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Page 8 of 20 Puhlmann et al. Microbiome Res Rep 2024;3:28 https://dx.doi.org/10.20517/mrr.2024.04

Figure 3. Microbiota composition observed during fecal batch in vitro fermentations using a donor low in Bifidobacterium spp.. Mean
relative abundance (%) of common genera (at least 1% relative abundance and 50% prevalence in all samples) detected in in vitro
fermentations over time (t = 0, 6, 24 and 48 h in the control (Control; only inoculum) and for inulin (Inulin) vs. dried chicory root powder
(Powder) or cubes (Cubes). The lower panel zooms in on the common taxa, omitting the contribution of Escherichia-Shigella spp., which
dominated the community between 0 and 6 h of fermentation.

[Figure 4]. The pH decreased in all conditions from 0 to 6 and 24 h, but then slightly re-increased at 48 h.
Fermentation of inulin induced the largest pH decrease, especially at 6 and 24 h [Figure 4]. Consequently,
inulin pH levels at 24 and 48 h were statistically significantly lower than those of the control, the dried
chicory root powder and cubes [Supplementary Figure 8 and Supplementary Table 4]. Gas production
increased rapidly and peaked at 24 h with inulin, resulting in the highest gas production between 0 and 48 h,
which was statistically significantly higher than control and dried chicory root products [Figure 4 and
Supplementary Table 4]. Along with pH and gas, we measured the SCFAs acetate, propionate, and butyrate.
Butyrate started to increase between 6 and 24 h, but most butyrate was produced between 24 and 48 h for
inulin and dried chicory root products. The highest butyrate levels were recorded at 48 h for dried chicory
root cubes with (mean ± SE) 5.22 ± 0.21 mM, which differed statistically significantly from control (3.67 ±
0.18 mM, P = 0.031) and inulin (4.21 ± 0.11 mM, P = 0.049). Additionally, the increase in butyrate between
24 to 48 h was the highest for dried chicory root cubes (+3.13 ± 0.16 mM; Supplementary Table 4).
Compared to inulin, the butyrate increase between 6 to 24 h was also statistically significantly higher for
dried chicory root cubes (P = 0.009) and powder (P = 0.009; Supplementary Table 4). In contrast to the
dynamics observed for butyrate, propionate increased substantially already between 0 to 6 h. At 6 h,
propionate levels were statistically significantly highest for dried chicory root cubes but then at 24 h for
inulin. At 48 h, propionate levels remained higher for inulin despite not differing anymore from dried
chicory root products [Figure 4 and Supplementary Table 4]. Similar to propionate, acetate also increased
rapidly from 6 h onwards, reaching the highest levels at 48 h, but not differing between inulin and dried