Milk oligosaccharides: A review

doi: 10.1111/1471-0307.12209

REVIEW

Milk oligosaccharides: A review DIANA L OLIVEIRA,1,2 R ANDREW WILBEY,1 ALISTAIR S G R A N D I S O N 1 and L U
IS A B R O S E I R O 2 * 1

Department of Food and Nutritional Sciences, University of Reading, Whiteknights Reading RG6 6AP, UK, and Laborat orio Nacional de Energia e Geologia (LNEG), Unidade de Bioenergia, Edifıcio K2, Estrada do Pacßo do Lumiar 22, Lisboa 1649-036, Portugal 2

Milk oligosaccharides (OSs) confer unique health benefits to the neonate. Although human digestive enzymes cannot degrade these sugars, they support specific commensal microbes and act as decoys to prevent the adhesion of pathogenic micro-organisms to gastrointestinal cells. The limited availability of human milk oligosaccharides (HMOs) impedes research into these molecules and their potential applications in functional food formulations. Recent studies show that complex OSs with fucose and N-acetyl neuraminic acid (key structural elements of HMO bioactivity) also exist in caprine milk, suggesting a potential source of bioactive milk OSs suitable as a functional food ingredient. Keywords Carbohydrates, Prebiotics, Caprine milk.

INTRODUCTION

*Author for correspondence. E-mail: [email protected] © 2015 Society of Dairy Technology

The significant contribution of dairy components as physiologically functional foods was first recognised by the Japanese in the 1980s. At that time, functional food was defined as ‘any food ingredient that may provide a health benefit beyond the traditional nutrients that it contains’ (Macfarlane et al. 2006). During the last two decades, functional foods have received increasing attention (Bhat and Bhat 2010), particularly in respect of positive effects on host health and/ or well-being beyond their nutritional value (Bhat and Bhat 2011). Milk can be considered the model ‘nutraceutical’, that is food that conveys immunological and other health benefits together with the nutritional contribution. Significant protection of babies by feeding human milk has been demonstrated in relation to diarrhoeal diseases, respiratory tract infections, bacteraemia and meningitis (Morrow et al. 2005). For over 30 years, it has been accepted that breastfed infants are better protected against infectious agents than formula-fed infants, attributable to the evolution of milks to fit the needs of that species. Various factors present in milk are known to modulate the developing microbiota within the infant gastrointestinal tract (GIT), including Vol 68 International Journal of Dairy Technology

immunoglobulins, lactoferrin, lysozyme, bioactive lipids, leucocytes and various milk glycans (glycolipids, glycoproteins and free oligosaccharides) (Newburg et al. 2005). Of the functional ingredients, oligosaccharides (OSs) are arguably the most important, as they function as prebiotics. PREBIOTICS Prebiotics are defined as ‘a selectively fermented ingredient that allows specific changes, in both the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health’ (Gibson et al. 2004; Roberfroid 2007a). This definition requires that prebiotics are resistant to gastric acidity, hydrolysis by host enzymes and gastrointestinal absorption. Others define prebiotics as ‘a carbohydrate that results in changes in numbers of key bacterial genera in the colon, for example bifidobacteria’ (Palframan et al. 2003). The latter definition not only considers the microflora changes in the colonic ecosystem of humans but also in the whole GIT and, as such, extrapolates the definition into other areas that may benefit from a selective targeting of particular micro-organisms. Macfarlane et al. (2006) stated that prebiotics are short-chain carbohydrates that have unusual 1

