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Page 14 of 20 Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04

richness increased again at 48 h for dried chicory root cubes and powder but not inulin. This coincided with
higher levels of the pectin-degraders Monoglobus spp. and bacteria from the Eubacterium eligens group, as
well as the butyrate-producing Roseburia spp. in both the fermentation liquid and mucin-covered beads.
The re-increase in gut bacteria richness and appearance of specialist bacteria suggests that the more intact
plant cell matrix of dried chicory root results in prolonged fermentation, preventing substrate depletion at a
later time point. Interestingly, lactate consumption and butyrate production were also highest between 24
and 48 h, resulting in butyrate being the only SCFA to notably increase during this time period and to reach
the highest levels for dried chicory root cubes. Increased butyrate production levels after prolonged
fermentation have been previously reported for large wheat bran particles [18,21] . It appears that the three-
[60]
dimensional organization plays a role herein, as exemplified by alginate-entrapped starch and wheat plant
[58]
cell walls , leading to higher butyrate production than their extracted single fiber alone. Especially larger
particle sizes (> 2 mm) often result in higher butyrate and lower acetate proportions , which is attributed
[20]
to reaching a fermentation plateau and allowing more time for lactate/acetate-to-butyrate conversion
through cross-feeding . Lactate conversion into propionate or butyrate is limited in vitro at a more acidic
[20]
pH (5.5 vs. 6.5) , but it is debatable whether the slightly lower pH in inulin (5.75) compared to dried
[61]
chicory root (5.89) fermentations, was physiologically relevant to be the main driver of higher lactate
consumption and butyrate production. However, we observed for dried chicory root cubes, the largest
lactate production and consumption coincided with the largest butyrate production. This suggests that
lactate-to-butyrate conversion could play an essential role in the breakdown of the dried chicory root cubes.

Finally, we observed a lower increase in BCFAs and ammonium for inulin compared to the dried chicory
root products and control. This may be attributed to the significantly greater pH decrease for inulin, as the
formation of these protein fermentation compounds is known to be less favored at lower pH . In the in
[11]
vivo situation, the production of BCFAs is linked to the availability of fiber substrate in the proximal versus
distal colon, and inulin, due to its simple structure, is believed to be fermented more proximally [11,12] .
Consequently, BCFA formation may still occur in vivo in the distal colon due to substrate depletion from
inulin but not from dried chicory root as a consequence of its slower breakdown.

Butyrate produced by gut bacterial fiber fermentation can be used by colonocytes as an energy source and
thereby potentially strengthen the colonic epithelium. Studying this interaction relies on the use of
cultivated cell-line models (possibly combined with mimicking mucus secretion) or the removal of in vivo
colonic tissues (yet losing the protective mucus layer). Previous studies using colonic biopsies revealed acute
distinct effects of fiber on permeability in biopsies of healthy and/or diseased donors [62,63] , but also
considerable heterogeneity in the individual responses . Here, we applied a similar acute model to study
[63]
the interaction between the individuals’ gut microbial fiber breakdown products and their human colonic
epithelium. To model this, we investigated whether previous exposure to dried chicory root fermentation
supernatants could acutely counteract or diminish stressor-induced impairments in the gut barrier function
similar to a low-grade inflammatory state. We did not observe an acute, uniform difference between human
biopsies stressed with SDC alone and those that were previously exposed to the fermentation supernatant,
despite a less rapid decrease in TER over time in the latter. Based on the measured SCFA concentration in
fermentation supernatants and dilution in the Ussing chamber, we estimated butyrate concentration to be
0.25 mM. Previous studies using sodium butyrate at a concentration of 5 and 25 mM (factor 20 to 100
higher, representing physiological concentrations) also failed to demonstrate a protective acute effect
against stressor-induced impairments using the mast cell degranulator Compound 48/80 (C48/80) in
[40]
healthy donors’ biopsies . It is possible that the acute exposure in this model was too short to positively
modulate gut barrier integrity. Butyrate has a considerable history of well-reported improvements on
[64]
human colonic function in vivo and two-week in vivo exposure to butyrate using direct delivery by