Carbohydrates on human intestinal cell surface are important recognition sites for pathogenic bacterial binding that initiates infection. They are also the major components of human milk oligosaccharides (HMO). Depending on the lactation cycle, human milk contains >4 g/L of these complex and heterogeneous mixture of oligosaccharides [1]. HMO are composed by serial arrangement of D-glucose, D-galactose, N-acetylglucosamine, L-fucose and N-acetylneuraminic acid (Neu5 Ac). Despite this large combinatorial potential human milk contains just over 200 oligosaccharides [1]. HMO contain a Lactose (Ga1β1-4G1c) moiety at their reducing end with lactoN-biose I units (LNB; Ga1β1-3G1cNAc) or lactosamine (Ga1β1 4G1cNAc) elongated from a β1-3 or β1-6 linkage to the lactosyl terminus. A peculiar characteristic of HMO is their terminal fucosylation via a1-2/3/4 linkages and/or by a2-3/6 sialylation. In the absence of fucosidase and sialidase activities, these residues obstruct HMO core structures from microbial fermentation. HMO are not digested by infant gastrointestinal enzymes and remain largely intact until they reach the large intestine, where they can be used as fermentable substrate by the resident bacteria. One of their functions is to act as selective substrate to stimulate the colonic growth and proliferation of specific bacteria, such as Bifidobacteria [2].
HMOs are a class of indigestible oligosaccharides functioning as prebiotics, or “selectively fermented ingredients that allow specific changes, both in the composition and/or activity in the gatrointestinal microbiota that confers benefits upon host well-being and health” [3]. The large intestine of breast-fed infants is continuously exposed to copious amounts of HMO from mothers' milk and is characterized by a microbiota dominated by bifidobacterial species. The role of HMO is to selectively nourish the growth of specific strains of bifidobacteria priming the development of a unique gut microbiota in breast milk fed infants [4, 5, 6].
Recent studies investigating the catabolism and fermentation of HMOs by individual strains of infant-borne bifidobacteria have shown that Bifidobacterium longum subsp. infantis can grow extensively on HMOs as a sole carbon source, while adult-borne bifidobacterial species exhibited a more restricted growth profile [26]. Not all Bifidobacteria can grow on HMOs, for example within the closely related B. longum subspecies only strains belonging to subsp. infantis are capable of growth on HMOs. Limited HMO capacity has been shown for B. bifidum, while B. B. adolescentis and B. animalis subsp. lactis are unable to metabolize these complex oligosaccharides. These results suggest that HMOs may selectively promote the growth of certain bifidobacterial strains in the colonic lumen frequently isolated from breast-fed infants [23, 24].
It has been recently shown that compared to their nonautistic siblings, the fecal microbiome of children with Autism Spectrum Disorders (ASD) contain increased diversity of Clostridia spp. and higher cell counts of Clostridium histolyticum group [9]. The lack of a highly specific prebiotic, substrates, such as HMO, has hindered the development of infants and children's therapies to displace Clostridia spp. populations with beneficial, non-pathogenic Bifidobacteria spp. populations.
Enteric infections are responsible for ˜2.1 million deaths per year and are the leading cause of children and infant mortality in developing countries [10]. Frequent occurrences of diarrhea are common among C-section, preterm, and formula-fed infant populations, and their cost on the healthcare system is between $400 and $1600 per infant treated [11, 12]. Exclusively breastfed infants possessing a bifidobacteria-rich infant colonic microbiota have dramatically lower rates of enteric infections, necrotizing enterocolitis (NEC), and gastroenteritis [11, 13, 14]. There is strong evidence for the use of probiotics and Bifidobacteria to prevent NEC in preterm infants [15].
Fucosylated oligosaccharides are abundant in human milk [16] and are known to inhibit the binding of pathogenic bacteria. HMO and in particular the fucosylated HMOs, share common structural motifs with glycans on the infant's intestinal epithelia known to be receptors for pathogens. Such structures imply that their presence in milk provides its host with a defensive strategy, with a1,2-fucosylated HMO acting as a barrier to prevent binding of pathogens such as Campylobacter jejuni and caliciviruses to epithelial cells, thereby protecting infants from disease [4, 17]. HMOs, and in particular fucosylated HMOs are important functional constituent of human breast milk, and hold the promise for their use as a class of active ingredients for therapeutics specifically aimed at improving gut health. Unfortunately to date, a source of fucosylated oligosacchardes similar to those in human milk remains yet to be identified, for example bovine milk has been thought to be rich in sialylated oligosaccharides but not fucosylated ones. Interestingly, human milk only contains only about 20% of sialylated oligosaccharides.
At present, the only source of HMO is human milk, and the structural complexity of these oligosaccharides has hindered their commercial production. Attempts of reproducing HMO include the chemical synthesis of 2′- and 3′-Fucosyllactose as described in WO/2005/055944, and in transgenic non-human mammals (U.S. Pat. No. 5,750,176).
Milk oligosaccharides have also been characterized in domesticated animals including cow and goat, although they are generally lower in abundance and vary in prevalence of specific oligosaccharide compositions. An important distinction between human milk and other domesticated animals is the presence in the latter of N-glycolylneuraminic acid residues, these are absent in HMO consistent with the lost the ability of humans to synthesize this sialic acid [18]. These sources of milk oligosaccharides are therefore not suitable prebiotic oligosaccharides.
Prebiotics used to mimic the prebiotic effect of HMO include fruto-oligosaccharides (FOS), extracted from chicory roots and galacto-oligosaccharides (GOS) enzymatically synthesized from dairy-derived galactose [74]. FOS is broadly bifidogenic and is utilized by most bifidobacteria. FOS and GOS are added to some infant formulas (e.g. Similac Early Shield in the U.S.), and have found use as prebiotics in a wide range of food products. However these prebiotics lack the structural complexity of HMOs, such as the presence of terminal fucose or sialic acid moieties, and therefore unlikely provide the full spectrum of bioactivities of HMOs. FOS and GOS are therefore unlikely to retain the immunological and pathogen inhibition functions of HMOs. Moreover, current nutraceutical milk oligosaccharide mimetics, such as GOS and FOS do not reflect the genomic and physiological links between infant-type bifidobacteria and HMO; instead they target the bifidobacterial population nonspecifically.
There are currently no prebiotic oligosaccharides that can fully mimic the biological, structural, and glycomic functionalities of HMO. Analogues and mimics of HMOs could protect the mucosal surfaces in the infant gastrointestinal tract from pathogens, while at the same time act as a highly selective prebiotic substrate to target specific infant-type bifidobacterial populations, such as the presence of terminal fucose or sialic acid moieties, and therefore unlikely provide the full spectrum of bioactivities of HMOs.