Human milk oligosaccharides (HMOs) are a family of structurally diverse unconjugated glycans that are highly abundant in and unique to human milk. Originally, HMOs were discovered as a prebiotic "bifidus factor" that serves as a metabolic substrate for desired bacteria and shapes an intestinal microbiota composition with health benefits for the breast-fed neonate. Today, HMOs are known to be more than just "food for bugs". An accumulating body of evidence suggests that HMOs are antiadhesive antimicrobials that serve as soluble decoy receptors, prevent pathogen attachment to infant mucosal surfaces and lower the risk for viral, bacterial and protozoan parasite infections. In addition, HMOs may modulate epithelial and immune cell responses, reduce excessive mucosal leukocyte infiltration and activation, lower the risk for necrotizing enterocolitis and provide the infant with sialic acid as a potentially essential nutrient for brain development and cognition. Most data, however, stem from in vitro, ex vivo or animal studies and occasionally from association studies in mother-infant cohorts. Powered, randomized and controlled intervention studies will be needed to confirm relevance for human neonates. The first part of this review introduces the pioneers in HMO research, outlines HMO structural diversity and describes what is known about HMO biosynthesis in the mother's mammary gland and their metabolism in the breast-fed infant. The second part highlights the postulated beneficial effects of HMO for the breast-fed neonate, compares HMOs with oligosaccharides in the milk of other mammals and in infant formula and summarizes the current roadblocks and future opportunities for HMO research.

Human milk oligosaccharides: Every baby needs a sugar mama

Research on human milk oligosaccharides (HMOs) has received much attention in recent years. However, it started about a century ago with the observation that oligosaccharides might be growth factors for a so-called bifidus flora in breast-fed infants and extends to the recent finding of cell adhesion molecules in human milk. The latter are involved in inflammatory events recognizing carbohydrate sequences that also can be found in human milk. The similarities between epithelial cell surface carbohydrates and oligosaccharides in human milk strengthen the idea that specific interactions of those oligosaccharides with pathogenic microorganisms do occur preventing the attachment of microbes to epithelial cells. HMOs may act as soluble receptors for different pathogens, thus increasing the resistance of breast-fed infants. However, we need to know more about the metabolism of oligosaccharides in the gastrointestinal tract. How far are oligosaccharides degraded by intestinal enzymes and does oligosaccharide processing (e.g. degradation, synthesis, and elongation of core structures) occur in intestinal epithelial cells? Further research on HMOs is certainly needed to increase our knowledge of infant nutrition as it is affected by complex oligosaccharides.

Oligosaccharides in Human Milk: Structural, Functional, and Metabolic Aspects

The human gut microbiota is engaged in multiple interactions affecting host health during the host's entire life span. Microbes colonize the neonatal gut immediately following birth. The establishment and interactive development of this early gut microbiota are believed to be (at least partially) driven and modulated by specific compounds present in human milk. It has been shown that certain genomes of infant gut commensals, in particular those of bifidobacterial species, are genetically adapted to utilize specific glycans of this human secretory fluid, thus representing a very intriguing example of host-microbe coevolution, where both partners are believed to benefit. In recent years, various metagenomic studies have tried to dissect the composition and functionality of the infant gut microbiome and to explore the distribution across the different ecological niches of the infant gut biogeography of the corresponding microbial consortia, including those corresponding to bacteria and viruses, in healthy and ill subjects. Such analyses have linked certain features of the microbiota/microbiome, such as reduced diversity or aberrant composition, to intestinal illnesses in infants or disease states that are manifested at later stages of life, including asthma, inflammatory bowel disease, and metabolic disorders. Thus, a growing number of studies have reported on how the early human gut microbiota composition/development may affect risk factors related to adult health conditions. This concept has fueled the development of strategies to shape the infant microbiota composition based on various functional food products. In this review, we describe the infant microbiota, the mechanisms that drive its establishment and composition, and how microbial consortia may be molded by natural or artificial interventions. Finally, we discuss the relevance of key microbial players of the infant gut microbiota, in particular bifidobacteria, with respect to their role in health and disease.

The First Microbial Colonizers of the Human Gut: Composition, Activities, and Health Implications of the Infant Gut Microbiota

Most studies involving prebiotic oligosaccharides have been carried out using inulin and its fructo-oligosaccharide (FOS) derivatives, together with various forms of galacto-oligosaccharides (GOS). Although many intestinal bacteria are able to grow on these carbohydrates, most investigations have demonstrated that the growth of bifidobacteria, and to a lesser degree lactobacilli, is particularly favoured. Because of their safety, stability, organoleptic properties, resistance to digestion in the upper bowel and fermentability in the colon, as well as their abilities to promote the growth of beneficial bacteria in the gut, these prebiotics are being increasingly incorporated into the Western diet. Inulin-derived oligosaccharides and GOS are mildly laxative, but can result in flatulence and osmotic diarrhoea if taken in large amounts. However, their effects on large bowel habit are relatively minor. Although the literature dealing with the health significance of prebiotics is not as extensive as that concerning probiotics, considerable evidence has accrued showing that consumption of GOS and FOS can have significant health benefits, particularly in relation to their putative anti-cancer properties, influence on mineral absorption, lipid metabolism, and anti-inflammatory and other immune effects such as atopic disease. In many instances, prebiotics seem to be more effective when used as part of a synbiotic combination.

Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics

This review describes the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to carbohydrate analysis and covers the period 1991-1998. The technique is particularly valuable for carbohydrates because it enables underivatised, as well as derivatised compounds to be examined. The various MALDI matrices that have been used for carbohydrate analysis are described, and the use of derivatization for improving mass spectral detection limits is also discussed. Methods for sample preparation and for extracting carbohydrates from biological media prior to mass spectrometric analysis are compared with emphasis on highly sensitive mass spectrometric methods. Quantitative aspects of MALDI are covered with respect to the relationship between signal strength and both mass and compound structure. The value of mass measurements by MALDI to provide a carbohydrate composition is stressed, together with the ability of the technique to provide fragmentation spectra. The use of in-source and post-source decay and collision-induced fragmentation in this context is described with emphasis on ions that provide information on the linkage and branching patterns of carbohydrates. The use of MALDI mass spectrometry, linked with exoglycosidase sequencing, is described for N-linked glycans derived from glycoproteins, and methods for the analysis of O-linked glycans are also covered. The review ends with a description of various applications of the technique to carbohydrates found as constituents of glycoproteins, bacterial glycolipids, sphingolipids, and glycolipid anchors.

Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates.

Human milk oligosaccharides, representing the third largest fraction of human milk, have been assigned important protective functions for newborns acting as bifidogenic substrates or as inhibitory agents towards pathogens. Using high-pH anion-exchange chromatography and an enzyme test kit, twenty oligosaccharides and lactose were determined in milk samples of German women from days 3 to 90 postpartum. Twenty-two secretor mothers with Lewis blood group Le(a 2 b þ ) synthesised all twenty oligosaccharides, and could be assigned to milk group 1. Five non-secretor mothers (Le(a þ b 2 )) produced all oligosaccharides with the exception of a1,2-fucosylated compounds (milk group 2), whereas three secretor mothers with blood type Le(a 2 b 2 ) lacked a1,4-fucosyloligosaccharides, corresponding to milk group 3. Secretor women of milk groups 1 and 3 synthesised significantly higher amounts of total neutral oligosaccharides and of several total core structures (e.g. lacto-N-tetraose) than non-secretor women. Generally, these oligosaccharides significantly decrease during the first 3 months postpartum. By comparing fucosyloligosaccharides within and among the three milk groups, insight into their biosynthesis could be gained. Six acidic oligosaccharides without fucose residues were detected in milk samples of all mothers. Regression analysis confirmed that total acidic oligosaccharides declined threefold during the study period. Milk samples corresponding to the three milk groups exhibited significant qualitative and quantitative differences during the first 3 months of lactation. It can be assumed that particularly milk of non-secretor women (milk group 2) exerts a modified biological protection in the babies in comparison with milks of secretors (groups 1 and 3). Oligosaccharides: Lactation: Milk groups: Fucosyltransferases: Sialyltransferases

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Variation of human milk oligosaccharides in relation to milk groups and lactational periods

Oligosaccharides from human milk as revealed by matrix-assisted laser desorption/ionization mass spectrometry.

Context Oligosaccharides 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. Objective The 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 sources A 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 selection The 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 extraction Oligosaccharide concentrations, both mean values and single values, reported in the literature were sorted by gestational age, secretor status of mothers, and defined lactation periods. Results Mean 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. Conclusions Worldwide 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.

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Systematic review of the concentrations of oligosaccharides in human milk.

Neutral oligosaccharides in human milk samples from approximately 50 women were analysed applying a recently developed high-pH anion-exchange chromatographic method. Three different oligosaccharide patterns could be detected in accordance with milk groups that had been already described. These oligosaccharide groups correspond to the Lewis blood types Le(a−b+), Le(a+b−) and Le(a−b−). In addition to these oligosaccharide patterns, a new carbohydrate pattern was detected in a milk sample from a Le(a−b−) individual. Here, only nonfucosylated oligosaccharides and compounds bearing a1,3 linked fucosyl residues were found, whereas structures with a1,2 and a1,4 fucosyl linkages were missing. This finding led to the hypothesis that there are four different oligosaccharide milk groups that fit well to the genetic basis of the Lewis blood group system.

Stephan Thurl

Papers

Variation of human milk oligosaccharides in relation to milk groups and lactational periods

Oligosaccharides from human milk as revealed by matrix-assisted laser desorption/ionization mass spectrometry.

Systematic review of the concentrations of oligosaccharides in human milk.

Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides

Quantification of Individual Oligosaccharide Compounds from Human Milk Using High-pH Anion-Exchange Chromatography