Feeding Type Shapes Infant Gut Microbiota and Metabolite Profiles in a Simulated Colonic Model

The establishment of the infant gut microbiota is strongly influenced by feeding type, with human milk (HM) and infant formula (IF) driving distinct microbial profiles. This study compared the effects of donated raw human milk (HM) and Holder-pasteurised HM (Past-HM) on microbial composition using a 4-stage in vitro gut model inoculated with pooled faecal samples from five healthy 3-6-month-old infants. Colonic digesta were sampled over a 48-h period following introduction of raw HM, Past-HM and IFs for microbial diversity by quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing, and short-chain fatty acid (SCFA) analysis by gas chromatography. Microbial profiling revealed 2026 operational taxonomic units (OTUs) across eight phyla and 165 genera. Past-HM and goat milk-based IF (GIF) promoted <i>Bifidobacterium</i> dominance and produced higher levels of total SCFA, especially acetic acid, compared to HM. Spearman correlation linked <i>Bifidobacterium</i> with acetic acid and <i>Escherichia-Shigella</i> with propionic acid. PCA showed OTU composition (Dim1, 72.6%) and SCFA profiles (Dim2, 19.8%) and distinguished control and milk-inoculated groups. Feeding type was the dominant factor shaping microbiota and metabolite profiles, exerting a stronger influence than incubation time (PERMANOVA <i>p</i> < 0.05). These findings underscore the pivotal role of early feeding choices in directing both microbial colonisation and microbial metabolic activity in the infant gut.