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effects in the gut, altering the composition, or balance, of the microbiota. Prebiotics also act as carbon and energy sources for bacterial growth. Recognising these advantages and possible health benefits, prebiotics are now being used in many situations, mainly in infant feed. Douglas and Sanders (2008) defined prebiotics as ‘food ingredients that promote the growth or activity of a limited number of bacterial species for the benefit of host health; in general terms, prebiotics are food for bacterial species that are considered beneficial for health and well-being’. The target genera are lactobacilli and bifidobacteria. However, prebiotic success has predominantly been with the latter, probably because there are usually more bifidobacteria in the human colon than lactobacilli, and they exhibit a preference for OSs (Gibson et al. 2010). Reports from in vitro studies demonstrated that prebiotics can modify the gastrointestinal microflora by increasing bifidobacteria and/or lactobacilli numbers, thus improving the health of the human gut and enhancing nonspecific immune responses (Wang and Gibson 1993; Rhoades et al. 2008). Most prebiotics and prebiotic candidates identified today are nondigestible carbohydrates, mainly OSs, obtained either by extraction from plants or by synthesis from mono- and di-saccharides such as sucrose or lactose. Lactose is the major carbohydrate in milks and has many uses in foods and pharmaceuticals (Schaafsma 2008). Concentrations vary between species, for example 41 g/L in caprine milk, 49 g/ L in ovine milk, 47 g/L in bovine milk and 69 g/L in human milk (Park et al. 2007). Lactose is a disaccharide of galactose and glucose, usually present as a mixture of a- and b-forms. It is a valuable nutrient also because, apart from providing energy, it favours the intestinal absorption of calcium, magnesium and phosphorous, plus the utilisation of vitamin C (G€anzle et al. 2008). Asp (2004) stated that the main differences between carbohydrates are between those absorbed in the small intestine and others that provide a substrate for the microflora in the large intestine. This is the physiological basis for the dietary fibre concept, OSs being recognised as dietary fibres in most countries (Wang et al. 2009). Daddaoua et al. (2006) explained a way of manipulating the colon microflora by avoiding antibiotics and using probiotics (health-promoting bacteria) or prebiotics (products that favour the balance towards ‘anti-inflammatory’ microflora). OLIGOSACCHARIDES The International Union of Pure and Applied Chemistry (IUPAC) define OSs thus as follows: ‘Oligosaccharides are compounds containing three to nine monomeric sugar residues, and the number of these residues determines the degree of its polymerisation’. Fructo-oligosaccharides (FOSs) are widely found in many edible plants and fruits, while galacto-oligosaccharides (GOSs) are produced from 2

lactose by action of a galactosyl transferase and act as a prebiotic with a sweet taste and low energy value. Also, lactose and its derivatives appear to be valuable ingredients with a wide range of nutritional benefits, particularly in the fields of gut health promotion (G€anzle et al. 2008). HUMAN MILK OLIGOSACCHARIDES Human milk oligosaccharides (HMOs) are believed to stimulate the growth of bifidobacteria in the gastrointestinal tract (GIT) while protecting against enteric pathogens (Daddaoua et al. 2006). These OSs contain lactose at the reducing end and typically a fucose or a sialic acid at the nonreducing end, the 2’- fucosyllactose (2’- FL) and lacto-N-fucopentaose-I (LNF-I), both of which contain an a1,2-linked fucose, being the most common. HMOs are believed to have many roles in a developing infant, in addition to putative prebiotic functions, and may possess anti-adhesive effects that reduce the binding of pathogenic bacteria to colonocytes (Lane et al. 2010). They also have modulating effects on immunological processes at the level of gut-associated lymphoid tissue (Guarner 2009) plus may also decrease intestinal permeability in preterm infants in a dose-related manner in the first post-natal month (Taylor et al. 2009). The structure of HMOs, compromising more than 200 different molecular structures (Coppa et al. 2004; Bode 2006; Ninonuevo et al. 2006), differs significantly from plantderived FOSs or enzymically synthesised GOSs (Fanaro et al. 2005a). To date, the variable OS content of human milk cannot be successfully reproduced on a large scale for inclusion in infant formulae (Sarney et al. 2000; Manning and Gibson 2004; German et al. 2008; Guarner 2009; Taylor et al. 2009). Therefore, an HMO-like product would be valuable as a supplement for infant or even adult nutrition.

Origins of HMO Research Interest in HMO started with the observation that OSs might be growth factors for the Bifidobacterium bifidus flora in breastfed infants (Coppa et al. 2004, 2006). Lars Bode (2012) has recently published a review on HMO introducing the pioneers in HMO research. According to this author, the discovery of HMO was driven by scientists and physicians with two very different perspectives and interests. Paediatricians and microbiologists were trying to understand the observed health benefits associated with human milk feeding, while chemists were trying to characterise the carbohydrates uniquely found in human milk. Already at the end of the 19th century, when overall infant first-year mortality rates were as high as 30%, it was observed that breastfed infants had a much higher chance of survival and had lower incidences of infectious diarrhoea and many other diseases than ‘bottle-fed’ infants. At that time, Theodore Escherich, an Austrian paediatrician and microbiologist, had just discovered a relationship between intestinal bacteria and the © 2015 Society of Dairy Technology

