Exploring galectin interactions with human milk oligosaccharides and blood group antigens identifies BGA6 as a functional galectin-4 ligand.

Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as ß-galactoside binding lectins. However, certain members of this family have shown selective affinity toward specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7, and -12) toward a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I versus type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity toward poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance experiments. Particularly, we identified oligosaccharide blood group A antigen tetraose 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited interleukin-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses.

Dietary human milk oligosaccharides reduce allergic airway inflammation by modulating SCFAs level and ILC2 activity.

Group 2 innate lymphoid cells (ILC2s) play a crucial role in the progression of asthma, yet the regulatory mechanisms modulating ILC2 responses in asthma remain underexplored. Human milk oligosaccharides (HMOs), vital non-nutritive components of breast milk, are known to significantly shape immune system development and influence the incidence of allergic diseases. However, their impact on ILC2-driven asthma is not fully understood. Our research reveals that dietary HMOs act as potent inhibitors of ILC2 responses and allergic airway inflammation. Treatment with 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) significantly reduced ILC2-related airway inflammation induced by papain or Alternaria alternata in mice, evidenced by decreased eosinophil (EOS) infiltration and lower IL-5 and IL-13 levels in BALF. Notably, while ILC2 expresses HMO receptors, HMO did not act directly on ILC2 but potentially modulated their activity through alterations in gut microbiota derived SCFAs. HMO treatments alleviated airway inflammation in SCFA-dependent manners, with SCFA depletion or receptor blocking reversing these beneficial effects. This study reveals the potential of dietary HMOs in managing asthma through modulation of ILC2 activity and the gut-lung axis, proposing a new therapeutic avenue that utilises the immunomodulatory capacities of nutritional components to combat respiratory diseases.

Selective human milk oligosaccharide utilization by members of the Bifidobacterium pseudocatenulatum taxon.

Human milk oligosaccharides (HMOs) are essentially unaffected by the digestive enzymes of the nursling and are known for their ability to enrich certain microbial species in the infant gut microbiota, in particular bifidobacteria. HMO metabolism has been studied in various bifidobacterial species such as B. breve, B. bifidum, and B. longum subsp. infantis. In the current study, we describe differential growth abilities elicited by twenty-three newly isolated Bifidobacterium pseudocatenulatum strains on particular HMOs, such as 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). Through gene-trait matching and comparative genome analysis, we identified genes involved in the degradation of fucosylated HMOs in this strain set, while we employed a transcriptomic approach to facilitate the identification and characterization of genes and associated enzymes involved in LNT metabolism by strain B. pseudocatenulatum MM0196. A total of 252 publicly available genomes of the B. pseudocatenulatum taxon were screened for homologs of the glycosyl hydrolases (GHs) identified here as being required for selected HMO metabolism. From this analysis, it is clear that all members of this species possess homologs of the genes involved in LNT degradation, while genes required for degradation of fucosylated HMOs are variably present.IMPORTANCEOur findings allow a better understanding of the complex interaction between Bifidobacterium and its host and provide a roadmap toward future applications of B. pseudocatenulatum as a probiotic with a focus on infant health. Furthermore, our investigations have generated information on the role of HMOs in shaping the infant gut microbiota, thus also facilitating applications of HMOs in infant nutrition, with potential extension into the mature or adult gut microbiota. Supplementation of HMOs is known to result in the modulation of bacterial communities toward a higher relative abundance of bifidobacteria, which in turn enforces their ability to modulate particular immune functions and strengthen the intestinal barrier. This work may therefore inspire future studies to improve the formulation of neonatal nutritional products, aimed at facilitating the development of a healthy digestive and immune system and reducing the differences in gut microbiota composition observed between breastfed and formula-fed babies or full-term and preterm infants.

Infant Microbiota Communities and Human Milk Oligosaccharide Supplementation Independently and Synergistically Shape Metabolite Production and Immune Responses in Healthy Mice.

