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Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04 Page 13 of 20

The cell matrix of plant foods is a complex structure made of cellulose strengthened by hemicelluloses and
pectin intrinsically intertwined into plant cell walls that encapsulate other non-structural carbohydrates,
macro- and micronutrients. Dried chicory root is a food product that is particularly high in fiber due to its
intra-cellular inulin content being part of the intrinsic plant cell matrix [13,17] . While inulin is a fiber known to
be easily fermentable by the human gut microbiota, the presence of the plant cell wall in dried chicory root
forms a physical barrier that gut bacteria need to open to access intracellular inulin and other cellular
components. Consequently, the breakdown of dried chicory root differs from that of isolated inulin. Dietary
fibers are, by definition, not digested in the upper gastrointestinal tract, but they can still be affected by the
prolonged incubation in digestive juices and gastric and small intestinal pH changes leading to, for instance,
the dissolution of pectin [54,55] . We observed that an estimated 15% of the total pectin leaked from the dried
chicory root matrix during the in vitro gastric and small intestinal digestion. This was nearly twice as high
for chicory root powder than cubes, likely due to the larger damage of the plant cell matrix induced by
milling, breaking more plant cells open. Pectin that leaks out from plant foods is believed to be mainly
soluble pectin from the intercellular space, which glues the plant cells together, enforcing the overall plant
cell matrix [16,56] . Indeed, concomitant with the leakage of pectin over time, we observed an overall weakening
of the macrostructure. Nonetheless, no obvious damage to the plant cell matrix in the form of cracks or
holes in plant cell walls was visible, which indicates that dried chicory root cubes are likely to arrive in the
lower gastrointestinal tract as intact particles. A weakened plant cell matrix may favor the release of
intracellular inulin, and we observed higher amounts of fructose-monomers and fructo-oligosaccharides,
together with longer-chain fructan-polymers, for dried chicory root powder, which we hypothesize to
represent and relate to the higher plant cell damage. Thus, it is likely that dried chicory root cubes function
as a delivery system of inulin and pectin that remain primarily encapsulated inside the intact plant cells to
reach the distal parts of the colon.

This plant matrix intactness challenges the breakdown by the gut microbiota as the opening of the plant cell
wall requires the degradation of the chemically more complex pectins and hemicelluloses. Chemical
complexity selects for the action of specialist bacteria that have the functional machinery to access and
[7]
metabolize diverse sugar constituents , and the presence of different dietary fibers slows their gut microbial
breakdown [57-59] . Therefore, we hypothesized that the kinetics of microbiota-mediated fiber breakdown may
differ in dried chicory roots compared to inulin.

After an initial rapid modulation in overall gut microbiota composition and a decrease in gut bacterial
richness within six hours, distinct differences between fiber products started to emerge between 6 to 24 h
when most of the gas and total SCFAs were produced. Inulin resulted in the largest pH decrease and highest
gas production, with propionate production surpassing that from dried chicory root cubes and powder at
24 h. While acetate and total SCFA production did not differ, both dried chicory root products produced
significantly more butyrate than inulin. This was paralleled by significantly larger lactate production (up to
6 h) and consumption (from 6 h onwards) for dried chicory root cubes compared to hardly any
consumption between 6 to 24 h for inulin, and little changes for dried chicory root powder. Comparing
dried chicory root particle sizes, gut bacterial richness decreased less rapidly for powder than cubes. This
may be due to the presence of more readily available substrate as the plant cell matrix in powder is more
damaged. Consequently, more fibers are exposed (intracellular inulin alongside pectin and hemi-/cellulose
cell wall fibers) and more surface area is created for bacterial adherence compared to the cubes, where
bacteria have to diffuse between the plant cells to break them down from the outside.

Between 24 and 48 h, the overall gut microbiota community composition hardly changed, but we observed
remarkable distinctions in butyrate production and relative abundances of individual taxa. Gut bacteria