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physiology of digestion in infants. Also, Ernst Moro, one of Escherich’s former students, and Henri Tissier, a graduate student in Paris, independently found differences in the bacterial composition in the faeces of breastfed compared to ‘bottle-fed’ infants. It remained unknown which components in human milk determined the bacterial composition in the infant’s intestine until more than half a century later. Eschbach had noted in 1888 that human milk contained ‘a different type of lactose’ than bovine milk. Shortly after that, it was found that lactose in human and bovine milk is the same, but that human milk contains an additional unknown carbohydrate fraction. It was more than 40 years later that Michel Polonowski and Albert Lespagnol were able to characterise this carbohydrate fraction and called it ‘gynolactose’. In 1926, Herbert Sch€ onfeld reported that the whey fraction of human milk contains a growth-promoting factor for Lactobacillus bifidus (later reclassified as Bifidobacterium bifidus) referred as the ‘bifidus factor’. A connection between these findings led later to the conclusion that the ‘bifidus factor’ indeed consisted of OSs (Bode 2012). Given the evolution of milk as a product of epithelial secretions nourishing mammalian offspring, the presence of nondigestible OSs would appear to be paradoxical. For decades, the question of why milk would contain indigestible material has challenged scientists studying milk. The presence and particularly the remarkable abundance of OSs in human milk (Table 1) have led investigators to propose biological, physiological and protective functions for these molecules (Coppa et al. 1993; Kunz et al. 2000; Bode 2006). Certainly, the number and structural diversity of these molecules would allow for more than one function. However, to date, the detailed structural basis of these functions is not fully understood. Recently, HMOs have been demonstrated to selectively nourish the growth of highly specific strains of bifidobacteria, thus establishing the means to guide the development of a unique gut microbiota in infants fed breast milk (Harmsen et al. 2000; Ward et al. 2006; Ninonuevo et al. 2007). Certain OSs derived from the mammalian epithelial cells of the mother also share common epitopes on the infant’s intestinal epithelia known to be receptors for pathogens. The presence of such structures in milk has been hypothesised to have evolved to provide a direct defensive strategy acting as decoys to prevent binding of pathogens to epithelial cells, thereby protecting infants from disease (Newburg et al. 2005).

HMO composition Consistent with the potential for multiple nutritional and biological functions, human milk is comprised of a complex mixture of OSs that differs in size, charge and abundance (Ninonuevo et al. 2005). HMOs are composed of both neutral and anionic structures with building blocks of five monosaccharides: D-glucose (Glc), D-galactose (Gal), N-acetylglucosamine (GlcNAc), L-fucose (Fuc) and N-acetylneuraminic © 2015 Society of Dairy Technology

Table 1 Average composition of basic nutrients in human, bovine, caprine and ovine milk (adapted from Daddaoua et al. 2006 and Park et al. 2007) Composition (g/L)

Human

Bovine

Caprine

Ovine

Protein Casein Albumin, globulin Fat Lactose Ash Solids nonfat Calories/100 mL Oligosaccharides

12.0 4.0 7.0 40 69 3.0 89 68 8

32 26 6.0 36 47 7.0 90 69 0.03–0.06

34 24 6.0 38 41 8.0 89 70 0.25–0.30

62 42 10 79 49 9.0 120 105 0.02–0.04

acid (NeuAc or Neu5Ac, also named as NANA or sialic acid). The HMOs are usually divided into two groups according to their chemical structures: • Neutral: contain no charged monosaccharide residues (Glc, Gal, GlcNAc) and often Fuc linked to a lactose (Galb1-4Glc) core and • Acidic: negatively charged and include the same sugars plus often the same core structures, and N-acetylneuraminic acid (NeuAc) (Mehra and Kelly 2006; Bao et al. 2007). The basic structure of HMOs includes a lactose (Lac) core at the reducing end and is elongated by NAc units, with greater structural diversity provided by extensive fucosylation and/or sialylation wherein Fuc and NeuAc residues are added at the terminal positions (Newburg et al. 2005). HMOs are characterised by an enormous structural diversity (Chaturvedi et al. 2001a). The ability to understand the diversity of biological functions of HMOs has been hindered by their complexity. About 200 molecular species have been identified in a pooled human milk sample (Ninonuevo et al. 2006). All HMOs contain lactose (Gal 1-4 Glc) at their reducing end, which can be elongated by the addition of 1-3- or 1-6-linked lacto-N-biose (Gal 1-3GlcNAc-, type 1 chain) or N-acetyllactosamine (Gal 1-4 GlcNAc-, type 2 chain). A linear chain is formed via a b1–3 linkage attached to the core structure of GlcNAc, whereas a branched chain results when two GlcNAc units are added on both the b1–3 and b1–6 positions (Wu et al. 2010). Branched structures are designed as iso-HMO and linear structures without branches as paraHMO. Some HMOs occur in several isomeric forms, for example lacto-N-fucopentaose (LNFP) or sialyl-lactoseN-tetraose (LST) (Bode 2012). The smallest oligosaccharides are generated either when Fuc is added to Lac, thus generating two forms of the trisaccharide, fucosyllactose (2-FL; Fuca1–2, Galb1–4Glc, and 3-FL; Gal b1–4[Fuc a1–3] Glc), or when NeuAc is added 3

