Human Milk Oligosaccharides as Prebiotics

How far is it from infant formula to human milk? A look at the human milk oligosaccharides

Background: Infant formula has a similar composition to breast milk, especially on key components like proteins, fats, and functional oligosaccharides. However, significant differences exist between bovine milk-based formula and human breast milk, particularly in the abundance and diversity of human milk oligosaccharides (HMOs). HMOs are pivotal in shaping the infant gut microbiome and immune system, with potential implications for infant health and development.Objectives: The literature review aimed to explore the role of HMOs in shaping the gut microbiome and strengthening the intestinal immune system in infants.Methods: Descriptive research was carried out in May-August 2023 using the literature review method. The design and implementation of this study referred to the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) for Randomized Controlled Trials (RCTs). Literature sources were obtained from PubMed, Elsevier, Medline, Nature, PLOS, MPDI, JAMA, and Scopus publication databases with a limitation of the last ten years (2013-2023). One hundred twenty articles were obtained with these keywords and then selected in stages according to predetermined criteria. Six articles that met the criteria were retrieved from 2013-2023. The articles obtained were compiled, analyzed, and concluded by looking for similarities and dissimilarities, giving views, comparing, and summarizing.Results: The review revealed that HMOs significantly influenced the composition and function of the infant's intestinal microbiota, inhibiting harmful pathogens in the intestine. Therefore, HMOs as prebiotics were crucial in promoting intestinal immune system health in infants.Conclusion: Human Milk Oligosaccharides (HMOs) in infant formula substantially impacted the gut microbiome composition and the infant's intestinal immune system. Studies consistently showed that HMOs influenced the growth and diversity of gut bacteria, leading to outcomes similar to those seen in breastfed infants.

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.

Human milk is universally recognized as the optimal and most natural source of nutrition for newborns, offering benefits that extend far beyond basic energy and macronutrient provision. Among its complex constituents, human milk oligosaccharides (HMOs) represent the third most abundant solid component, surpassed only by lactose and lipids. HMOs are distinguished by their exceptionally high structural diversity—over 200 distinct structures have been identified to date. This structural complexity underlies the extensive biological functions HMOs perform within the infant’s body. HMOs play a pivotal role in promoting healthy growth, development, and overall well-being in infants and young children, functioning as indispensable bioactive molecules. Their key physiological activities include: immunomodulation and allergy prevention by promoting immune tolerance and reducing the risk of allergic diseases; potent anti-inflammatory and antioxidant effects that protect vulnerable infant tissues; support for brain development and cognitive enhancement through multiple mechanisms; anti-pathogenic properties, acting as soluble receptor analogs or “decoy” molecules to competitively block viral, bacterial, and other pathogen adhesion, thereby preventing colonization and infection in the gastrointestinal tract; and functioning as blood group substances. At the translational and application level, HMO research is actively driving cross-disciplinary innovation. Building on a deep understanding of their immunological and neurodevelopmental benefits, certain structurally defined HMOs have been successfully incorporated into infant formula. These HMO-supplemented formulas have received regulatory approval and are now commercially available worldwide, providing a nutritional alternative that more closely resembles human milk for infants who are not exclusively breastfed. This represents a significant step toward narrowing the compositional gap between formula and breast milk. Simultaneously, research into the symbiotic relationship between HMOs and the gut microbiota—particularly their role as selective prebiotic substrates promoting the growth of beneficial bacteria—has catalyzed the development of novel functional foods, dietary supplements, and microbiome-targeted therapies. These include advanced synbiotic formulations that combine specific probiotic strains with HMOs to synergistically optimize gut health and function. Furthermore, the intrinsic qualities of HMOs—including their natural origin, safety profile, biocompatibility, and proven antioxidant properties—have attracted growing interest in the emerging field of high-performance cosmetics. They are increasingly being explored as innovative functional ingredients in skincare products aimed at reducing oxidative stress and supporting skin health. This review aims to systematically synthesize recent advancements in HMO research, offering a comprehensive analysis centered on their complex composition and structural diversity; the molecular and cellular mechanisms underlying their diverse biological functions; their translational potential across sectors such as nutrition, medicine, and consumer care (including cosmetics); and the major challenges that persist in the field. It critically examines both foundational discoveries and recent breakthroughs. By integrating these interconnected themes, the review provides a holistic and up-to-date perspective on the scientific landscape of HMOs, highlighting their essential role in early-life nutrition and their expanding relevance across health and wellness applications. It also outlines promising directions for future research, with the goal of advancing evidence-based innovation in infant health and beyond.

