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Page 6 of 20 Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04

Figure 2. Scanning electron microscopy micrographs of fresh and digested dried chicory root. (A-C) plant cell matrix of (A) fresh chicory
root with (B) intact plant cells and (C) intracellular inulin as indicated by red arrows; (D) intact plant cell matrix of dried chicory root
cubes after rehydration prior to upper gastrointestinal digestion; (E) plant cell matrix after gastric digestion of dried chicory root cubes;
(F) plant cell matrix after gastric and small intestinal digestion of dried chicory roots. While the macrostructure appeared to weaken
over time [Supplementary Figure 2], the overall structure remained visually unchanged throughout the gastric and small intestinal
phases, with no observed holes or cracks in the plant cell walls. Note the different magnifications in panels (B) and (C).

root pieces [Figure 2A-C] and rehydrated chicory root cubes [Figure 2D]. Plant cells were open [Figure 2B]
with a small level of damage most likely resulting from cutting the plant particles for image preparation.
Within the plant cells, inulin was clearly visible as a crystalline structure due to precipitation in ethanol
[Figure 2C]. Next, we hydrated the chicory root products to mimic their consumption and conducted an
upper in vitro digestion with an oral, gastric, and small intestinal phase using the well-established
INFOGEST procedure [30,31] . After oral and gastric digestion with a pH lowered from 3 to 2 (to mimic the
fasted state/end of gastric digestion), the overall plant structure remained largely intact, with densely packed
plant cells filled with inulin [Figure 2E]. Similarly, at the end of the small intestinal phase, no obvious
damage to plant cells in the form of holes or cracks in the cell wall was observed [Figure 2F]. The same was
confirmed by light microscopy [Supplementary Figure 1]. However, throughout the gastric and small
intestinal phase, the macrostructure [Supplementary Figure 2] weakened overall, with plant cells appearing
less round and robust [Figure 2E and F]. To estimate how much pectin was potentially leaking from the
plant cell structure of the dried chicory cubes compared to the powder, we measured uronic acid content as
a proxy for pectin in the liquid part of gastric and small intestinal digesta. Estimated based on the total
uronic acid (UA) content in dried chicory [Supplementary Figure 3], we found that on average (mean ±
SD), 16.71% ± 2.91% of pectin (UA: 8.36 ± 1.46 mg/g product) leaked from dried chicory root powder, which
was slightly higher than for chicory root cubes with 10.20% ± 0.70% of pectin (UA: 5.10 ± 0.35 mg/g
product). Throughout the small intestinal phase, the leaked pectin increased for powder to 27.56% ± 0.73%
(UA: 13.78 ± 0.37 mg/g product), which was nearly twice as high as for chicory root cubes with 14.48% ±
1.25% (UA: 7.24 ± 0.63 mg/g product). Besides pectin, we also investigated differences in inulin of various
chain lengths (DP) detected in the digesta of dried chicory root powder and cubes by HPAEC
chromatograms [Supplementary Figure 4]. We observed a consistently higher total area under the curve of
the HPAEC chromatograms for dried chicory root powder compared to the cubes, representing all detected
mono-/disaccharides and fructan oligomers and polymers. No changes in chain length distribution
throughout the digestion phases were observed, but dried chicory root powder had the highest amounts of