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Page 4 of 19 Klaassens et al. Microbiome Res Rep 2024;3:38 https://dx.doi.org/10.20517/mrr.2024.13

Short-chain fatty acid determination
For the in vitro experiments, short-chain fatty acid (SCFA) quantification has been performed by
Probisearch SLU (Spain). Quantities of SCFAs were analyzed by gas chromatography (GS-MS), using the
column Supelcowax 10 (Supelco) and the following analytical standards: Acetic acid (Sigma), Butanoic acid
(Sigma), Propionic acid (Sigma), and 4Methylpentanoic acid as internal standard (IS) (Sigma). Samples
were analyzed immediately to avoid any acid loss. All samples were adjusted to a pH range between 3-3.5 in
order to coagulate the sample, resulting in separation into two phases: solid phase (discarded) and aqueous
liquid phase, which is the target phase. The target phase was enriched with the IS at a final concentration of
100 mg/L. Linearity was evaluated by preparing three calibration curves of acid acetic, propionic acid, and
butyric acid, respectively, in an aqueous matrix, with 100 mg/L of isobutyl acetic acid as IS. Recovery
coefficients of studied SCFAs were measured in order to evaluate the complete method, by spiking in each
matrix at medium-level concentrations (Medium level concentration: Acetic acid: 150 mg/L; propionic and
butyric acids 10 mg/L). A GCMS Agilent Technology was used with a short wax column to separate the
SCFAs. Temperatures were increased from 40 to 240 °C in 36 min. In the GCMS analysis, the identification
of detected compounds is performed in manual mode, by comparing their mass spectra with those available
in the database of the library (library Wiley Nist 9 + 8) W9N8.

For the ex vivo experiments, SCFA quantification has been carried out by ProDigest (Belgium) in
accordance with the methodology described by De Weirdt et al. . The gas phase composition was analyzed
[18]
with a Compact GC4.0 (Global Analyser Solutions, Breda, the Netherlands), equipped with a Molsieve 5A
pre-column and Porabond Q column (CH , O , H and N ) and a Rt-Q-bond pre-column and column (CO ,
2
4
2
2
2
N O and H S). Concentrations of gases were determined by means of a thermal conductivity detector.
2
2
®
SHIME gut model with infant fecal microbiome
The ex-vivo experiments were carried out by ProDigest, Ghent, Belgium. Formula matrix-dependent effects
were tested in an in vitro simulator model of the human digestion system. Therefore, the Simulator of the
Human Intestinal Microbial Ecosystem (SHIME) model was used as a multi-compartment dynamic
®
®
[19]
simulator of the human gut . The SHIME model mimics different segments of the gastrointestinal tract
and the colon, so that detailed information on the fermentation profile, including the localization of the
effects along the intestinal tract, can be assessed. Setup conditions and nutritional media were used
according to [20,21] . The nutritional medium was setup according to Marsaux et al. (final concentration of
K HPO 4.7 g/L; KH PO 14.7 g/L; NaHCO 1.8 g/L; yeast extract 1.8 g/L; peptone 1.8 g/L; mucin 0.9 g/L;
4
2
4
2
3
[22]
[20]
cysteine 0.5 g/L; polyoxyethylene sorbitan monooleate 1.8 mL/L in the reactors) . To show microbiota-
inherent metabolic activity, a setup without the addition of probiotics (Blank) was also tested.
The infant donor microbiota from 9 exclusively breastfed infants - 4 of which were born via cesarean
section (CS) and 5 of which were born by vaginal delivery (VD) - were used. Fecal material was collected
with parental consent and ethical approval of the University Hospital Ghent (reference number
B670201836585). To give a general overview of the kinetic of metabolic activity, samples were taken at
baseline, as well as after 6, 24, and 48 h of colonic fermentation with the donor microbiota.
IF with intact or hydrolyzed protein were subjected to a full passage through the oral, gastric, and small
[23]
intestinal phases, the latter involving absorption as described previously . Fecal suspensions were prepared
in phosphate buffer, to which reducing agents were added (K HPO 8.8 g/L; KH PO 6.8 g/L; sodium
2
4
4
2
thioglycolate 0.1 g/L; sodium dithionite 0.015 g/L). The obtained suspensions were mixed with a
[24]
cryoprotectant [modified version of in a 1:1 (v:v) ratio, so that 7.5% fecal suspensions were obtained]. The
fecal suspensions were flash-frozen and then preserved at -80 °C (cryostock). Just before an experiment,