Breastfeeding plays a protective role against infections, partially through the prebiotic effect of human milk oligosaccharides (HMOs). Aiming to mimic these beneficial capacities, there is an ongoing search to make infant formula closer to human milk, including by adding oligosaccharides. Over the past two decades, multiple studies have been published on different types of prebiotics and their role in reducing infection rates in infants. This review aims to answer the question of whether there is evidence that the addition of oligosaccharides to infant formula decreases the prevalence of infection, and whether the effect is influenced by the kind of oligosaccharide added. The review of the literature reveals an important heterogeneity, including different types and dosages of prebiotics, different intervention periods and inclusion criteria, etc., making it impossible to formulate a consensus about the efficacy of adding prebiotics to infant formula. We would cautiously suggest that supplementation with galactooligosaccharides (GOSs)/fructooligosaccharides (FOSs) seems to have a beneficial effect on infection rates. For HMOs, more studies about the different types of HMOs are necessary to make any deductions. GOSs alone, inulin, and MOSs (bovine-milk-derived oligosaccharides) do not reduce the incidence of infections. The combination of GOSs and PDX (polydextrose) was found to play a protective role in one study. The evidence of the effect of prebiotics in reducing the use of antibiotics is low. The many lacunas in the direction of study uniformity offer many opportunities for further research.

Objective:Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2’-Fucosyllactose (2’-FL) and 6’-Sialyllactose (6’-SL), can reduce NEC through inhibition of TLR4 signaling.Design:NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2’-FL, 6’-SL or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, human tissue explants, and via in silico modeling.Results:Supplementation of infant formula with either 2’-FL and/or 6’-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2’-FL and 6’-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2’-FL and 6’-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling.Conclusion:2’-FL and 6’-SL, but not lactose, prevent NEC in mice and piglet models, and attenuate NEC-inflammation in human ileum, in part through TLR4 inhibition.

Human milk oligosaccharides (HMOs) have garnered significant attention as one of the bioactive components in human milk, with growing applications in infant formula and food products. HMOs enhance butyrate production, which is produced by butyrate-producing bacteria such as Faecalibacterium prausnitzii and contributes to gut health through its diverse biological functions. However, the specific mechanisms by which individual HMOs promote butyrate production remain unclear. In this study, we conducted in vitro co-culture experiments of F. prausnitzii and Bifidobacterium bifidum, examining their relative abundance, fatty acid production, residual sugar levels, and gene expression. Our results revealed that B. bifidum utilizes HMOs and provides the constituent sugars to F. prausnitzii, thereby promoting butyrate production by F. prausnitzii. Furthermore, we found that the underlying mechanisms vary depending on the structure of the HMOs. Specifically, 2′-fucosyllactose and 3′-sialyllactose enhance the butyrate production efficiency of F. prausnitzii, while 6′-sialyllactose primarily promotes the growth of F. prausnitzii. These findings not only deepen our understanding of how HMOs influence infant gut health but also suggest new directions for developing nutritional products that leverage the distinct functional properties of each HMO.

