Page 42 - Read Online

Page 16 of 20 Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04

relative levels of Roseburia spp. (single donor) or Butyricicoccus spp., both known butyrate producers [75,76] .
[75]
However, neither Roseburia spp. nor Butyricicoccus spp. reportedly use lactate for butyrate production .
We hypothesize that the ongoing activity of known lactate-utilizing bacteria from the Eubacterium hallii
group (renamed to Anaerobutyricum spp.) [49,77-79] contributed to butyrate formation from lactate despite
their relative levels not continuously increasing. The same has been observed in synthetic communities
where the gene expression of the lactate-to-butyrate pathway was highly increased rather than their cell
numbers . Our previous in vivo results demonstrated that the increase in Bifidobacterium spp. was
[78]
concomitant with a three-fold increase in the well-known butyrate-producing Anaerostipes spp. having the
same lactate-to-butyrate pathway as Anaerobutyricum spp. [75,79] . Then, we demonstrated using a synthetic
community that representative members with the canonical functionality of these genera formed a trophic
[27]
chain yielding butyrate from dried chicory root . Hence, it is possible that similar trophic chains involving
lactate/acetate producers and butyrate producers were formed here, too, but represent donor and model-
specific cross-feeding networks.

In this study, we demonstrate how the presence and intactness of the plant cell matrix in dried chicory root
affect the bacterial breakdown of its dietary fiber, contrasting it with isolated inulin. Dried chicory root,
particularly in the form of cubes, resulted in lower gas and more butyrate production compared to isolated
inulin, although cumulating into similar final total levels of SCFAs. Lower gas production has been
postulated to be a beneficial outcome in vivo especially in fiber-related therapies for irritable bowel
[12]
syndrome . The observed butyrate production throughout the later stage of fermentation, together with
the detection of a re-increase in pectin-degraders, may translate in vivo into a prolonged fermentation,
during which chicory root fibers are transported into the distal colon where butyrate production could
benefit gut health. This would also explain the high levels of butyrate and other SCFAs in the fecal samples
of our previous dried chicory root randomized-controlled in vivo trial. We were not able to demonstrate a
uniformly strengthening effect of butyrate containing fermentation supernatants in the Ussing chamber
using biopsies stressed with SDC, but observed donor-specific effects for overall gut integrity. However,
high production of butyrate from the dried chicory root cubes could still benefit gut barrier integrity in
vivo. In conclusion, dried chicory root is an intrinsic fiber product containing high amounts of inulin that
remain encapsulated within its plant cell matrix in the upper gastrointestinal tract - this affects its
breakdown kinetics by the human gut microbiota rationalizing a more distal fermentation and production
of butyrate benefitting human health in vivo.

DECLARATIONS
Acknowledgments
We thank ProDigest and especially Cindy Duysburgh at ProDigest for her effort and input. We thank Henk
Schols for his indispensable advice regarding the upper gastrointestinal digestion of dried chicory root and
analysis of dissolved pectin and inulin, as well as Margaret Bosveld for the inulin measurements and Natalia
Hutnik for the pectin measurements. We thank Steven Aalvink for taking the SEM pictures and the
Wageningen Electron Microscopy Centre for making it possible to use their facilities. We thank Norbert de
Ruijter for his advice on the visual imaging of the chicory root and Shoreh Keshtkar for her assistance with
the light microscopy. We thank Laura Vandionant, Merlijn van Gaal and Ineke Heikamp- de Jong for
performing and assisting with the DNA extraction, PCR and library preparation of the fermentation
samples for gut microbiota analysis. We thank the whole Nutrition Gut Brain Axis group of Örebro
University that made it possible to execute the Ussing chamber experiment in their facility and the human
participants that donated their fecal microbiota as well as colonic biopsies.