BACKGROUND: Multiple studies have demonstrated associations between the early-life gut microbiome and incidence of inflammatory and autoimmune disease in childhood. Although microbial colonization is necessary for proper immune education, it is not well understood at a mechanistic level how specific communities of bacteria promote immune maturation or drive immune dysfunction in infancy. OBJECTIVES: In this study, we aimed to assess whether infant microbial communities with different overall structures differentially influence immune and gastrointestinal development in healthy mice. METHODS: Germ-free mice were inoculated with fecal slurries from Bifidobacterium longum subspecies infantis positive (BIP) or B. longum subspecies infantis negative (BIN) breastfed infants; half of the mice in each group were also supplemented with a pool of human milk oligosaccharides (HMOs) for 14 d. Cecal microbiome composition and metabolite production, systemic and mucosal immune outcomes, and intestinal morphology were assessed at the end of the study. RESULTS: The results showed that inoculation with a BIP microbiome results in a remarkably distinct microbial community characterized by higher relative abundances of cecal Clostridium senu stricto, Ruminococcus gnavus, Cellulosilyticum sp., and Erysipelatoclostridium sp. The BIP microbiome produced 2-fold higher concentrations of cecal butyrate, promoted branched short-chain fatty acid (SCFA) production, and further modulated serotonin, kynurenine, and indole metabolism relative to BIN mice. Further, the BIP microbiome increased the proportions of innate and adaptive immune cells in spleen, while HMO supplementation increased proliferation of mesenteric lymph node cells to phorbol myristate acetate and lipopolysaccharide and increased serum IgA and IgG concentrations. CONCLUSIONS: Different microbiome compositions and HMO supplementation can modulate SCFA and tryptophan metabolism and innate and adaptive immunity in young, healthy mice, with potentially important implications for early childhood health.

Analysis of human milk oligosaccharides from women with gestational diabetes mellitus.

Human milk oligosaccharides (HMOs) are bioactive components which play an important role in infant health. HMO composition is vulnerable to changes of maternal conditions including lactation stages and maternal phenotypes. Pregnant diseases such as gestational diabetes mellitus (GDM) are commonly found in women during pragnancy, and may cause disorder in maternal physiological metabolism which is harmful to infants. Unfortunately, anlysis of oligosaccharides from women with GDM is limited. To address this issue, we analyzed HMO compositions and profiles in breast milk from women with GDM using an established 96-well plate permethylation platform and MALDI-TOF-MS. We enrolled 127 women with GDM, and investigated HMO abundances in colostrum, transition milk, and mature milk respectively. We found that GDM affected HMO compositions in breast milk, and the level of fucosylation became higher over the course of lactation for all the women with GDM. Interestingly, the relative abundances of fucosylated HMOs in different lactation stages were affected differentially by GDM, with the most pronounced effect in colostrum. In particular, the relative abundances of H3N1F1 and H3N1F2 sharply decreased over time, showing very low levels in late lactation. These differences in our findings need further investigation to develop optimal feeding for mothers with GDM.

Human milk oligosaccharides and the association with microbiota in colostrum: a pilot study.

HMOs (Human milk oligosaccharide) has an impact on maternal and infant health. Colostrum samples of 70 breastfeeding women in China were collected and recorded clinical characteristics. The major oligosaccharides and microbiota were quantitated in colostrum. The concentration of fucosylated HMOs in primipara was higher than that of multipara (p = 0.030). The concentration of N-acetylated HMOs in vaginal delivery milk was less than that of cesarean (p = 0.038). Non-fucosylated HMOs of breastfeeding women were less than that of breast pump (p = 0.038). Meanwhile, the concentration of LNT was positively correlated with Lactobacillus (r = 0.250, p = 0.037). DS-LNT was negatively correlated with Staphylococcus (r = - 0.240, p = 0.045). There was a positive correlation of Streptococcus with LNFP II (r = 0.314, p = 0.011) and 3-SL (r = 0.322, p = 0.009). In addition, there was a negative correlation between 2'-FL and 3-FL (r = - 0.465, p = 0.001). There was a positive correlation between LNT and LNnT (r = 0.778, p = 0.001). Therefore, the concentration of HMOs is related to number of deliveries, delivery mode, lactation mode and perinatal antibiotic. The concentration of HMOs is related to Lactobacillus, Streptococcus and Streptococcus in colostrum. In addition, there are connections between different oligosaccharides in content. The study protocol was also registered in the ClinicalTrails.gov (ChiCTR2200064454) (Oct. 2022).

Sialic acid in human milk and infant formulas in China: concentration, distribution and type.

