Shaping of the Infant Gut Microbiome by Milk Oligosaccharides

Oligosaccharides in Human Milk and Bacterial Colonization

Human milk microbiota (HMM) and human milk oligosaccharides (HMOs) are crucial components of breast milk that influence infant health and development. This study provides long-term insights into the dynamic changes in HMM and HMOs and their interactions among Taiwanese lactating mothers. A cohort of 90 mothers with singleton, full-term infants contributed 319 breast milk samples across eight lactation stages over 52 weeks. HMOs were quantified using mass spectrometry, while milk microbiota composition was analyzed through 16S rRNA sequencing. Our findings reveal that HMO concentrations steadily declined from birth to 52 weeks postpartum. The most abundant HMO was 2'-fucosyllactose (2'-FL, 18.8%), followed by 3-fucosyllactose (3-FL, 16.5%). Notably, 2'-FL levels decreased over time, while 3-FL concentrations increased. Other HMOs exhibited a general downward trend throughout lactation. HMM composition also shifted dynamically, with significant variations in α-diversity and β-diversity across lactation stages. The predominant bacterial genera-Streptococcus, Pseudomonas, and Staphylococcus-showed distinct bimodal temporal patterns. Correlation analyses revealed that tetratose and sialylated HMOs were positively associated with Bacteroides abundance, while multiple HMOs negatively correlated with Streptococcus. These findings underscore the complex interactions between breast milk oligosaccharides and microbial communities, highlighting their potential implications for infant gut health. Further research is needed to explore the clinical significance of these transformations and their role in shaping the early-life microbiome. PRACTICAL APPLICATIONS: This study shows how breast milk changes over time, with certain sugars (HMOs) decreasing and others shifting, while beneficial bacteria also fluctuate. These changes may influence an infant's gut health and development. Understanding these patterns can help improve infant nutrition, guide breastfeeding recommendations, and support the design of better infant formulas or probiotics.

While ample research on independent associations between infant cognition and gut microbiota composition and human milk (HM) oligosaccharides (HMOs) has been reported, studies on how the interactions between gut microbiota and HMOs may yield associations with cognitive development in infancy are lacking. We aimed to determine how HMOs and species of Bacteroides and Bifidobacterium genera interact with each other and their associations with cognitive development in typically developing infants. A total of 105 mother-infant dyads were included in this study. The enrolled infants [2.9-12 months old (8.09 ± 2.48)] were at least predominantly breastfed at 4 months old. A total of 170 HM samples from the mothers and fecal samples of the children were collected longitudinally. Using the Mullen Scales of Early Learning to assess cognition and the scores as the outcomes, linear mixed effects models including both the levels of eight HMOs and relative abundance of Bacteroides and Bifidobacterium species as main associations and their interactions were employed with adjusting covariates; infant sex, delivery mode, maternal education, site, and batch effects of HMOs. Additionally, regression models stratifying infants based on the A-tetrasaccharide (A-tetra) status of the HM they received were also employed to determine if the associations depend on the A-tetra status. With Bacteroides species, we observed significant associations with motor functions, while Bif. catenulatum showed a negative association with visual reception in the detectable A-tetra group both as main effect (value of p = 0.012) and in interaction with LNFP-I (value of p = 0.007). Additionally, 3-FL showed a positive association with gross motor (p = 0.027) and visual reception (p = 0.041). Furthermore, significant associations were observed with the interaction terms mainly in the undetectable A-tetra group. Specifically, we observed negative associations for Bifidobacterium species and LNT [breve (p = 0.011) and longum (p = 0.022)], and positive associations for expressive language with 3'-SL and Bif. bifidum (p = 0.01), 6'-SL and B. fragilis (p = 0.019), and LNFP-I and Bif. kashiwanohense (p = 0.048), respectively. Our findings suggest that gut microbiota and HMOs are both independently and interactively associated with early cognitive development. In particular, the diverse interactions between HMOs and Bacteroides and Bifidobacterium species reveal different candidate pathways through which HMOs, Bifidobacterium and Bacteroides species potentially interact to impact cognitive development in infancy.

