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Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04 Page 7 of 20

fructose mono- and oligomers (DP2-5) and longer-chain inulin (> DP16) compared to dried chicory cubes.

Differences in gut microbiota response to different particle sizes of dried chicory root compared to
inulin
Following the assessment of changes in the upper gastrointestinal tract, dried chicory root cubes and
powder were subjected to breakdown by the gut microbiota and compared to inulin using a fecal batch
fermentation experiment. At baseline, all samples had a similar microbiota composition as determined by
16S rRNA gene amplicon sequence analysis [Supplementary Figure 5]. In line with the selection of a donor
with a low bifidobacteria count, no Bifidobacterium spp. were detected at baseline [Figure 3]. Following
inoculation, we observed a rapid increase in Escherichia-Shigella spp. in all conditions (control, dried
chicory root cubes and powder and inulin), which was highest at 6 h, making up approximately half the
microbial community, and decreasing to about 30% at 48 h [Supplementary Table 1]. These changes were
not statistically significantly different between conditions [Supplementary Table 2] and likely represent a
model-induced outgrowth of this facultative aerobic bacteria taxon.

When addressing changes in the overall microbiota composition, we observed a rapid modulation in all
conditions within 6 h, which peaked at 24 h before leveling off at 48 h (between-sample β-diversity analysis
based on Bray-Curtis dissimilarity, Supplementary Figure 5 and within-sample α-diversity analysis based on
gut bacteria richness, Supplementary Figure 6). These time-dependent changes were reflected in the changes
in relative abundance levels of individual gut microbiota members over time [Figure 3]. At the genus level,
numerous taxa in all conditions decreased rapidly within 6 h and leveled off at 24 h of fermentation,
resulting in the largest observed fold changes in this time period [Supplementary Table 1]. Particularly,
Streptococcus spp. peaked at 6 h with significantly higher levels for dried chicory root cubes (14.9%)
compared to inulin (11.8%). In addition, Parasutterella spp. increased to their highest levels at 6 h in dried
chicory root cubes (3.3%) and powder (3.0%; Supplementary Table 1) and had still statistically significantly
higher levels at 24 h compared to inulin [Supplementary Table 2]. A number of genera first decreased
between 0 to 6 h but then re-increased between 6 and 24 and 48 h. Notably, bacteria from the Eubacterium
[49]
hallii group (now also known as Anaerobutyricum spp. ) decreased in all conditions and were not detected
at 6 or 24 h, but then re-increased above baseline levels at 48 h in inulin (4.5-fold) and dried chicory root
powder (1.8-fold) and cubes (2.2-fold). Monoglobus spp. also decreased in all conditions, but re-increased
from 6 to 48 h only for dried chicory root powder and cubes, despite reaching (slightly) lower levels than at
baseline. The same was true for bacteria from the Eubacterium eligens group and Roseburia spp.
[Supplementary Table 1], which were, after 48 h, significantly higher in powder and cubes due to their
virtual absence in inulin and control fermentations [Supplementary Table 2]. Taking all increases in all taxa
together, we observed the highest number of significantly increasing genera at 48 h for dried chicory root
cubes, with 15 genera increasing in relative abundance compared to inulin with 12 genera increasing and
powder and control with 11 genera [Supplementary Table 3].

The used system with mucin-covered beads also allowed for the analysis of the mucus-adhering microbiota,
of which notably Roseburia spp. had significantly higher levels in dried chicory root powder and cubes after
48 h of fermentation [Supplementary Figure 7 and Supplementary Table 2]. The use of mucin-covered
beads also explains the increase in relative abundance of mucin-degrading Akkermansia spp. in control
incubations lacking additional fermentable substrates.

Differences in pH, gas production and fermentation metabolites of different particle sizes of dried
chicory root compared to inulin
Concomitantly, with the time-dependent development in the microbiota composition, we also observed
changes in pH, gas production, and metabolites such as SCFAs and other organic acids produced