Background Necrotising enterocolitis (NEC) is one of the most common and fatal intestinal disorders in preterm infants. Breast-fed infants are at lower risk for NEC than formula-fed infants, but the protective components in human milk have not been identified. In contrast to formula, human milk contains high amounts of complex glycans. Objective To test the hypothesis that human milk oligosaccharides (HMO) contribute to the protection from NEC. Methods Since human intervention studies are unfeasible due to limited availability of HMO, a neonatal rat NEC model was used. Pups were orally gavaged with formula without and with HMO and exposed to hypoxia episodes. Ileum sections were scored blindly for signs of NEC. Two-dimensional chromatography was used to determine the most effective HMO, and sequential exoglycosidase digestions and linkage analysis was used to determine its structure. Results Compared to formula alone, pooled HMO significantly improved 96-hour survival from 73.1% to 95.0% and reduced pathology scores from 1.98±1.11 to 0.44±0.30 (p<0.001). Within the pooled HMO, a specific isomer of disialyllacto-N-tetraose (DSLNT) was identified to be protective. Galacto-oligosaccharides, currently added to formula to mimic some of the effects of HMO, had no effect. Conclusion HMO reduce NEC in neonatal rats and the effects are highly structure specific. If these results translate to NEC in humans, DSLNT could be used to prevent or treat NEC in formula-fed infants, and its concentration in the mother’s milk could serve as a biomarker to identify breast-fed infants at risk of developing this disorder.

Higher concentrations of non-3'-SL HMOs were associated with protection against postnatal HIV transmission independent of other known risk factors. Further study of these novel, potentially anti-HIV components of breast milk is warranted.

f Human milk oligosaccharides (HMO), which constitute a major component of human milk, promote the growth of particular bacterial species in the infant's gastrointestinal tract. We hypothesized that HMO also interact with the bacterial communities present in human milk. To test this hypothesis, two experiments were conducted. First, milk samples were collected from healthy women (n ‫؍‬ 16); culture-independent analysis of the bacterial communities was performed, HMO content was analyzed, and the relation between these factors was investigated. A positive correlation was observed between the relative abundance of Staphylococcus and total HMO content (r ‫؍‬ 0.66). In a follow-up study, we conducted a series of in vitro growth curve experiments utilizing Staphylococcus aureus or Staphylococcus epidermidis and HMO isolated from human milk. HMO exhibited stimulatory effects on bacterial growth under various nutritional conditions. Analysis of culture supernatants from these experiments revealed that HMO did not measurably disappear from the culture medium, indicating that the growth-enhancing effects were not a result of bacterial metabolism of the HMO. Instead, stimulation of growth caused greater utilization of amino acids in minimal medium. Collectively, the data provide evidence that HMO may promote the growth of Staphylococcus species in the lactating mammary gland. Human milk oligosaccharides (HMO) are complex glycans highly abundant (ϳ5 to 15 g/liter) in human milk (5,6,24). Representing a diverse collection of structures, almost 150 distinct forms of HMO have been identified (42,43). Based upon their structures, HMO can be classified as simple (sialylated or fucosylated lactose compounds made of three sugar units) or complex (composed of differentially sialylated or fucosylated repeats of lacto-N-biose or N-acetyllactosamine linked to a lactose motif). Interestingly, HMO patterns are highly unique to individual women, and their concentrations vary over the course of lactation, with levels peaking in colostrum and lower concentrations in mature milk (8,12,15,39).From an evolutionary standpoint, it is logical that because the mammary gland exerts a substantial amount of energy to produce these compounds, HMO likely promote the fitness of the offspring even though the infant is largely unable to metabolize them (1, 14, 16). Research from several groups suggests the possibility that HMO are capable of interacting with an infant's gastrointestinal microbiota to promote his or her health (10,27,30,35,38). Indeed, some of the first studies in this area noted the capacity of HMO to promote growth of Bifidobacterium bifidum, a species overrepresented in the gastrointestinal tract of the breast-fed, but not formula-fed, infant (17,29,40). Over 50 years later, research has confirmed that the genetic capacity to metabolize these compounds is conserved among bifidobacteria species common in the infant's gastrointestinal tract (38). These findings suggest that HMO may serve a prebiotic function in selectively promoting the...