Novel oligosaccharides in human milk: understanding mechanisms may lead to better prevention of enteric and other infections

Human milk oligosaccharides (HMOs), the third most abundant solid component in human milk, vary significantly among women due to factors such as secretor status, race, geography, season, maternal nutrition and weight, gestational age, and delivery method. In recent studies, HMOs have been shown to have a variety of functional roles in the development of infants. Because HMOs are not digested by infants, they act as metabolic substrates for certain bacteria, helping to establish the infant's gut microbiota. By encouraging the growth of advantageous intestinal bacteria, these sugars function as prebiotics and produce short-chain fatty acids (SCFAs), which are essential for gut health. HMOs can also specifically reduce harmful microbes and viruses binding to the gut epithelium, preventing illness. HMO addition to infant formula is safe and promotes healthy development, infection prevention, and microbiota. Current infant formulas frequently contain oligosaccharides (OSs) that differ structurally from those found in human milk, making it unlikely that they would reproduce the unique effects of HMOs. However, there is a growing trend in producing OSs resembling HMOs, but limited data make it unclear whether HMOs offer additional therapeutic benefits compared to non-human OSs. Better knowledge of how the human mammary gland synthesizes HMOs could direct the development of technologies that yield a broad variety of complex HMOs with OS compositions that closely mimic human milk. This review explores HMOs' complex nature and vital role in infant health, examining maternal variation in HMO composition and its contributing factors. It highlights recent technological advances enabling large-scale studies on HMO composition and its effects on infant health. Furthermore, HMOs' multifunctional roles in biological processes such as infection prevention, brain development, and gut microbiota and immune response regulation are investigated. The structural distinctions between HMOs and other mammalian OSs in infant formulas are discussed, with a focus on the trend toward producing more precise replicas of HMOs found in human milk.

BackgroundThe 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.MethodsThe 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.ResultThe 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.DiscussionOur findings support the recognition of human milk as a synbiotic comprising beneficial bacteria and prebiotic HMOs.

Breastfeeding represents a strong selective factor for shaping the infant gut microbiota. Besides providing nutritional requirements for the infant, human milk is a key source of oligosaccharides, human milk oligosaccharides (HMOs), and diverse microbes in early life. This study aimed to evaluate the influence of human milk microbiota and oligosaccharides on the composition of infant faecal microbiota at one, three, and nine months postpartum. We profiled milk microbiota, HMOs, and infant faecal microbiota from 23 mother–infant pairs at these time points. The predominant genera in milk samples were Streptococcus, Staphylococcus, and an unclassified Enterobacteriaceae genus-level taxon (Enterobacteriaceae uncl.), whereas the infant faecal microbiota was predominated by Bifidobacterium, Bacteroides, and Enterobacteriaceae uncl. Mother–infant dyads frequently shared bacterial amplicon sequence variants (ASVs) belonging to the genera Bifidobacterium, Streptococcus, Enterobacteriaceae uncl., Veillonella, Bacteroides, and Haemophilus. The individual HMO concentrations in the milk showed either no change or decreased over the lactation period, except for 3-fucosyllactose (3-FL), which increased. Neither maternal secretor status nor HMO concentrations were significantly associated with microbiota composition at the different ages or the bacterial ASVs of maternal milk and infant faeces. This study suggests an age-dependent role of milk microbes in shaping the gut microbiota, while variations in HMO concentrations show limited influence.

