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Page 8 of 19 Klaassens et al. Microbiome Res Rep 2024;3:38 https://dx.doi.org/10.20517/mrr.2024.13

Figure 1. Growth and metabolic activity of probiotic strains in single and co-culture conditions in the presence of prebiotics or
intact/hydrolyzed protein. (A) Growth of Bb (top) and Lf (bottom) in co-culture (1:1 ratio), in the presence of iPF without prebiotics
(green), with GOS (blue), or eHF with GOS (orange); (B) Formation of Acetate; (C) Propionate; (D) Butyrate; or (E) Lactate or (F)
volume of gas formation in ml after 24 and/or 48 h after growth of Bb as single culture (open bars), Lf as single culture (dashed lines
bar) or both strains as co-culture (filled bars) in the different formulae. Error shown is the standard deviation. n = 3 per time point.
* ** *** ****
Selection of significant differences are marked with a P < 0.05, P < 0.01, P < 0.001, P < 0.0001. iPF: Intact protein formula; GOS:
galactooligosaccharide, eHF: extensively hydrolyzed formula; Bb: B. breve DSM 32583; Lf: L. fermentum CECT 5716.
Effect of infant formula matrix on the metabolic activity of donor infant intestinal microbial
ecosystems ex vivo
To investigate the effects of an intact and hydrolyzed protein formula matrix on the donor ecosystem, the
®
SHIME model was inoculated with fecal suspension of nine exclusively breastfed infants. Subsequently, iPF
+ GOS or eHF + GOS formula was added to the system. Acetate levels were rising within the first 6 h of
colonic fermentation, and a trend toward higher acetate levels in eHF + GOS was observed for all inocula
compared to iPF + GOS (P > 0.05; Figure 2A).

Propionate and butyrate levels increased at later time points during colonic incubations. High inter- and
intra-individual variations were observed and these were not influenced by the type of formula or birth
mode [Figure 2B and C]. Total lactate levels were already increasing at 6 h and decreased again over time,
independent of the formula matrix [Figure 2D]. D-lactate was also formed by the infant donor microbiota
[Figure 2E]. In contrast, L-Lactate was almost depleted by 48 h of colonic fermentation, showing cross-
feeding mechanisms in the complex bacterial ecosystem [Figure 2F]. In summary, the formation of total
SCFA increased over time, with acetate as the quantitatively most abundant SCFA produced by the
microbiota of exclusively breastfed infants [Figure 2G]. All samples showed only a non-significant trend
toward slightly increased SCFA levels (P > 0.05) in eHF + GOS, independent of birth mode. Increased SCFA
formation is also mirrored in a drop in pH with infant microbiome inoculum [Figure 2H]. Formation of gas
by the un-supplemented (i.e., without probiotic supplementation) microbiota showed a wide range between