| Summary: | Whey protein and its hydrolysate are ubiquitously consumed as nutritional supplements. This study aimed to evaluate the potential effect of whey protein hydrolysate (WPH) on the infant gut microbiome, which is more variable than that of adults. Colonic fermentation was simulated through a static digestion model and fecal culture fermentation, using feces from normal infants aged from 1–3 years old. During in vitro gut fermentation, measurements of short-chain fatty acids (SCFA) concentrations and 16S rRNA amplicon sequencing were performed. Additionally, the growth curves of cultivated probiotics were analyzed to evaluate the prebiotic potential of WPH. Besides the decline of pH in fermentation, the addition of WPH induced a significant increase in the SCFA production and also the relative abundance of Proteobacteria, <i>Bacteroides</i>, and <i>Streptococcus</i> (<i>p</i> < 0.05). The lower ratio of Firmicutes/Bacteroidetes in WPH-supplemented samples indicated the positive modulation of WPH on the gut microbiota, which could benefit the energy balance and metabolism of infants. The stimulation effect of WPH on the probiotics (particularly <i>Lactobacillus acidophilus</i> NCFM) during cultivation implied the prebiotic potential as well. Our findings shed light on WPH as a valuable dietary supplement with not only enriched resources of essential amino acids but also the potential to restore the infant gut microbiome.
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