Human milk is the gold standard for infant feeding. Human milk oligosaccharides (HMOs) are a unique group of oligosaccharides in human milk. Great interest in HMOs has grown in recent years due to their positive effects on various aspects of infant health. HMOs provide various physiologic functions, including establishing a balanced infant's gut microbiota, strengthening the gastrointestinal barrier, preventing infections, and potential support to the immune system. However, the clinical application of HMOs is challenging due to their specificity to human milk and the difficulties and high costs associated with their isolation and synthesis. Here, the differences in oligosaccharides in human and other mammalian milk are compared, and the synthetic strategies to access HMOs are summarized. Additionally, the potential use and molecular mechanisms of HMOs as a new food bioactive component in different diseases, such as infection, necrotizing enterocolitis, diabetes, and allergy, are critically reviewed. Finally, the current challenges and prospects of HMOs in basic research and application are discussed.

ContextOligosaccharides are the third largest solid component in human milk. These diverse compounds are thought to have numerous beneficial functions in infants, including protection against infectious diseases. The structures of more than 100 oligosaccharides in human milk have been elucidated so far.ObjectiveThe aim of this review was to identify the main factors that affect the concentrations of oligosaccharides in human milk and to determine whether it is possible to calculate representative and reliable mean concentrations.Data SourcesA comprehensive literature search on oligosaccharide concentrations in human milk was performed in 6 electronic databases: BIOSIS, Current Contents Search, Embase, Lancet Titles, MEDLINE and PubMed.Study SelectionThe initial search resulted in 1363 hits. After the elimination of duplicates, the literature was screened. The application of strict inclusion criteria resulted in 21 articles selected.Data ExtractionOligosaccharide concentrations, both mean values and single values, reported in the literature were sorted by gestational age, secretor status of mothers, and defined lactation periods.ResultsMean concentrations, including confidence limits, of 33 neutral and acidic oligosaccharides reported could be calculated. Concentrations of oligosaccharides in human milk show variations that are dependent on both the secretor type of the mother and the lactation period as examined by analyses of variance. In addition, large interlaboratory variations in the data were observed.ConclusionsWorldwide interlaboratory quantitative analyses of identical milk samples would be required to identify the most reliable methods of determining concentrations of oligosaccharides in human milk. The data presented here contribute to the current knowledge about the composition and quantities of oligosaccharides in human milk and may foster greater understanding of the biological functions of these compounds.

Human milk contains a unique profile of oligosaccharides (OS) and preliminary evidence suggests they impact brain development. The objective of this study was to assess the impact of bovine and/or human milk oligosaccharides (HMO) (2′-fucosyllactose and Lacto-N-neotetraose) on cognition, brain development, and hippocampal gene expression. Beginning on postnatal day (PND) 2, male pigs received one of four milk replacers containing bovine milk oligosaccharides (BMOS), HMO, both (BMOS + HMO), or neither. Pigs were tested on the novel object recognition task using delays of 1- or 48-h at PND 22. At PND 32–33, magnetic resonance imaging procedures were used to assess structural brain development and hippocampal tissue was collected for analysis of mRNA expression. Pigs consuming only HMO exhibited recognition memory after a 1-h delay and those consuming BMOS + HMO exhibited recognition memory after a 48-h delay. Both absolute and relative volumes of cortical and subcortical brain regions were altered by diet. Hippocampal mRNA expression of GABRB2, SLC1A7, CHRM3, and GLRA4 were most strongly affected by diet. HMO and BMOS had distinct effects on brain structure and cognitive performance. These data suggest different mechanisms underlie their influence on brain development.

Early life is an essential period for the establishment of the gut microbiota and is considered a critical window for intervention. Breast milk serves as a natural food for babies and contains a variety of bioactive ingredients that promote healthy growth. The active ingredients in breast milk that are closely related to infant gut development, such as human milk microbiota (HMM), are the primary source of infant gut microbiota. Human milk oligosaccharides (HMOs) can reach the infant colon intact and serve as fermentable substrates for specific gut microbiota, thereby helping shape the infant gut microbiota. The absence of these active ingredients in milk formula is strongly associated with health problems and abnormal gut microbiota in infants. This review provides an overview of the changes in the gut microbiota of the host at various stages of development, summarizing recent research on the active ingredients of early-life ingested food-breast milk, including HMM and HMOs, their origin, structure, impact factors, and contribution to infant health benefits. It also reviews major strategies for utilizing HMOs for infant gut probiotics and next-generation potential probiotics, providing a reference for the later development of formula milk and related nutritional products.

