Bifidogenic Effect of 2'-Fucosyllactose (2'-FL) on the Gut Microbiome of Healthy Formula-Fed Infants: A Randomized Clinical Trial

Abstract

Breast milk is rich in bioactive components, especially human milk oligosaccharides (HMOs), which are crucial for establishing gut microbiota. The 2'-FL (2-Fucosyllactose), one of the most abundant oligosaccharides in breast milk, functions as a selective prebiotic. Objective: To examine the effect of adding 2'-FL (2-Fucosyllactose) to an infant formula containing prebiotic galacto-oligosaccharides (GOSs) and fructo-oligosaccharides (FOSs) on the gut microbiome of healthy formula-fed infants. Methods: This study enrolled infants from three groups: an HMO experimental group (n = 29), a GOS/FOS control group (n = 30), and an exclusively breastfed (breast milk [BM]) reference group (n = 28). Fecal samples from the three groups in the first and fourth months of life were analyzed. The V3 and V4 regions of the 16S rRNA gene were amplified and sequenced on the Illumina MiSeq. ANOVA, Kruskal-Wallis, richness indices (Chao1, Shannon), UniFrac distances, and the Adonis tests were used to perform statistical analyses on the relative abundance of phyla and genera, as well as the alpha and beta-diversity of the gut microbiota. Results: After intervention, Actinobacteriota emerged as the predominant phylum in both the HMO (60.4%) and BM (46.6%) groups. Bifidobacterium and Escherichia-Shigella were identified as the two most abundant bacterial genera in both groups. Nevertheless, the statistical analysis showed that the relative abundance of Bifidobacterium in the HMO formula-fed group after intervention was similar to that in the BM group (p > 0.05). Infants in the HMO and GOS/FOS groups showed higher relative abundance of [Ruminococcus]_gnavus_group bacteria compared to those in the BM group. Groups fed with infant formula demonstrated higher alpha-diversity of gut microbiota compared to breastfed infants (p < 0.05), at the time of admission as well as after the intervention. Beta-diversity was significantly different among the three groups, according to type of feeding. Infants fed a 2'-FL-supplemented infant formula exhibited growth comparable to that of breastfed infants throughout the intervention period, demonstrating that the formula was both safe and well tolerated. Conclusions: Adding 2'-FL to an infant formula containing 4 g/L of GOS + FOS resulted in a stronger bifidogenic effect compared to the formula without 2'-FL.

Keywords: 2-fucosyllactose; breast milk; fructo-oligosaccharides; galacto-oligosaccharides; gut microbiome; human milk oligosaccharides; infant formula.

Figure 1

Subject disposition for the randomized controlled trial with formula-fed infants and the companion study with human milk-fed infants serving as the reference group. (HMO) experimental group, (GOS/FOS) control group, and (BM) breast milk-fed infants.

Figure 2

Histogram of the number of DNA sequences in the samples (n = 178) from the three groups included in the study, collected at admission and after the intervention period.

Figure 3

Upon enrollment: Panel (A) Relative abundance of the most prevalent phyla. (*) = p values. Panel (B) Relative abundance of the most prevalent genera. (*) = p values. Kruskal–Wallis test followed by Dunn’s test for multiple comparisons: a. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; GOS/FOS x BM p < 0.05. Panel (C) Graphical representation (“boxplot”) of alpha-diversity using Chao1 and Shannon indices, with the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. ns = not significant; ** p < 0.01 for Shannon index HMO x BM; *** p< 0.01 for Chao1 index HMO x BM and GOS/FOS x BM; **** p< 0.01 for Shannon index GOS/FOS x BM. Panel (D) is the final weighted UniFrac distance by feeding type (p = 0.48); sex (p = 0.57); delivery mode (p < 0.001 *). Panel (E) Final unweighted UniFrac distance by feeding type (p = 0.004 *), sex (p = 0.01 *), and delivery mode (p < 0.02 *). (*) = statistical significance. HMO, experimental group; GOS/FOS, control group; BM, breast milk-fed infants.

Figure 3

Upon enrollment: Panel (A) Relative abundance of the most prevalent phyla. (*) = p values. Panel (B) Relative abundance of the most prevalent genera. (*) = p values. Kruskal–Wallis test followed by Dunn’s test for multiple comparisons: a. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; GOS/FOS x BM p < 0.05. Panel (C) Graphical representation (“boxplot”) of alpha-diversity using Chao1 and Shannon indices, with the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. ns = not significant; ** p < 0.01 for Shannon index HMO x BM; *** p< 0.01 for Chao1 index HMO x BM and GOS/FOS x BM; **** p< 0.01 for Shannon index GOS/FOS x BM. Panel (D) is the final weighted UniFrac distance by feeding type (p = 0.48); sex (p = 0.57); delivery mode (p < 0.001 *). Panel (E) Final unweighted UniFrac distance by feeding type (p = 0.004 *), sex (p = 0.01 *), and delivery mode (p < 0.02 *). (*) = statistical significance. HMO, experimental group; GOS/FOS, control group; BM, breast milk-fed infants.

