been shown to reduce oxidative stress in the colonic mucosa .

In line with an expected heterogeneity in the individual responses, we observed donor-specific differences,
which became apparent in unstressed biopsies. Notably, for two donors (donors 2 and 4), exposure to the
dried chicory root fermentation supernatant did not compromise, but maintained overall gut integrity
measured by TER compared to the changes observed in control biopsies (%TER change). These donors also
had the highest final relative levels of Bifidobacterium spp. at 48 h of fermentation. Bifidobacteria can
positively impact gut epithelium proliferation via direct interaction of their tight adherence pili with colonic
[65]
cells in mice . Moreover, bifidobacteria are known to strengthen the gut mucosa (despite being absent in
this acute model) via the proposed interaction of the neurotransmitter GABA (γ-aminobutyric acid) and the
[66]
SCFA acetate with goblet cells . Therefore, it is possible that a beneficial effect of dried chicory root
fermentation on gut barrier integrity in such an acute model may become more apparent by using donors
who previously consumed the dried chicory root product. Furthermore, studying a larger number of donors
would enhance our understanding of the overall significance of the present observation.

Understanding time-dependent digestive changes in food products relies on in vitro systems, with static
(batch) incubations that are commonly used due to cost-effectiveness. However, these systems only model
and do not represent human digestive processes . Here, we used the standardized upper gastrointestinal in
[67]
vitro INFOGEST model [30,31] tailored (as recommended) to our high dietary fiber product indigestible to
human endogenous enzymes. The observed minimal structural changes supported the simplified
incubations under anoxic, sterile conditions before in vitro fermentations. Additionally, we used simple
fecal batch in vitro fermentations, which are common for individual and pooled microbiota response
assessments. However, these batch fermentations are prone to known model-induced shifts in bacterial
taxa , limiting in vivo generalizability. Particularly, we noticed an increase in the common fast-growing,
[68]
aerotolerant sugar-fermenter Escherichia-Shigella spp. in the control fermentations. It remains to be
assessed how these higher relative levels in the control would reflect in absolute numbers as we did not
measure bacterial load. Various other studies have observed this Enterobacteriaceae overgrowth [69-74] , which
reflects the model’s limitations including the nature of the inoculum (low bacterial cell numbers favoring
Escherichia-Shigella spp.) and potential residual or re-entering oxygen. It is unlikely that the 4% mono-/
[69]
disaccharides naturally present in the dried chicory root products substantially contributed to fermentation,
although they might be absorbed in vivo in the small intestine. Moreover, we used two in vitro fermentation
systems that differed in their set-up. The Ussing chamber fermentation was used to produce fermentation
supernatants and contained a higher amount of fecal inoculum (1:5 vs. 1:13) than the single donor
experiment. This exposed more bacterial cells to the same amount of fiber substrate, reaching the
fermentation plateau faster, as observed from a higher amount and rate of butyrate production. However, in
spite of these limitations and different model configurations, remarkably similar responses were obtained
across donors, resulting in increased levels of microbial butyrate producers and butyrate levels, which is in
line with previous in vivo results . This demonstrates the donors’ individuality in gut microbiota
[27]
composition and, at the same time, the potential of dried chicory root to increase butyrate production
across gut bacterial communities from different donors.

In all donors, lactate increased at 6 h and was subsequently consumed throughout the fermentation
coinciding with butyrate production. For the low bifidobacteria donor, lactate originated possibly by the
action of Streptococcus spp. , while for the donors, in the Ussing chamber fermentations, Bifidobacterium
[75]
spp. increased by seven-fold and coincided with lactic acid production. Previously, we showed that dried
[27]
chicory root had a strong bifidogenic effect, increasing Bifidobacterium spp. by four-fold in vivo . For all
donors, we also observed an increase in butyrate and propionate production with significant changes in the