ABSTRACTThe gastrointestinal microbiota members produce α-l-fucosidases that play key roles in mucosal, human milk, and dietary oligosaccharide assimilation. Here, 36 open reading frames (ORFs) coding for putative α-l-fucosidases belonging to glycosyl hydrolase family 29 (GH29) were identified through metagenome analysis of breast-fed infant fecal microbiome. Twenty-two of those ORFs showed a complete coding sequence with deduced amino acid sequences displaying the highest degree of identity with α-l-fucosidases from Bacteroides thetaiotaomicron, Bacteroides caccae, Phocaeicola vulgatus, Phocaeicola dorei, Ruminococcus gnavus, and Streptococcus parasanguinis. Based on sequence homology, 10 α-l-fucosidase genes were selected for substrate specificity characterization. The α-l-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on α1,3/4-linked fucose present in Lewis blood antigens and the human milk oligosaccharide (HMO) 3-fucosyllactose. In addition, Fuc1584 also hydrolyzed fucosyl-α-1,6-N-acetylglucosamine (6FN), a component of the core fucosylation of N-glycans. Fuc35A and Fuc193 showed activity on α1,2/3/4/6 linkages from H type-2, Lewis blood antigens, HMOs and 6FN. Fuc30 displayed activity only on α1,6-linked l-fucose, and Fuc5372 showed a preference for α1,2 linkages. Fuc2358 exhibited a broad substrate specificity releasing l-fucose from all the tested free histo-blood group antigens, HMOs, and 6FN. This latest enzyme also displayed activity in glycoconjugates carrying lacto-N-fucopentaose II (Lea) and lacto-N-fucopentaose III (Lex) and in the glycoprotein mucin. Fuc18, Fuc19A, and Fuc39 also removed l-fucose from neoglycoproteins and human α-1 acid glycoprotein. These results give insight into the great diversity of α-l-fucosidases from the infant gut microbiota, thus supporting the hypothesis that fucosylated glycans are crucial for shaping the newborn microbiota composition.IMPORTANCE α-l-Fucosyl residues are frequently present in many relevant glycans, such as human milk oligosaccharides (HMOs), histo-blood group antigens (HBGAs), and epitopes on cell surface glycoconjugate receptors. These fucosylated glycans are involved in a number of mammalian physiological processes, including adhesion of pathogens and immune responses. The modulation of l-fucose content in such processes may provide new insights and knowledge regarding molecular interactions and may help to devise new therapeutic strategies. Microbial α-l-fucosidases are exoglycosidases that remove α-l-fucosyl residues from free oligosaccharides and glycoconjugates and can be also used in transglycosylation reactions to synthesize oligosaccharides. In this work, α-l-fucosidases from the GH29 family were identified and characterized from the metagenome of fecal samples of breastfed infants. These enzymes showed different substrate specificities toward HMOs, HBGAs, naturally occurring glycoproteins, and neoglycoproteins. These novel glycosidase enzymes from the breast-fed infant gut microbiota, which resulted in a good source of α-l-fucosidases, have great biotechnological potential.

