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Klaassens et al. Microbiome Res Rep 2024;3:38 https://dx.doi.org/10.20517/mrr.2024.13 Page 9 of 19

Figure 2. Effect of formula protein matrix on the metabolic activity of infant fecal donor microbiota. (A) Acetate; (B) propionate; (C)
butyrate; (D) total lactate; (E) D-lactate; (F) L-lactate; (G) total SCFA production; (H) pH; and (I) volume of gas production during
simulated colonic fermentation with donor infant microbiota and either iPF (blue) or formula with eHF (orange). Both formulae
contained prebiotic GOS. Samples from the same donor are linked with a line. Donor samples of infants born by caesarian section are
shown as triangles, and donor samples of infants born via VD mode are shown as circles. n.s.: Not significant; iPF: intact protein formula;
GOS: galactooligosaccharide; eHF: extensively hydrolyzed formula; SCFA: short-chain fatty acid.

the individuals and increased with time and was independent of the formula matrix and mode of birth
[Figure 2I].

Effect of probiotic supplementation on the metabolic activity of an ex-vivo infant intestinal microbial
ecosystem
Changes in the bacterial ecosystem are associated with changes in the overall metabolic activity. Therefore,
changes in intestinal metabolites upon probiotic supplementation of formulae with either intact or
hydrolyzed protein were assessed [Figure 3]. The simulated colonic incubation was done with either single
probiotic supplementation [Bb (green) or Lf (grey)] or double probiotic supplementation (Bb + Lf; blue).
Values of un-supplemented conditions (samples shown in Figure 2A-I) are shown again for comparison
(Blank; white).

The formation of individual SCFA seemed to be strain-specific. Supplementation with Bb or the
combination of Bb + Lf yielded significantly higher levels of acetate, compared to non-supplemented
(Blank) or Lf-supplemented conditions [Figure 3A]. Levels were independent of birth mode, as seen by
similar distribution of children born by VD (circles) and CS (triangles). In contrast, patterns for propionate,
butyrate, and lactate (neither D- nor L-lactate) showed high individual variation and did not differ between
the added probiotics [Figure 3B-F]. Overall, Bb and Bb + Lf supplementation showed a tendency to increase
the total amount of SCFA, independent of the formula matrix, compared to respective controls (P > 0.05)
[Figure 3G]. Production of different acids seems to reduce pH levels, though changes were not significant