A safety evaluation of mixed human milk oligosaccharides in rats

To investigate the relationship between human milk oligosaccharides(HMOs) levels in healthy mothers and infant physical growth. Based on a nationally representative, multi-stage stratified sampling cross-sectional survey on human milk composition, 195 lactating women within 6 months postpartum and their infants were enrolled. Human milk oligosaccharide(HMOs) concentrations were quantified using ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS). Infant anthropometric measurements(height, weight, and head circumference) were collected to calculate Z-scores. Associations between HMOs and infant growth were analyzed via general linear models. Total HMOs and total sialylated HMOs in mature breast milk showed negative correlations with weight for length Z-score(WLZ), weight for age Z-score(WAZ), and BMI for age Z-score(BAZ)(β=-0.80 to-0.25, P&lt;0.05). Specifically, 6'-sialyllactosamine(6'-SL), sialyllacto-N-tetraose c(LSTc), and disialyllacto-N-tetraose(DSLNT) demonstrated significant negative associations with WAZ(β=-0.36 to-0.25, P&lt;0.05). Total neutral non-fucosylated HMOs and lacto-N-tetraose(LNT) were also negatively correlated with length for age Z-score(LAZ) and WAZ(β=-0.33 to-0.25, P&lt;0.05). Among secretor+/Lewis+(Se+Le+) mothers, the negative effects of total HMOs and sialylated HMOs on infant WLZ, WAZ, and BAZ were further strengthened(β=-1.22 to-0.48, P&lt;0.05). Specific HMO concentrations in human milk during the first 6 months postpartum are associated with infant growth outcomes. Total HMOs, sialylated HMOs, and neutral non-fucosylated HMOs demonstrate negative associations with weight indicators.

The intestinal immune barrier is considered to be the gatekeeper of the human body and rapidly develops directly after birth. Many pre- and postnatal factors influence the development of the gut-barrier, which is composed of the microbiota, the mucus, the epithelial layer and the mucosal immune system. Even minor disturbances during barrier development can have consequences for health far into adulthood. Here we critically discuss the current knowledge on which pre- and postnatal factors influence development, maturation, and maintenance of the gut immune barrier. Human milk has a unique composition and is the gold standard for adequate development of the intestinal immune barrier. Not only the influence of human milk oligosaccharides (HMOs) but also that of glycoproteins (HMGPs) is reviewed. We discuss the influence of maternal genetic factors, such as the secretor and Lewis phenotypes on breast milk fucosylation and sialylation of HMOs and HMGPs. This diversity in HMOs and HMPGs influences microbiota composition and also the development of the immune barrier. Cow milk-derived infant formula is often being used as an alternative for human breast milk. The consequences of this for proper development of the intestinal immune barrier and, in particular, the differences in the type of oligosaccharides and glycosylation patterns (sialic and fucose composition) between cow and human milk are critically discussed. Current and prospective strategies to promote proper gut-immune maturation are proposed. These might include more personalized infant formulas when breast milk is not an option.

Breastfeeding is integral in the proper maturation of the intestinal barrier and protection against inflammatory diseases. When human milk (HM) is not available, supplementation with HM bioactives like Human Milk Oligosaccharides (HMOs) may help in providing breastfeeding barrier-protective benefits. An increasing HMO variety is becoming industrially available, enabling approaching the HMO complexity in HM. We aimed at assessing the impact of blends of available HMOs on epithelial barrier function in vitro. The capacity of individual [2′-Fucosyllactose (2′FL), Difucosyllactose, Lacto-N-tetraose, Lacto-N-neotetraose, 3′-Siallylactose and 6′-Siallylactose] or varying combinations of 3, 5 and 6 HMOs to modulate fluorescein-isothiocyanate (FITC)-labelled Dextran 4 KDa (FD4) translocation and/or transepithelial resistance (TEER) was characterized in Caco-2: HT29- methotrexate (MTX) cell line monolayers before and after an inflammatory challenge with TNF-α and IFN-γ. The six HMO blend (HMO6) dose-dependently limited the cytokine-induced FD4 translocation and TEER decrease and increased TEER values before challenge. Similarly, 3 and 5 HMO blends conferred a significant protection against the challenge, with 2′FL, one of the most abundant but most variable oligosaccharides in HM, being a key contributor. Overall, our results suggest differential ability of specific HMOs in modulating the intestinal barrier and support the potential of supplementation with combinations of available HMOs to promote gut health and protect against intestinal inflammatory disorders.