This study compared the concentrations, types and distributions of sialic acid (SA) in human milk at different stages of the postnatal period with those in a range of infant formulas. Breast milk from mothers of healthy, full-term and exclusively breastfed infants was collected on the 2nd (n 246), 7th (n 135), 30th (n 85) and 90th (n 48) day after birth. The SA profiles of human milk, including their distribution, were analysed and compared with twenty-four different infant formulas. Outcome of this observational study was the result of natural exposure. Only SA of type Neu5Ac was detected in human milk. Total SA concentrations were highest in colostrum and reduced significantly over the next 3 months. Approximately 68·7­76·1 % of all SA in human milk were bound to oligosaccharides. Two types of SA, Neu5Ac and Neu5Gc, have been detected in infant formulas. Most SA was present in infant formulas combined with protein. Breastfed infants could receive more SA than formula-fed infants with the same energy intake. Overall, human milk is a preferable source of SA than infant formulas in terms of total SA content, dynamics, distribution and type. These SA profiles in the natural state are worth to be considered by the production of formulas because they may have a great effect on infant nutrition and development.

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

Combining 2'-fucosyllactose and galactooligosaccharides exerts anti-inflammatory effects and promotes gut health.

This study investigated the potential of 2'-Fucosyllactose (2'-FL) and galactooligosaccharides (GOS) combinations as a novel and cost-effective substitute for human milk oligosaccharides (HMOs) in promoting gut health and reducing inflammation. In vitro studies using Caco-2 cells showed that 2'-FL and GOS combinations (H1: GOS:2'-FL ratio of 1.8:1; H2: ratio of 3.6:1) reduced lipopolysaccharide-induced inflammation by decreasing pro-inflammatory markers, while individual treatments had no significant effects. In a mouse model of dextran sulfate sodium (DSS)-induced colitis, combined 2'-FL and GOS supplementation alleviated symptoms, improved gut permeability, and enhanced intestinal structure, with the GH1 group (H1 combo with DSS) being the most effective. 2'-FL and GOS combinations also enhanced short-chain fatty acid production in infant fecal batch fermentation and mouse fecal analysis, with GH1 showing the most promising results. GH1 supplementation altered gut microbiota in mice with DSS-induced colitis, promoting microbial diversity and a more balanced Firmicutes to Bacteroidota ratio. Infant formula products (IFPs) containing 2'-FL and GOS combinations (IFP2: 174 mg GOS and 95 mg 2'-FL per 14 g serving, 1.8:1 ratio; IFP3: 174 mg GOS and 48 mg 2'-FL per 14 g serving, 3.6:1 ratio) demonstrated gastrointestinal protective and anti-inflammatory properties in a coculture model of Caco-2 and THP-1 cells. These findings suggest that 2'-FL and GOS combinations have potential applications in advanced infant formulas and supplements to promote gut health and reduce inflammation.

Ameliorating effect of 2'-fucosyllactose and 6'-sialyllactose on lipopolysaccharide-induced intestinal inflammation.

Human milk oligosaccharides (HMO) affect gut microbiota during neonatal development, particularly with respect to the immune system. Bovine milk-based infant formulas have low oligosaccharide contents. Thus, efforts to fortify infant formulas with HMO are being undertaken. Two major HMO, 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL), exert anti-inflammatory effects; however, the associations between anti-inflammatory effects induced by 2'-FL and 6'-SL cotreatment and gut microbiota composition and metabolite modulation remain unclear. Therefore, in this study, we evaluated the effects of a mixture of these HMO. To determine the optimal HMO ratio for anti-inflammatory effects and elucidate its mode of action, LPS-induced inflammatory HT-29 epithelial cells and intestinal-inflamed suckling mice were treated with various mixtures of 2'-FL and 6'-SL. A 2'-FL:6'-SL ratio of 5:1 was identified as the most effective pretreatment HMO mixture in vitro; thus, this ratio was selected and used for low-, middle-, and high-dose treatments for subsequent in vivo studies. In vivo, high-dose HMO treatment restored LPS-induced inflammation symptoms, such as BW loss, colon length reduction, histological structural damage, and intestinal gene expression related to inflammatory responses. High-dose HMO was the only treatment that modulated the major phyla Bacteroidetes and Firmicutes and the genera Ihubacter, Mageeibacillus, and Saccharofermentans. These changes in microbial composition were correlated with intestinal inflammation-related gene expression and short-chain fatty acid production. To our knowledge, our study is the first to report the effects of Ihubacter, Mageeibacillus, and Saccharofermentans on short-chain fatty acid levels, which can subsequently affect inflammatory cytokine and tight junction protein levels. Conclusively, the HMO mixture exerted anti-inflammatory effects through changes in microbiota and metabolite production. These findings suggest that supplementation of infant formula with HMO may benefit formula-fed infants by forming unique microbiota contributing to neonatal development.

Biochemical characterization of a novel C-terminally truncated ß-galactosidase from Paenibacillus antarcticus with high transglycosylation activity.

