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

cells encapsulating other non-structural fibers, such as inulin [13,14] . This complex network does not dissolve
into its isolated compounds in the human gastrointestinal tract but rather functions as a vehicle
transporting these fiber structures to the lower gut [15,16] . For these reasons, fibers originally present in the
plant matrix have been termed intrinsic fibers to distinguish them from isolated fibers extracted from the
plant cells .
[14]
Chicory root is a root vegetable with a plant cell matrix encapsulating particularly high amounts of inulin
inside the plant cell vacuoles. While chicory roots are nowadays mainly used for the production of isolated
[17]
inulin, they have long been used as both a medicinal and a culinary root vegetable . In its dried form,
chicory root consists of up to 85% fiber, 70% of which is inulin, making it an excellent source of intrinsic
[17]
dietary fiber . We hypothesize that the presence of the plant cell wall can potentially function as a physical
barrier, shielding inulin from immediate contact with the gut microbiota, thereby impacting intrinsic fiber’s
breakdown kinetics and location in the human gut. Food processing steps such as particle size reduction [18-22]
or thermal treatment [19,23] have the potential to affect gut bacteria accessibility and related breakdown
kinetics due to the induced damage of the plant cell matrix. Until now, in vitro assessments of plant food
breakdown kinetics have focused on pectin-, starch- and lipid-containing whole foods [20,24-26] but never
inulin-rich vegetables like chicory root. Previously, we have shown in a placebo-controlled human trial that
the intake of dried chicory root particles dramatically modulated gut microbiota composition by stimulating
a trophic chain involving members of Bifidobacterium spp. and Anaerostipes spp. toward butyrate
production and improved both gut and metabolic health . We attributed these changes to a slow release of
[27]
fibers from dried chicory root particles. This would prolong the fermentation of the dried chicory root
particles rationalizing a rather distal location of their breakdown. In humans, slow and gradual fiber
fermentation has been hypothesized to benefit gut health by distributing fiber fermentation from the
proximal throughout the distal colon . Moreover, SCFAs delivered to the distal colon have been shown to
[12]
confer more pronounced systemic health benefits compared to a proximal delivery [28,29] .

Our aim was to assess whether the plant cell matrix of dried chicory root remains intact in the upper
gastrointestinal tract and how its presence affects lower gut microbial composition and fermentation
kinetics, as well as the potential effect of dried chicory root fermentation supernatants on gut barrier
integrity. For this purpose, we executed a series of experiments using in vitro and ex vivo models for the
upper and lower gastrointestinal tract that were primed with dried chicory root particles with two different
degrees of cell wall intactness in the form of cubes and milled into powder [Figure 1].

METHODS
Dried chicory root
Dried chicory root was provided by WholeFiber BV (Emmeloord, the Netherlands). The product is made
from chicory roots that have been washed, cut, and dried, producing cube-like pieces of approximately
3 mm rib. The final product has a dry weight of 93%w/w, of which 70%w/w is native inulin, 10%w/w pectin,
5%w/w hemicellulose and cellulose, and 4%w/w mono- and disaccharides, 5%w/w proteins, and remaining
minerals, polyphenols, and vitamins. To assess whether dried chicory root in its structure was similar to
fresh, unprocessed chicory root, fresh chicory root was also provided by WholeFiber BV. To study the effect
of particle size, dried chicory root cubes were ground to a mean particle size of < 0.5 mm.

Upper gastrointestinal digestion model
To assess potential physical changes during upper gastrointestinal digestion, we mimicked digestive
processes during the oral, gastric, and small intestinal phases using an adapted version of the INFOGEST
protocol [30,31] . Experiments were executed in triplicate and samples were taken at the end of the oral phase