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to lactose, generating the sialyllactoses (3-SL; NeuAca1– 3Gal b1–4Glc and 6-SL; NeuAca1–6Galb1–4Glc) (Espinosa et al. 2007). However, these small OSs are generally less abundant than the larger, more complicated structures. HMOs are especially rich in the type 1 oligosaccharides. Lacto-N-biose (LNB; Galb1–3GlcNAc) is a building unit of the three type 1 HMOs, such as lacto-N-tetraose (LNT; Galb1–3GlcNAcb1–3Galb1–4Glc), lacto-N-fucopentaose-I (LNFP; Fuca1–2Galb1–3GlcNAcb1–3Galb1–4Glc) and lacto-N-difucohexaose I (Fuca1–2Galb1–3[Fuca1–4] GlcNAcb1–3Galb1–4Glc)]. More detail on the structures and functions of selected HMOs is available in a review by Bode (2006).

Metabolism Once ingested, HMOs resist the low pH in the infant’s stomach as well as digestion by pancreatic and brush border enzymes based on data from in vitro degradation studies (Engfer et al. 2000; Gnoth et al. 2000). Data obtained in the 1980s and 90s showed that HMOs reached the distal small intestine and colon in an intact form and were excreted in the infant’s faeces (Sabharwal et al. 1984, 1988a,b, 1991; Chaturvedi et al. 2001b; Coppa et al. 2001). More recent studies with CE and laser-induced fluorescence detection and MS confirmed and refined these original observations and suggested multi-stage HMO processing and degradation that depended on infant age, blood group and feeding regime (Albrecht et al. 2010, 2011a,b). In the first stage between birth and about 2 months of life, faeces of breastfed infants contained sialylated and nonsialylated HMOs that are similar but not identical to those in the corresponding milk samples. In the subsequent second stage, the faeces contained mainly HMO degradation and processing products that were different to the HMOs in the corresponding milk samples. In the third stage, starting from when other than human milk was introduced, HMOs entirely disappeared from the infant’s faeces (Albrecht et al. 2011a). Rudloff et al. (1996) were the first to show that intact HMOs appeared in the urine of preterm breastfed infants, but not in formula-fed infants. These results suggested that HMOs are absorbed in the infant’s intestine and reach the systemic circulation. In the following years, the same group used a set of elegant and elaborate in vivo C-labelling studies to further investigate HMO metabolism (Rudloff et al. 1996, 2012; Obermeier et al. 1999). Lactating women received an oral bolus of C-labelled Gal, and their mammary glands incorporated the label during HMO synthesis. The breastfed infant then ingested the in vivo labelled HMOs with the mother’s milk, and approximately 1% appeared in the infant’s urine with the C-label still at the same position as in the orally administered Gal, indicating direct incorporation and minimal rearrangement. While nonsialylated HMOs cross monolayers of cultured intestinal epithelial cells by receptor-mediated transcytosis and paracytosis, sialylated HMOs use paracytosis only (Gnoth et al. 2001). It 4

remains unknown which receptors facilitate absorption and how rapidly HMOs are absorbed, appear in the circulation and are cleared from the system. Osborn and Khan (2000) described the complexity of these molecules and also their biological role on the human body, including cell growth, cell–cell adhesion and immune defence. HEALTH BENEFITS OF HMO Originally identified as the ‘bifidus factor’ in human milk, HMO had mostly been recognised for their ‘bifidogenic’ or prebiotic effects. However, recent evidence suggests that HMOs are more than just a substrate to promote the growth of desired bacteria in the infant’s intestine. Ignatova-Ivanova et al. (2010) concluded that the presence of GOSs contributes to higher growth rates and production of antimicrobial substances. Martin-Sosa et al. (2002) also suggested that HMOs reduced urinary tract infections, while Abrahams and Labbok (2011) reported that breastfed infants were less likely to develop otitis caused by Streptococcus pneumonia and also displayed a lower risk of developing respiratory syncytial virus (RSV), as human milk often covers the mucosal surfaces in the infant’s nasopharyngeal regions and occasionally reaches the upper respiratory tract during suckling.