The third-most solid component in breast milk is human milk oligosaccharides (HMOs)—crucial for the growth and development of infants. HMOs are divided into three main categories: fucosylated, nonfucosylated, and sialylated HMOs. However, comparative studies evaluating the variations of the impact of in vitro fermentation of these typical or dominant HMOs on the metabolic functions of an infant's gut microbiome remain limited. Therefore, we systematically analyzed the role of three typical HMOs—2′-fucosyl lactose, lactose-N-tetrose, and 3′-sialyllactose—in the structural reshaping of the infant intestinal flora; we also explored their impact on short-chain fatty acid (SCFA) production and metabolism, as well as their overall metabolic spectrum. We found that HMOs aided the growth of beneficial microorganisms, such as Bifidobacterium, Lactobacillus, and Enterococcus, while also reducing the number of harmful bacteria, including Escherichia-Shigella. Further, HMOs had a substantial impact on amino acid, purine, and lipid metabolic pathways and significantly increased SCFA levels. Correlation analyses revealed significant associations between Bifidobacterium and multiple lipid metabolites. Positive correlations between Lactobacillus and amino acid derivatives, as well as close links between Bacteroides and acetate production, were detected. Overall, these findings indicate that the gut flora-metabolite interplay is central to HMO function, and different HMOs variably regulate the gut microbiota composition and metabolic pathways. This study provides a theoretical basis for optimizing HMOs' fortification strategies in infant formulas.

Human milk oligosaccharides (HMOs) promote the development of the infant gut microbiota, notably through the growth of beneficial bacteria. However, the interaction between maternal diet, HMOs and the offspring's gut microbiota remains incompletely understood. We hypothesize that a high-fat (HF) diet influences the composition of HMOs and shapes gut microbiota in offspring. In lactating rat mothers fed with a HF or a low-fat (LF) diet, we measured at mid-lactation, the levels of milk sialylated oligosaccharides (SL-MOs) and the expression of enzymes for synthesizing SL-MOs in the mammary gland. In rat pups from LF or HF mothers, we analysed the caecal microbiota and measured levels of faecal short-chain fatty acids (SCFAs). In addition, in humans, we quantified 24 HMOs in the colostrum of lean and obese mothers. In rat mothers, HF feeding significantly reduced the breast milk 6'SL level but not the 3'SL one and increased the gene-expression level of the St3Gal1 enzyme in the mammary gland. In HF rat pups, the caecal microbiota was drastically modified with increases in α- and β-diversities as well as in the abundance of Parabacteroides and Lachnospiraceae. Levels of faecal SCFAs including acetate, butyrate, isobutyrate, valerate and isovalerate were also increased in HF pups. In humans, maternal obesity only slightly increased DFLNHa concentration in the colostrum. In conclusion, our data suggest that a hyperlipidic diet during lactation may reduce the level of sialylated oligosaccharides in breast milk and alter the abundance of several groups of bacteria in the infant's gut. We also demonstrated that, in humans, obesity did not highly affect levels of HMOs in the colostrum reinforcing previous findings suggesting that being overweight does not influence drastically the HMO profile in breast milk.

Human milk is elixir for neonates and is a rich source of nutrients and beneficial microbiota required for infant growth and development. Its benefits prompted research into probing the milk components and their use as prophylactic or therapeutic agents. Culture-independent estimation of milk microbiome and high-resolution identification of milk components provide information, but a holistic purview of these research domains is lacking. Here, we review the current research on bio-therapeutic components of milk and simplified future directions for its efficient usage. Publicly available databases such as PubMed and Google scholar were searched for keywords such as probiotics and prebiotics related to human milk, microbiome and milk oligosaccharides. This was further manually curated for inclusion and exclusion criteria relevant to human milk and clinical efficacy. The literature was classified into subgroups and then discussed in detail to facilitate understanding. Although milk research is still in infancy, it is clear that human milk has many functions including protection of infants by passive immunization through secreted antibodies, and transfer of immune regulators, cytokines and bioactive peptides. Unbiased estimates show that the human milk carries a complex community of microbiota which serves as the initial inoculum for establishment of infant gut. Our search effectively screened for evidence that shows that milk also harbours many types of prebiotics such as human milk oligosaccharides which encourage growth of beneficial probiotics. The milk also trains the naive immune system of the infant by supplying immune cells and stimulatory factors, thereby strengthening mucosal and systemic immune system. Our systematic review would improve understanding of human milk and the inherent complexity and diversity of human milk. The interrelated functional role of human milk components especially the oligosaccharides and microbiome has been discussed which plays important role in human health.