The transgalactosylase activity of ß-galactosidases offers a convenient and promising strategy for conversion of lactose into high-value oligosaccharides, such as galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs). In this study, we cloned and biochemically characterized a novel C-terminally truncated ß-galactosidase (PaBgal2A-D) from Paenibacillus antarcticus with high transglycosylation activity. PaBgal2A-D is a member of glycoside hydrolase (GH) family 2. The optimal pH and temperature of PaBgal2A-D were determined to be pH 6.5 and 50°C, respectively. It was relatively stable within pH 5.0-8.0 and up to 50°C. PaBgal2A-D showed high transglycosylation activity for GOS synthesis, and the maximum yield of 50.8% (wt/wt) was obtained in 2 h. Moreover, PaBgal2A-D could synthesize lacto-N-neotetraose (LNnT) using lactose and lacto-N-triose II (LNT2), with a conversion rate of 16.4%. This study demonstrated that PaBgal2A-D could be a promising tool to prepare GOS and LNnT.

Preclinical Safety Evaluation of the Human-Identical Milk Oligosaccharide Lacto-N-Triose II.

Lacto-N-triose II (LNT II), an essential human milk oligosaccharide and precursor to lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), was evaluated for safety. Genotoxicity was assessed through in vitro tests including Bacterial Reverse Mutation Test and mammalian cell micronucleus test, and a subchronic oral gavage toxicity study was conducted on juvenile Sprague-Dawley rats. In this study, LNT II was administered at dose levels of 0, 1,500, 2,500, or 5,000 mg/kg body weight (bw)/day for 90 days, followed by a 4-week treatment-free recovery period. LNT II was non-genotoxic in the in vitro assays. No compound-related effects were observed across all dosage levels based on various measures, including clinical observations, body weight gain, feed consumption, clinical pathology, organ weights, and histopathology. Consequently, the highest dosage of 5,000 mg/kg bw/day was established as the no-observed-adverse-effect-level (NOAEL). These results suggest the safe use of LNT II in young children formula and as a food ingredient, within the limits found naturally in human breast milk.

Human milk oligosaccharides promote intestinal epithelium regeneration independent of the microbiota during necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) is a severe intestinal disease primarily affecting premature infants, marked by impaired epithelial regeneration. Breastfed infants are less susceptible to NEC than formula-fed ones, and human milk oligosaccharides (HMO) found in breast milk have prebiotic properties that can protect against NEC. However, it is unclear how HMOs influence intestinal epithelium regeneration in relation to the gut microbiota. METHODS: Broad-spectrum antibiotics were administered to pregnant dams to reduce the microbiota in offspring. NEC was induced through administration of hyperosmolar formula, lipopolysaccharide, and hypoxia from postnatal days (p) 5-9. Intestinal epithelial organoids were derived from p9 mice. HMOs were isolated from human donor breast milk and then solubilized in the formula for each feed or culture media for organoids. RESULTS: HMOs did not alter the microbiota profile in the presence of a normal or reduced microbiota. In the reduced microbiota, HMO treatment decreased NEC intestinal injury, and increased proliferation and stem cell activity. Additionally, in the complete absence of the microbiota, HMOs stimulated intestinal organoid growth. CONCLUSION: This study demonstrates that HMOs promoted intestinal epithelial regeneration independent of the gut microbiota. These findings provide further insight into the various benefits HMOs may have in the protection against NEC.

α-L-Fucosidases from an Alpaca Faeces Metagenome: Characterisation of Hydrolytic and Transfucosylation Potential.

In various life forms, fucose-containing glycans play vital roles in immune recognition, developmental processes, plant immunity, and host-microbe interactions. Together with glucose, galactose, N-acetylglucosamine, and sialic acid, fucose is a significant component of human milk oligosaccharides (HMOs). Fucosylated HMOs benefit infants by acting as prebiotics, preventing pathogen attachment, and potentially protecting against infections, including HIV. Although the need for fucosylated derivatives is clear, their availability is limited. Therefore, synthesis methods for various fucosylated oligosaccharides are explored, employing enzymatic approaches and α-L-fucosidases. This work aimed to characterise α-L-fucosidases identified in an alpaca faeces metagenome. Based on bioinformatic analyses, they were confirmed as members of the GH29A subfamily. The recombinant α-L-fucosidases were expressed in Escherichia coli and showed hydrolytic activity towards p-nitrophenyl-α-L-fucopyranoside and 2'-fucosyllactose. Furthermore, the enzymes' biochemical properties and kinetic characteristics were also determined. All four α-L-fucosidases could catalyse transfucosylation using a broad diversity of fucosyl acceptor substrates, including lactose, maltotriose, L-serine, and L-threonine. The results contribute insights into the potential use of α-L-fucosidases for synthesising fucosylated amino acids.