Modulators of intestinal epithelial cell responses Human milk oligosaccharides may also directly modulate host intestinal epithelial cell responses (Bode 2012). Studies have confirmed that HMOs directly modulate intestinal epithelial cell responses, reduce cell growth and induce differentiation and apoptosis in cultured intestinal epithelial cells by altering growth-related cell cycle genes (Angeloni et al. 2005). These in vitro studies strongly suggest that HMOs can directly interact with the infant’s intestinal epithelial cells, affect gene expression and reprogramme the cell cycle as well as cell surface glycosylation. Prevention of pathogen adhesion Many viral, bacterial or protozoan pathogens first need to adhere to mucosal surfaces to colonise or invade the host to cause disease. Pathogen adhesion is often initiated by lectin–glycan interactions. HMOs also reduce microbial infections by acting as anti-adhesive antimicrobials (Knol et al. 2005; Newburg et al. 2005). Some HMOs resemble mucosal cell surface glycans, serving as soluble decoy receptors to prevent pathogen binding and thus reducing the risk of infections. HMOs serve as soluble ligand analogues and block pathogen adhesion (Newburg et al. 2005). The chemical structures of HMOs are homologous to the carbohydrate units of glycoconjugates, especially of glycolipids, on cell surfaces of mammalian epithelial cells. For example, binding of Escherichia coli, Streptococcus pneumonia, Campylobacter jejuni, Helicobacter pylori and © 2015 Society of Dairy Technology

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Vibrio cholerae was inhibited by the glycoconjugates present in HMOs. Fucosylated and acidic OSs from human milk, in particular a-1,2-linked fucose, were elucidated (Ruiz-Palacios et al. 2003; Newburg et al. 2004a,b) and demonstrated to inhibit adhesion of pathogens that cause gastrointestinal disorders, for example Campylobacter jejuni and Escherichia coli (Morrow et al. 2005; Newburg et al. 2005), thus protecting the infant against infections, including diarrhoea – one of the most common causes of infant mortality (Meyrand et al. 2013). Infants fed with human milk having low concentrations of 2-FL may be more susceptible to diarrhoea than babies fed the breast milk containing high concentrations of 2-FL.-fucosyllactosamine (Ruiz-Palacios et al. 2003; Morrow et al. 2004, 2005; Leach et al. 2005; Newburg et al. 2005; Bode 2009). It is also possible that HMOs can have glycomemodifying effects through changing of the expression of intestinal epithelial cell surface glycans. Angeloni et al. (2005) demonstrated that Caco-2 cells change their surface glycan profile after exposure to 3-SL, a constituent of HMOs. In that study, the expression of a2–3- and a2–6linked sialic acid residues in Caco-2 cells was significantly reduced. Thus, this particular HMO appears to modify the glycan content of the epithelial cell surface and the receptor sites for some pathogens. The same researchers further confirmed that the adhesion of enteropathogenic E. coli (EPEC) was reduced upon treatment with 3-SL.

Role of OSs in the development of the immune system While HMO-mediated changes in the infant’s microbiota or intestinal epithelial cell response may indirectly affect the infant’s immune system, results from in vitro studies suggest that HMOs also directly modulate immune responses. HMOs may either act locally on cells of the mucosa-associated lymphoid tissues or on a systemic level as approximately 1% of the HMOs are absorbed and reach the systemic circulation (Rudloff et al. 1996; Gnoth et al. 2001; Ruhal and Choudhury 2012). The end products of bacterial metabolism may possibly play a role in colon cancer prevention (Van Loo et al. 1999). Chichlowski et al. (2011) cited previous researchers such as Velupillai and Harn (1994), Eiwegger et al. (2004) and Newburg (2009), who demonstrated that HMOs directly affected the immune system. Many sialylated and fucosylated HMOs may have significant effects on the progression of inflammatory responses (Kunz et al. 1999). A more recent study has also shown that HMOs can inhibit transfer of HIV-1 virus to CD4+ lymphocytes (Hong et al. 2009). Eiwegger et al. (2010) demonstrated a novel, direct immuno-modulatory effect of acidic fraction of HMO when compared with a similar fraction from cow’s milk. In this study, acidic HMOs stimulated production of IFN-c and IL10, directing the neonatal Th-2-type T-cell phenotype © 2015 Society of Dairy Technology

towards a Th-0-type profile blood-derived mononuclear cell in the umbilical cord. This effect also reduced a Th-2-type immune response in allergen-specific T cells from peanutallergic individuals. Both results strongly suggest anti-allergic properties of certain acidic HMOs.