Figure 4

At the end of the study: Panel (A) Relative abundance of the most prevalent phyla. (*) = p values. The Kruskal–Wallis test followed by Dunn’s test for multiple comparisons: a. HMO x GOS/FOS p < 0.05; HMO x BM p > 0.05; BM x GOS/FOS p > 0.05; b. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; BM x GOS/FOS p > 0.05. Panel (B) Relative abundance of the most prevalent genera. (*) = p values. The Kruskal–Wallis test followed by Dunn’s test for multiple comparisons: c. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; BM x GOS/FOS p < 0.05; d. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; BM x GOS/FOS p < 0.05; e. HMO x GOS/FOS p < 0.05; HMO x BM p > 0.05; BM x GOS/FOS p > 0.05; f. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; BM x GOS/FOS p > 0.05; g. HMO x GOS/FOS p > 0.05; HMO x BM p > 0.05; BM x GOS/FOS p < 0.05. Panel (C) Graphical representation (“box-plot”) of alpha-diversity using Chao1 and Shannon indices, with the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. ns = not significant; ** p < 0.01 for Shannon index HMO x BM; *** p < 0.01 for Chao1 index HMO x BM and GOS/FOS x BM; **** p < 0.01 for Shannon index GOS/FOS x BM. Panel (D) Final weighted UniFrac distance by type of feeding (p < 0.004 *); sex (p = 0.60); delivery mode (p = 0.45). Panel (E) Final unweighted UniFrac distance by feeding type (p = 0.001 *); sex (p = 0.45); delivery mode: (p = 0.81). (*) = statistical significance. HMO, experimental group; GOS/FOS, control group; BM, breast milk-fed infants.

Figure 4

At the end of the study: Panel (A) Relative abundance of the most prevalent phyla. (*) = p values. The Kruskal–Wallis test followed by Dunn’s test for multiple comparisons: a. HMO x GOS/FOS p < 0.05; HMO x BM p > 0.05; BM x GOS/FOS p > 0.05; b. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; BM x GOS/FOS p > 0.05. Panel (B) Relative abundance of the most prevalent genera. (*) = p values. The Kruskal–Wallis test followed by Dunn’s test for multiple comparisons: c. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; BM x GOS/FOS p < 0.05; d. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; BM x GOS/FOS p < 0.05; e. HMO x GOS/FOS p < 0.05; HMO x BM p > 0.05; BM x GOS/FOS p > 0.05; f. HMO x GOS/FOS p > 0.05; HMO x BM p < 0.05; BM x GOS/FOS p > 0.05; g. HMO x GOS/FOS p > 0.05; HMO x BM p > 0.05; BM x GOS/FOS p < 0.05. Panel (C) Graphical representation (“box-plot”) of alpha-diversity using Chao1 and Shannon indices, with the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. ns = not significant; ** p < 0.01 for Shannon index HMO x BM; *** p < 0.01 for Chao1 index HMO x BM and GOS/FOS x BM; **** p < 0.01 for Shannon index GOS/FOS x BM. Panel (D) Final weighted UniFrac distance by type of feeding (p < 0.004 *); sex (p = 0.60); delivery mode (p = 0.45). Panel (E) Final unweighted UniFrac distance by feeding type (p = 0.001 *); sex (p = 0.45); delivery mode: (p = 0.81). (*) = statistical significance. HMO, experimental group; GOS/FOS, control group; BM, breast milk-fed infants.

Figure 5

Beta-diversity: pairwise comparison between groups after intervention. (*) = statistical significance. Panel (A) UniFrac distance weighted at the end according to HMO x GOS/FOS groups p = 0.002 *. Panel (B) Final unweighted UniFrac distance for HMO vs. GOS/FOS groups p = 0.013 *. Panel (C) Final weighted UniFrac distance for HMO vs. BM groups p = 0.009 *. Panel (D) Final unweighted UniFrac distance for HMO vs. BM groups p = 0.001 *. Panel (E) Final weighted UniFrac distance according to the GOS/FOS x BM groups p = 0.185 *. Panel (F) Final unweighted UniFrac distance for GOS/FOS vs. BM groups p = 0.002 *. HMO, experimental group; GOS/FOS, control group; BM, Breast milk-fed infants.