The protein ingredient quality of infant formula influences colonic physiology and microbiota and the serum metabolomic profile in neonatal mini-piglets

Despite the WHO recommendations in favor of breastfeeding, most infants receive infant formulas (IFs), which are complex matrices involving numerous ingredients and processing steps. Our aim was to understand the impact of the quality of the protein ingredient in IFs on gut microbiota and physiology, blood metabolites and brain gene expression. Three IFs were produced using whey proteins (WPs) from cheese whey (IF-A) or ideal whey (IFs-C and -D) and caseins, either in a micellar form (IFs-A and -C) or partly in a non-micellar form (IF-D). Twenty-four Yucatan minipiglets received one of the IFs from 2- to 21-days of age. The piglets were then euthanized 84 min postprandially. Blood, ileal and colonic digesta and tissues, the liver and the hypothalamus were sampled. Gut microbiota composition and activity, the expression of intestinal and brain genes involved in barrier, immune, endocrine, nutrient carrier and neuronal functions and serum metabolite levels were determined. Intestinal paracellular permeability was assessed with an ex vivo Ussing chamber. The data were analyzed using multifactorial and univariate analyses. The colon was the main site to be physiologically affected by the quality of the dairy protein ingredients used in IFs. Colonic paracellular permeability was significantly higher in IF-D-fed piglets than in those receiving IFs-A and -C, in line with the expression of genes encoding tight junction proteins (OCLN, CLDN3 and CLDN4). IF-D up-regulated the colonic expression of genes involved in the immune function (SOCS3, PIGR and TNFα) when compared to IF-A. Although intestinal α- and β-diversities did not significantly differ among IFs, some specific differences were observed, such as the abundances of Campylobacterota phylum and Bacteroides genera and fecal butyrate production, which were increased with IFs-C and -D versus IF-A. The kynurenine pathway was favored in piglets fed IF-D compared to IF-A, based on colonic gene expression and serum metabolites. In addition, levels of some serum metabolites, particularly putrescine, spermidine and spermine, were higher in piglets fed IF-D than IFs-A and -C. Overall, IF-D, which combined ideal WPs and some non-micellar caseins, appeared to differ most from IF-A in terms of its physiological consequences, suggesting that both WP and casein ingredient quality may mediate the physiological properties of IFs, probably through changes to the colonic microbiota composition and activity.