Human milk microbiota and oligosaccharides in colostrum and mature milk: comparison and correlation

Abstract

Background: The interaction between the human breast milk microbiota and human milk oligosaccharides (HMOs) plays a crucial role in the healthy growth and development of infants. We aimed to clarify the link between the breast milk microbiota and HMOs at two stages of lactation.

Methods: The microbiota and HMOs of 20 colostrum samples (C group, 1-5 days postpartum) and 20 mature milk samples (S group, 42 days postpartum) collected from postpartum mothers were analyzed using 16S rRNA gene high-throughput sequencing and high-performance liquid chromatography-tandem mass spectrometry.

Result: The total average HMO content was significantly higher in the C group than in the S group (6.76 ± 1.40 g/L vs. 10.27 ± 2.00 g/L, p < 0.05). Among the HMOs, the average values of 2'-fucosyllactose (2'-FL, 1.64 ± 1.54 g/L vs. 3.03 ± 1.79 g/L), 3'-sialyllactose (3'-SL, 0.10 ± 0.02 g/L vs. 0.21 ± 0.06), 6'-SL (0.22 ± 0.09 g/L vs. 0.33 ± 0.11 g/L), and lacto-N-triaose 2 (LNT2, 0.03 ± 0.01 g/L vs. 0.16 ± 0.08 g/L) were significantly lower in the S group than in the C group (p < 0.05), while that of 3'-FL was significantly higher in the S group than in the C group (1.35 ± 1.00 g/L vs. 0.41 ± 0.43 g/L, p < 0.05). The diversity and structure of the microbiota in the S and C groups were also significantly different (p < 0.05). Comparative analysis of the microbial communities revealed that Proteobacteria and Firmicutes were the most abundant phyla, in both groups, with the keystone species (Serratia, Streptococcus and Staphylococcus) of breast milk closely interacting with HMOs, including 3'-SL, 6'-SL, and LNT2. In PICRUSt2 functional prediction analysis, the S group exhibited significant reduction in the expression of genes involved in several infectious disease pathways.

Discussion: Our findings support the recognition of human milk as a synbiotic comprising beneficial bacteria and prebiotic HMOs.

Keywords: breast milk; colostrum; human milk microbiota; human milk oligosaccharides; mature milk.

Figure 1

The proportion (A) and composition (B) of oligosaccharides in breast milk. C: the colostrum group (n = 20); S: the mature milk (42 days postpartum) group (n = 20). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the C group.

Figure 2

The microbial diversity and composition of the breast milk microbiota in the C and S group. (A) Alpha diversity (Ace and Sobs and Chao index) on the OTU level. (B) Hierarchical clustering tree on the OTU level. (C) Principal component analysis (PCoA) with cluster. (D) Non-metric multidimensional scaling analysis (NMDS) with cluster. (E) Venn diagram. C: the colostrum group (n = 20); S: the mature milk (42 days postpartum) group (n = 20). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the C group.

Figure 3

Analysis of the structure and communities of the breast milk microbiota in the N and M group. (A) Microbial distributions of different groups at the phylum, Family and Genus level. (B) The relative abundance of Firmicutes, Actinobacteria and Proteobacteria at the Phylum level. (C) The relative abundance of Alcaligenaceae, Bacillaceae, Gemellaceae, Pseudomonadaceae, Staphylococcaceae, and Yersiniaceae at the Family level. (D) Random forest at the genus level. Up (red) represented the S group raised; down (blue) represented the S group decreased (p < 0.05). (E) Linear discriminant analysis (LDA) distribution by analyzing Linear discriminate analysis effect size (LEfSe). The threshold of the LDA score was 4.0. C: the colostrum group (n = 20); S: the mature milk (42 days postpartum) group (n = 20). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the C group.

Figure 4

Functional prediction and co-abundance networks at the genus level between the C group and the S group. (A) STAMP analysis for the inferred metabolic pathway in level 2. (B) STAMP analysis for the inferred metabolic pathway in level 3. (C) The co-abundance networks. C: the colostrum group (n = 20); S: the mature milk (42 days postpartum) group (n = 20). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the C group.

Figure 5

Analysis of the correlation between oligosaccharides and the breast milk microbiota in the C group and the S group at the Genus level. C: the colostrum group (n = 20); S: the mature milk (42 days postpartum) group (n = 20). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.