Fecal microbiome and metabolome of infants fed bovine MFGM supplemented formula or standard formula with breast-fed infants as reference: a randomized controlled trial

Abstract

Human milk delivers an array of bioactive components that safeguard infant growth and development and maintain healthy gut microbiota. Milk fat globule membrane (MFGM) is a biologically functional fraction of milk increasingly linked to beneficial outcomes in infants through protection from pathogens, modulation of the immune system and improved neurodevelopment. In the present study, we characterized the fecal microbiome and metabolome of infants fed a bovine MFGM supplemented experimental formula (EF) and compared to infants fed standard formula (SF) and a breast-fed reference group. The impact of MFGM on the fecal microbiome was moderate; however, the fecal metabolome of EF-fed infants showed a significant reduction of several metabolites including lactate, succinate, amino acids and their derivatives from that of infants fed SF. Introduction of weaning food with either human milk or infant formula reduces the distinct characteristics of breast-fed- or formula-fed- like infant fecal microbiome and metabolome profiles. Our findings support the hypothesis that higher levels of protein in infant formula and the lack of human milk oligosaccharides promote a shift toward amino acid fermentation in the gut. MFGM may play a role in shaping gut microbial activity and function.

Figure 1

The core fecal microbiota of infants. The core microbiota is defined as highly prevalent microbes at the genus level present in >80% of samples.

Figure 2

Analysis of the community structure of fecal microbiota reveals differences between breast-fed (BF, green) and formula-fed (Experimental Formula, EF, orange; Standard Formula, SF, red) infants at 2, 4 and 6 months of age. (a) Principal Coordinates Analysis (PCoA) of the log transformed weighted Unifrac distance. The centroids of each cluster (centroid of mass) are calculated as the average of PC1 and PC2 for each group. The ellipses were constructed based on multivariate normal distribution at 95% confidence level. The effect size (R²) and significance (p-value) between dietary groups were evaluated using permutational MANOVA via the Adonis test (permutation = 999). (b) Divergence (the spread within the group) was significantly higher in the BF infants during the exclusive feeding period. The measurement of divergence is calculated as 1- the average spearman correlation between samples and the overall group-wise average. The group difference was evaluated using a Kruskal-Wallis H test follow by the post-hoc Dunn test at p < 0.05. (c,d) Significantly differentiating intestinal microbes between the breast-fed (BF, green) and formula-fed (FF, pink) infants. The group differences were evaluated using Analysis of Composition of Microbiomes (ANCOM) followed by FDR correction at p < 0.05.

Figure 3

(a) Fecal water % and Bristol score are significantly higher in the breast-fed (BF) infants during the exclusive feeding period. The group difference was evaluated using Kruskal-Wallis H test followed by a post-hoc Dunn test, p < 0.05. Fecal metabolite concentrations are associated with fecal water %. (b) Heatmap of the correlation coefficient (Pearson r > 0.4 or <−0.4, p < 0.05) of fecal metabolite concentrations correspond to fecal water %. Amount of water in infant stool is positively correlated with fecal concentration of (c) lactate, (d) 1,2-propanediol, (e) glucose and (f) galactose and is negatively correlated with (g) isovalerate and (h) isobutyrate. The correlation between fecal water % and metabolite concentration were evaluated using data collected from 2 months (baseline) and 4 and 6 months (without complementary food).

Figure 4

The fecal metabolome reveals differences between breast-fed (BF, green) and formula-fed (Experimental Formula, EF, orange; Standard Formula, SF, red) infants at 2, and 4 & 6 months of age. (a) Principal Coordinates Analysis (PCoA) of the Euclidean distance matrix of the generalized log transformed fecal metabolite concentration data. The centroids of each cluster (centroid of mass) were calculated as the average PC1 and PC2 of all samples for each group. The ellipses were constructed based on multivariate normal distribution at 95% confidence level. The effect size (R²) and significance (p-value) between dietary groups were evaluated using permutational MANOVA via the Adonis test (permutation = 999). (b) Divergence (the spread within each group) is significantly higher in BF infants during the exclusive feeding period. The measurement of divergence was calculated as 1- the average spearman correlation between samples and the overall group-wise average. The group difference was evaluated using Kruskal-Wallis H test follow by post-hoc Dunn test, p < 0.05.

Figure 5

Fecal metabolites that are significantly different between formula-fed infants who consumed standard formula (SF, red) and experimental formula (EF, orange).

Figure 6

Significantly differentiating fecal metabolites that are (a) higher and (b) lower in breast-fed (BF, green) than formula-fed (FF, red) infants. The group differences were evaluated using Kruskal-Wallis H test by FDR correction at p < 0.05. The complete list of differentiating metabolites is in SI Fig. 5. Abbreviation: 4-HPLA: 4-hydroxyphenyllactate, 4-HPA: 4-hydroxyphenylacetate.