Nutrients for brain development Breastfed preterm infants have superior developmental scores at 18 months of age and higher intelligence quotients at the age of 7 (Lucas et al. 1992). A body of evidence suggests that brain development and cognition partly depends on sialic acid-containing gangliosides and poly-sialic acidcontaining glycoproteins (Kunz et al. 1999; Wang et al. 2009). Sialic acid concentrations in the brain are more than double between a few months prior to birth and a couple of years after birth (Svennerholm et al. 1989). Animal studies suggest that dietary sialic acid is an essential nutrient to meet the high sialic acid demand during pre- and post-natal stages of brain development (Carlson 1985; Wang et al. 2007). Human milk is a rich source of sialic acid (Wang et al. 2001), and post-mortem analysis on human neonates showed that ganglioside- and protein-bound sialic acid concentrations are significantly higher in the brains of breastfed infants than infants fed with formula that contained lower amounts of sialic acid (Wang and Brand-Miller 2003). Sialylated HMOs contribute to the majority of sialic acid in human milk. It remains to be investigated whether sialylated HMOs are the primary sialic acid carrier that provides the developing brain with this seemingly essential nutrient and contribute to superior developmental scores and intelligence quotients in breastfed infants (Bode 2012). CHARACTERISATION OF OSS The analytical methods that are capable of separating and characterising the various sugar compositions and structures of oligosaccharides in human milk include high-performance liquid chromatography (HPLC), high pH anion exchange chromatography (HPAEC), capillary electrophoresis (CE) and various mass spectrometry (MS) platforms (Thurl et al. 1996; Chaturvedi et al. 1997; Charlwood et al. 1999; Nakhla et al. 1999; Shen et al. 2000; Suzuki and Suzuki 2001; Pfenninger et al. 2002; Sumiyoshi et al. 2003). Heinz Egge and co-workers were pioneers in the analysis of complex HMO using these methodologies, which are still the ones used today (Kunz 2012; Moreno et al. 2014). These methods are technically cumbersome and incapable of producing large quantities of highly purified isolated molecules, so there is little information on many of the basic biological properties of this class of molecule. There is also little information on the occurrence and variability of these structures between individuals. Combining the lack of basic information on the diversity of HMOs between different lactating humans, on changes in oligosaccharide compositions and 5

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abundances during the course of lactation and on the role of genetic, dietary and physiological determinants upon the structures and abundances of HMOs, it is currently difficult to predict to what extent variations in health outcomes for different breastfed infants can be attributed to variation in the oligosaccharides delivered in their milk. To establish the various functions associated with the diverse HMO structures, the details of variations in compositions and abundances of oligosaccharides among humans and during lactation need to be measured. Some characterisation of HMO has been accomplished using HPAEC and HPLC in combination with derivatisation techniques (Coppa et al. 1993; Chaturvedi et al. 2001a; Sumiyoshi et al. 2003; Musumeci et al. 2006; Asakuma et al. 2008). The identification and quantification of HMOs was based on the retention time of commercially available milk oligosaccharide standards. In one study, a decrease in the total concentration of oligosaccharides was observed from the first week postpartum to about half after 1 year; the absolute and relative concentrations of HMOs between individual donors and at different stages of lactation varied significantly (Chaturvedi et al. 2001a). Asakuma et al. (2008) analysed the levels of several neutral oligosaccharides in human milk colostrum for three consecutive days from 12 Japanese women. The concentrations of 2’-fucosyllactose and lactodifucotetraose on day one were found to be substantially higher than those on days two and three, while the lacto-N-tetraose concentration increased from days one to three. These data suggest that the structure and abundance of the various HMO are variable, and it now becomes of considerable scientific and practical interest to understand the underlying factors (genetics, diet, plus the physiological and pathological state of both the mother and infant). The arrival of HPLC-Chip TOF/MS technology provides the analytical means to adopt a new strategy to routinely profile HMOs. This analytical technique employs an integrated microfluidic chip coupled with a high-accuracy time-of-flight mass analyser. Using this analytical platform, daily profiles of oligosaccharides in human milk samples were determined for different individual human donors. The levels of HMOs and their heterogeneity were investigated both within the individual donor and among multiple donors at different stages of lactation. This approach is designed to provide basic knowledge on HMOs in normal humans as the key compositional basis for understanding the relationship between the levels of milk oligosaccharides and the specific functions these biomolecules contribute to maternal and infant health and development (Ninonuevo et al. 2005, 2006, 2007). Recently, application of novel analytical approaches such as porous graphite carbon LC-MS and selective reaction monitoring (SRM) or triple quadrupole MS is being developed and will greatly enhance the knowledge on OS characterisation (Ruhaak and Lebrilla 2012), particularly for those different from the HMOs. A very recent study (Mehra et al. 2014) just 6