Gut microbiota: A key role for human milk oligosaccharides in regulating host health early in life.

Human milk oligosaccharides (HMOs) are an evolutionarily significant advantage bestowed by mothers for facilitating the development of the infant's gut microbiota. They can avoid absorption in the stomach and small intestine, reaching the colon successfully, where they engage in close interactions with gut microbes. This process also enables HMOs to exert additional prebiotic effects, including regulating the mucus layer, promoting physical growth and brain development, as well as preventing and mitigating conditions such as NEC, allergies, and diarrhea. Here, we comprehensively review the primary ways by which gut microbiota, including Bifidobacteria and other genera, utilize HMOs, and we classify them into five central pathways. Furthermore, we emphasize the metabolic benefits of bacteria consuming HMOs, particularly the recently identified intrinsic link between HMOs and the metabolic conversion of tryptophan to indole and its derivatives. We also examine the extensive probiotic roles of HMOs and their recent research advancements, specifically concentrating on the unsummarized role of HMOs in regulating the mucus layer, where their interaction with the gut microbiota becomes crucial. Additionally, we delve into the principal tools used for functional mining of new HMOs. In conclusion, our study presents a thorough analysis of the interaction mechanism between HMOs and gut microbiota, emphasizing the cooperative utilization of HMOs by gut microbiota, and provides an overview of the subsequent probiotic effects of this interaction. This review provides new insights into the interaction of HMOs with the gut microbiota, which will inform the mechanisms by which HMOs function.

Enzymatic Synthesis of Disialyllacto-N-Tetraose (DSLNT) and Related Human Milk Oligosaccharides Reveals Broad Siglec Recognition of the Atypical Neu5Acα2-6GlcNAc Motif.

Sialic acids (Sias) are ubiquitously expressed on all types of glycans, typically as terminating residues. They usually link to galactose, N-acetylgalactosamine, or other Sia residues, forming ligands of many glycan-binding proteins. An atypical linkage to the C6 of N-acetylglucosamine (GlcNAc) has been identified in human milk oligosaccharides (HMOs, e.g., DSLNT) and tumor-associated glycoconjugates. Herein, describe the systematic synthesis of these HMOs in an enzymatic modular manner. The synthetic strategy relies on a novel activity of ST6GalNAc6 for efficient construction of the Neu5Acα2-6GlcNAc linkage, and another 12 specific enzyme modules for sequential HMO assembly. The structures enabled comprehensive exploration of their structure-function relationships using glycan microarrays, revealing broad yet distinct recognition by Siglecs of the atypical Neu5Acα2-6GlcNAc motif. The work provides tools and new insight for the functional study and potential applications of Siglecs and HMOs.

Human milk oligosaccharides reduce necrotizing enterocolitis-induced neuroinflammation and cognitive impairment in mice.

Necrotizing enterocolitis (NEC) is the leading cause of morbidity and mortality in premature infants. One of the most devastating complications of NEC is the development of NEC-induced brain injury, which manifests as impaired cognition that persists beyond infancy and which represents a proinflammatory activation of the gut-brain axis. Given that oral administration of the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'-FL) and 6'-sialyslactose (6'-SL) significantly reduced intestinal inflammation in mice, we hypothesized that oral administration of these HMOs would reduce NEC-induced brain injury and sought to determine the mechanisms involved. We now show that the administration of either 2'-FL or 6'-SL significantly attenuated NEC-induced brain injury, reversed myelin loss in the corpus callosum and midbrain of newborn mice, and prevented the impaired cognition observed in mice with NEC-induced brain injury. In seeking to define the mechanisms involved, 2'-FL or 6'-SL administration resulted in a restoration of the blood-brain barrier in newborn mice and also had a direct anti-inflammatory effect on the brain as revealed through the study of brain organoids. Metabolites of 2'-FL were detected in the infant mouse brain by nuclear magnetic resonance (NMR), whereas intact 2'-FL was not. Strikingly, the beneficial effects of 2'-FL or 6'-SL against NEC-induced brain injury required the release of the neurotrophic factor brain-derived neurotrophic factor (BDNF), as mice lacking BDNF were not protected by these HMOs from the development of NEC-induced brain injury. Taken in aggregate, these findings reveal that the HMOs 2'-FL and 6'-SL interrupt the gut-brain inflammatory axis and reduce the risk of NEC-induced brain injury.NEW & NOTEWORTHY This study reveals that the administration of human milk oligosaccharides, which are present in human breast milk, can interfere with the proinflammatory gut-brain axis and prevent neuroinflammation in the setting of necrotizing enterocolitis, a major intestinal disorder seen in premature infants.

