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Page 4 of 20 Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04

Figure 1. Schematic depiction of the study approach to understand how the plant cell matrix in dried chicory root cubes and powder
affects its upper digestion and lower gut microbial fermentation kinetics and subsequent interaction with the gut barrier. An upper in
vitro digestion model was used to assess the intactness of the plant cell matrix upon entering the lower intestine (colon), where, using an
in vitro fecal batch fermentation model, we studied gut microbial breakdown. Finally, we investigated how the fermentation metabolites
potentially interacted with the human colonic epithelium using an Ussing chamber. Created with BioRender.com.

(2 min) and after 30, 60, and 120 min of both the gastric and small intestinal digestion by separating the
liquid from the solid digesta using a sieve. Liquid digesta were heat-treated and snap frozen in liquid
nitrogen and solids were fixated in 70% ethanol for immediate image processing. We visualized the plant
cell matrix of fresh and dried undigested and digested chicory root cubes using scanning electron
microscopy (SEM) and light microscopy to assess structural changes. Potential leakage of pectin from the
plant cell matrix was estimated by measuring uronic acid concentration in the gastric and small intestinal
phase liquid digesta using an automated colorimetric m-hydroxydiphenyl assay and expressing measured
[32]
levels as a percentage of an expected total uronic acid content (5%w/w) in chicory root as determined
according to Ramasamy et al. . Potential differences between inulin from dried chicory root powder versus
[33]
cubes leaking into digestive fluids were estimated by measuring mono-/disaccharides and fructan-oligomers
and polymers of different chain lengths [degree of polymerization (DP)] using High Performance Anion
Exchange Chromatography (HPAEC) according to established methods . Details on the adapted
[34]
INFOGEST method and the pectin and inulin measurements can be found in the Supplementary Materials.

Lower gastrointestinal fermentation model
To investigate the effect of the presence and intactness of the plant cell wall matrix on the lower gut
microbial fermentation of the dried chicory root, a fecal in vitro batch fermentation experiment was
performed at ProDigest (Gent, Belgium) using dried chicory root cubes, powder, and isolated chicory inulin
as a comparison. For this purpose, the feces of a healthy human donor with low bifidobacteria count
(assessed by qPCR to be < 10 16S rRNA gene copies/g corresponding to a relative abundance of < 0.01%)
8
was chosen. This was done to exemplify how such a gut microbiota would respond to an intrinsic fiber
product since high baseline bifidobacteria levels reportedly affect related fiber responses [35-38] . Experiments
were executed in triplicate and samples were taken at baseline (t = 0 h) after 6, 24, and 48 h. Details on the
[39]
execution of the experiment including mucin-covered beads and the subsequent analysis of gut
microbiota composition in fermentation pellets, pH and gut microbial metabolites SCFAs, lactate,
ammonium, and branched-chain fatty acids (BCFAs) in fermentation supernatants, and gas production are