released the composition of BMOs by high-accuracy MALDI FT-ICR mass spectrometry. OLIGOSACCHARIDES IN INFANT FORMULA PRODUCTION Human milk currently remains the only known source of complex fucosylated and sialylated OSs: this limits further functional and translational research. The structural complexity of HMOs precludes chemical synthesis of all but the simplest HMO at present. Given the evidence for the beneficial effects of OSs in the diet of the neonate, a source of OSs similar to HMO would be of great value as a nutritional supplement for infant and even adult formulations (Meyrand et al. 2013). Potential sources are the milks of domesticated animals, plant material and synthesised compounds. Because the composition and structure of HMOs cannot yet be reproduced and there is limited production of OSs in dairy animals, OSs with simpler structures than HMO have been used as components in several dietary products to mimic the beneficial effects of HMOs (Mattila-Sandholm and Saarela 2003). Although not identical, there are some similar structural elements in the core molecules of the OSs. A very important element is the b-glycosidically bound Gal. The human intestine lacks enzymes to hydrolyse b-glycosidic linkages other than in lactose. Thus, b-glycosidically bound Gal is the structural element to protect these molecules from digestion during passage through the small intestine. In the neutral fraction of animal milk OSs, linkages to Fuc are, with few exceptions, very rare, whereas linkages to Gal or NAg are dominant (Boehm and Stahl 2007).

FOSs and GOSs Among the array of currently available and emerging prebiotics, relatively few have been examined for use in infant formulae. Stemming from the common observation of bifidobacteria in the faeces of breastfed infants, attempts have been made to reproduce this bifidogenic aspect in formulae by adding commercial prebiotics, in particular FOSs and GOSs, which are known to be broadly bifidogenic (Crittenden and Playne 2009). While HMOs are complex glycans composed of five different monosaccharides, these FOSs and GOSs are much simpler structures. FOSs are mostly b2-1-linked linear fructose oligomers of the inulin type, often extracted from Compositae spp. such as chicory (Roberfroid 2005, 2007b). The DP of chicory inulin varies between 2 and more than 60, and the polymers often carry Glc at the reducing end. FOSs are produced from inulin using an endo-inulinase that cleaves the polymers into smaller oligomers with or without glucose at the reducing end (Cho et al. 2001; Park and Yun 2001). FOSs can also be synthesised by transfructosylation using b-fructosidases from yeast or bacteria and sucrose as the substrate (Lafraya © 2015 Society of Dairy Technology