Chemical Synthesis of Human Milk Oligosaccharides: para-Lacto-N-hexaose and para-Lacto-N-neohexaose.

Human milk oligosaccharides (HMO) have emerged as a very active area of research in glycoscience and nutrition. HMO are involved in the early development of infants and may help to prevent certain diseases. The development of chemical methods for obtaining individual HMO aids the global effort dedicated to understanding the roles of these biomolecules. Reported herein is the chemical synthesis of two common core hexasaccharides found in human milk, i. e. para-lacto-N-hexaose (pLNH) and para-lacto-N-neohexaose (pLNnH). After screening multiple leaving groups and temporary protecting group combinations, a 3+3 convergent coupling strategy was found to work best for obtaining these linear glycans.

Stability of Human-Milk Oligosaccharide Concentrations Over 1 Week of Lactation and Over 6 Hours Following a Standard Meal.

BACKGROUND: Our previous studies revealed that human-milk oligosaccharides (HMOs) have health benefits for nursing infants and their concentrations change dynamically over 24 mo of lactation. Yet, the extent to which HMOs vary over the short term (days) and in response to acute factors such as maternal diet is unclear. OBJECTIVE: The purpose of this study was to determine the stability of HMO concentrations over 7 d and in response to a standard meal and sugar-sweetened beverage (SSB) over 6 h. METHODS: In this ancillary study, lactating mothers were enrolled at 6 wk postpartum. Participants received in-person instructions and materials to complete procedures at home. In the 1-wk experiment (n = 11), mothers pumped a milk sample at 07:00 h for 7 consecutive days. In the 6-h experiment (n = 35), mothers pumped a milk sample after an overnight fast at 06:00 h and then consumed a standard meal plus SSB provided by the study team. Mothers pumped a milk sample every hour for 6 consecutive hours. Samples were analyzed for the 19 most abundant HMOs. Repeated-measures ANOVA was used to test changes in HMO concentrations over time, reported as F(dftime, dferror) = F value, P value. RESULTS: Concentrations of all assayed HMOs were stable over 7 consecutive days, including, for example, the most widely studied HMOs in relation to infant health: 2'-fucosyllactose (2'FL) [F(2,17) = 0.39, P = 0.65], disialyl-lacto-N-tetraose (DSLNT) [F(4, 37) = 0.60, P = 0.66], and lacto-N-neotetraose (LNnT) [F(3, 32) = 1.5, P = 0.23]. Concentrations of all assayed HMOs were stable in response to a standard meal plus SSB. For example, fasted baseline concentrations of 2'FL, DSLNT, and LNnT were 2310 ± 1620 µg/mL, 560 ± 290 µg/mL, and 630 ± 290 µg/mL, respectively, and there were no changes in 2'FL [F(4, 119) = 1.9, P = 0.13], DSLNT [F(4, 136) = 0.39, P = 0.83], and LNnT [F(4, 120) = 0.64, P = 0.63] over 6 consecutive hours. CONCLUSIONS: HMO concentrations are stable over 1 wk of lactation and are not acutely affected by a standard meal plus SSB in mothers.

Recent progress in health effects and biosynthesis of lacto-N-tetraose, the most dominant core structure of human milk oligosaccharide.

Human milk oligosaccharides (HMOs), which are a group of complex carbohydrates highly abundant in human milk, have been recognized as critical functional biomolecules for infant health. Lacto-N-tetraose (LNT) is one of the most abundant HMO members and the most dominant core structure of HMO. The promising physiological effects of LNT have been well documented, including prebiotic property, antiadhesive antimicrobial activity, and antiviral effect. Its safety has been evaluated and it has been commercially added to infant formula as a functional ingredient. Because of great commercial importance of LNT, increasing attention has been paid to its highly efficient biological production. In particular, microbial synthesis based on metabolic engineering displays obvious advantages in large-scale production of LNT. This review contains important information about the recent progress in physiological effects, safety evaluation, and biosynthesis of LNT.