The oligosaccharide composition of breast milk may explain some of the benefits of breastfeeding in HEU children. HIV infection may modulate some of the consequences of HMOs on child survival.

No abstract

Human Milk Oligosaccharides (HMO) are a structurally divers group of complex glycans that are highly abundant in human milk but not in infant formula. The structural diversity of HMO, however, makes it difficult to study the molecular mechanisms underlying their potential benefits for the breast‐fed infant. More than 150 different HMO have been described ranging in size from low molecular weight trisaccharides to high molecular weight oligosaccharides with more than 30 monosaccharide monomers and ranging from being neutral to carrying several negative charges. Not all HMO may trigger the same effects. Here, we describe the isolation and purification of HMO subfractions and even individual HMO by two‐dimensional chromatography. In the first dimension we used anion‐exchange chromatography (QAE) to separate pooled HMO in a neutral and 5 negatively charged fractions containing 0 to 5 sialic acids per HMO molecule, respectively. In the second dimension we used automated size exclusion chromatography to separate HMO based on their molecular weight. Structural composition of the generated HMO fractions was analyzed by HPLC and mass spectrometry. Our approach generated structurally defined HMO subfractions and even individual HMO in quantities sufficient for in vitro and in vivo experiments aimed to elucidate HMO structure‐function relationships. (Supported by NIH K99/R00DK078668 to LB)

Human Milk Oligosaccharides (HMO) are complex glycans that are highly abundant in human milk but not in infant formula and are thought to benefit the breast‐fed infant. Here, we describe a fast, accurate and sensitive method to profile HMO from as little as 1uL human milk. First, milk samples were spiked with the non‐HMO trisaccharide raffinose for internal quantification. Then, samples were delipidated and deproteinated using centrifugation and chloroform/methanol extraction. Lactose, compromising more than 90% of all milk glycans but not being considered an HMO, was removed by incubating the samples with lactase, which was immobilized on magnetic beads. Samples were cleaned up with C18 and Carbograph microcolumns. Oligosaccharides were fluorescently tagged with 2‐aminobenzamide and analyzed by HPLC on an amide column with an ammonium formate/acetonitrile gradient and fluorescence detection (360/425nm). HPLC peaks were annotated according to standard retention times as well as online coupled mass spectrometry. Response factors were determined for all major HMO. The detection limit was less than 0.1 pmol with a linear response up to 400 pmol. Triplicate runs of the same milk samples yielded a relative standard deviation of the mean of less than 2% for total HMO and individual HMO. The described method will allow investigating genetic and environmental influences on HMO biosynthesis.

Human Milk Oligosaccharides (HMO), complex glycans highly abundant in human milk, are thought to protect the breast‐fed infant from infectious diseases and diarrhea. For example, the concentration of alpha1‐2‐fucosylated HMO in human milk is inversely correlated to the incidence of infant diarrhea. Diarrhea poses a particular threat to infants in developing countries, where mothers are also often malnourished. Here, we studied whether a low body mass index (BMI) in mothers had any effect on the HMO concentration in their milk. As part of the MINIMat project, milk samples from mothers in rural Bangladesh with a BMI of either 14–18 (n=8) or 24–28 (n=8) were collected at 8 weeks post‐partum. All milk samples were spiked with raffinose as internal standard. Oligosaccharides were isolated as described before, tagged with 2‐aminobenzamide, and analyzed by HPLC with fluorescence detection. Milk from mothers with a BMI of 14–18 contained 42.4% less total HMO than milk from mothers with a BMI of 24–28 (P<0.001), which may put the infant at higher risk of developing infectious diseases and diarrhea. Most intriguingly, 50% of all Bangladeshi samples were from Secretor‐negative mothers (lacking alpha1–2‐fucosylated HMO), which exceeds the 23% average reported for American/European mothers. This study is the first to show a correlation between the mother's nutritional status and the oligosaccharide concentration in her milk.