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et al. 2011; Tian et al. 2011). These -synthetic FOSs also usually carry Glc at the reducing end, and their DP is often less than 5. It is important to note that Gal and Fru oligomers do not naturally occur in human milk. Fructose is not found in human milk, and neither synthesised GOSs nor FOSs are fucosylated nor sialylated. FOSs are linear fructose polymers, whereas the basic structure of GOSs incorporates Lac at the reducing end that is typically elongated with up to six Gal residues, which can contain different branching, for example [Gal (b1 3/4/6)] 1–6Gal (b1–4)Glc. FOSs can be commercially produced through the reverse reaction of fructanases and sucrases or via enzymic hydrolysis of inulin (Espinosa et al. 2007). FOSs produced by the first method lacks a reducing end and contains one Glc residue and two or more fructose moieties, that is short-chain FOSs with degree of polymerisation (DP) 2–6 (Fanaro et al. 2005b), whereas hydrolysis of inulin produces free anomeric carbons and contains one fructose long-chain FOSs with DP 7–60 (Roberfroid 2005). Despite their structural differences when compared to HMO, ingestion of GOSs and FOSs still influences the microflora in the infant’s faeces and provides other benefits (Seifert and Watzl 2007; Boehm et al. 2008; Macfarlane et al. 2008). Falony et al. (2009) investigated FOS and inulin degradation by a wide range of Bifidobacterium spp., focusing on the presence of a preferential FOS breakdown mechanism. This revealed the existence of a limited number of phenotypically distinct clusters among those bifidobacterial strains tested. However, none of the species was able to degrade inulin or FOS completely, and both B. bifidum and B. breve, common infant isolates, degraded neither inulin nor FOS. Commercial GOS preparations are mostly produced by enzymatic treatment of Lac with b-galactosidases from different sources, such as fungi, yeast or bacteria, which results in a mixture of oligomers with various chain lengths (Fransen et al. 1998; Park and Oh 2010). Glc, Gal, Fru and mannose are examples of compounds that act as Gal acceptors for b-galactosidases, providing a virtually unlimited variety of OS preparations that may vary in technological properties such as flavour enhancement, sweetness, hygroscopicity and solubility (G€anzle et al. 2008). The preferred mode for GOS synthesis is by enzymic catalysis from lactose using glycosyltransferases (EC 2.4) or glycoside hydrolases (EC 3.2.1) (De Roode et al. 2003). These enzymes are responsible for the transfer of glycosyl moieties from a donor sugar to an acceptor (Ly and Withers 1999). Glycosyltransferases use sugar donors containing a nucleoside phosphate or a lipid phosphate remaining group (Coutinho et al. 2003; Lairson et al. 2008). Although highly regio-selective, stereo selective and efficient, these enzymes are not used for industrial GOS production due to their unavailability, prohibitive prices of commercial enzyme preparations and the need for specific sugar nucleotides as substrates (De Roode et al. 2003). © 2015 Society of Dairy Technology

Depending on enzyme source, GOSs contain b1–4 and b1–6, but also b1–2 or b1–3 linkages, leading to a variety of different structural isomers (Coulier et al. 2009). The DP is commonly 3–5 although Macfarlane et al. (2008) suggested that the upper DP limit for such GOSs was eight. However, Barboza et al. (2009) demonstrated that there were OSs with a DP of up to fifteen. The same researchers reported in vitro growth behaviour of different bifidobacterial strains of disaccharide- and monosaccharide-free fractions of GOS (pGOS). MALDI-FTICR MS analysis demonstrated that although all the strains tested were able to grow on the pGOS substrate, there were strain- and DP-specific bifidobacterial preferences for pGOS utilisation. In general, the neonate isolates (B. infantis and B. breve) were able to consume the GOS species with DP ranging from three to eight more efficiently, while B. adolescentis and B. longum subsp. longum were more selective. Previously, GOS consumption with specific DP preferences had been determined only for B. adolescentis DSM 20083 (Van Laere et al. 2000). The selective consumption of certain GOS structures by different Bifidobacterium spp. hints at the intriguing possibility of targeting GOS prebiotics to enrich selected bifidobacteria. Perhaps, the most studied prebiotic additive to infant formulae is an OS mixture with a GOS:FOS ratio of 9:1 (Fanaro et al. 2005a). This particular ratio has been shown to increase bifidobacteria counts in infant faeces (Boehm et al. 2002; Haarman and Knol 2005; Knol et al. 2005) and lower the incidence of pathogens (Knol et al. 2005). Other studies showed improved stool consistency and intestinal transit time with GOS/FOS (Mihatsch et al. 2006). Kapiki et al. (2007) showed that formula supplemented with FOS resulted in increased bifidobacteria and reduction in E. coli and enterococci. A study by Nakamura et al. (2009) demonstrated that faecal samples from infants fed formula supplemented with polydextrose, GOS and lactulose (8 g/L) contained significantly less bifidobacteria (20.7%) than faecal samples from infants fed breast milk (83.5%). The same study also confirmed that the prebiotic blend may have a greater impact on infant faecal bacterial populations in younger than in older infants. A defined mixture of GOS and FOS also reduced the incidence of atopic dermatitis during the first 6 months of life (Moro et al. 2006) and subsequent allergic manifestations and infections during the first 2 years of life (Arslanoglu et al. 2008). Long-term health benefits and risks of providing infants with significant amounts of these nonhuman milk glycans need to be further investigated.

Oligosaccharides from milk of other mammals Analysis of OS in the milk of New and Old World monkeys and apes indicated that, in general, the oligosaccharides in primate milk, including humans, are more complex and exhibit greater diversity compared to those in nonprimate milk (Goto 7

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et al. 2010; Tao et al. 2011). In humans, 50–80% of the OSs are fucosylated depending on the Se/L group, which is followed by chimpanzees at around 50% and gorillas with only 15%. Most other species show very low levels of fucosylation (