Human milk oligosaccharides (HMOs) represent the thirdmost abundantfraction of biomolecules in human milk (HM) and play a crucial rolein infant health and development. The unique contributions of HMOsto healthy development of breast-fed infants are assumed to rely onthe extraordinary complexity and diversity of HMO isomeric structures,which in turn still cause a huge analytical challenge. Many contemporaryanalytical methods aiming for more detailed HMO characterization combineion mobility (IM) with LC-MS for enhanced structural resolution butare typically lacking the robustness necessary for application toHM cohorts with hundreds of samples. To overcome these challenges,we introduce a novel, robust all-ion fragmentation (AIF) LC-ESI-IM-MSmethod integrating four analytical dimensions: high-resolution LCseparation, IM drift time, accurate mass precursor, and fragment ionmeasurements. This four-dimensional (4D) analytical characterizationis sufficient for resolving various HMO structural isomers in an efficientway. Thereby, up to 200 HMO compounds with a maximum degree of polymerizationof 13 could be simultaneously identified and relatively quantified.We devised two methods using this 4D analytical approach. One intendedfor in-depth characterization of multiple known but also novel HMOstructures and the second is designed for robust, increased-throughputanalyses. With the first approach, five trifucosyl-lacto-N-tetraose isomers (TF-LNTs), four of which were never detected beforein HM, as well as additional difucosyl-lacto-N-heaoseisomers (DF-LNHs), were revealed and structures fully elucidated byAIF and IM. This exemplifies the potential of our method for in-depthcharacterization of novel complex HMO structures. Furthermore, theincreased-throughput method featuring a shorter LC gradient was appliedto real-world HM samples. Here, we could differentiate the HM typesI–IV based on a broader range of partly new marker HMOs. Wecould also derive valuable new insights into variations of multipleand rare HMOs up to DP 11 across lactational stages. Overall, ourAIF LC-ESI-IM-MS approach facilitates in-depth monitoring and confidentidentification of a broad array of distinct and simple to very complexHMOs. We envision this robust AIF LC-ESI-IM-MS approach to advanceHMO research by facilitating the characterization of a broad rangeof HMOs in high numbers of HM samples. This may help to further extendour understanding about HMOs structure–function relationshipsrelevant for infants’ healthy development

Chapter 2 - Structures, Classification, and Biosynthesis of Human Milk Oligosaccharides

A mixture of three prebiotics does not affect vaccine specific antibody responses in healthy term infants in the first year of life

Human milk oligosaccharides (HMO) are one of the major components of human milk. HMO are non-digestible by the human gut, where they are known to play important functions as prebiotics and decoys for binding pathogens. Moreover, it has been proposed that HMO may provide sialic acids to the infant that are important in brain development, however this would require absorption of HMO into the bloodstream. HMO have consistently been found in the urine of humans and other mammals, suggesting systemic absorption. Here, we present a procedure for the profiling of milk oligosaccharides (MO) in plasma samples obtained from 13 term infants hospitalized for surgery for congenital heart disease. The method comprises protein denaturation, oligosaccharide reduction, and porous graphitized carbon solid phase extraction for purification followed by analysis using nHPLC-PGC-chip-TOF-MS. Approximately 15 free MO were typically observed in the plasma of human infants, including LNT, LDFP, LNFT, 3'SL, 6'SL, 3'SLN, and 6'SLN, of which the presence was confirmed using fragmentation studies. A novel third isomer of SLN, not found in human or bovine milk was also consistently detected. Differences in the free MO profiles were observed between infants that were totally formula-fed and infants that received at least some part breast milk. Our results indicate that free MO similar in structure to those found in human milk and urine are present in the blood of infants. The method and results presented here will facilitate further research toward the possible roles of free MO in the development of the infant.