Tadasu Urashima

Physiology of Consumption of Human Milk Oligosaccharides by Infant Gut-associated Bifidobacteria

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

Oligosaccharides of milk and colostrum in non-human mammals

Mammalian milk or colostrum usually contains, in addition to lactose, a variety of neutral and acidic oligosaccharides. Although the oligosaccharides of human milk have been reviewed in several recent publications, those of non-human mammals have received much less attention. This paper reviews the chemical structures and the variety of milk oligosaccharides in species other than humans, including placental mammals (e.g. primates, domestic herbivores, bears and other carnivores, the rat and the elephant) as well as monotremes (platypus and echidna) and marsupials (e.g. wallaby). The gastrointestinal digestion and absorption and the possible biological functions of these oligosaccharides are also discussed.

Structural characterisation of the exocellular polysaccharide produced by Streptococcus thermophilus OR 901

The exocellular polysaccharide of Streptococcus thermophilus OR 901, isolated from partially deproteinised whey, is a heteropolymer of D-galactopyranose and L-rhamnopyranose residues in the molar ratio 5:2. The structure was established by methylation analysis and 1D and 2D NMR spectroscopy of the native polysaccharide, in combination with characterisation of oligosaccharide fragments, obtained by partial acid hydrolysis, using methylation analysis and 1D 1H NMR spectroscopy. The polysaccharide has a branched heptasaccharide repeating unit with the following structure: [sequence: see text]

Recent Advances in Studies on Milk Oligosaccharides of Cows and Other Domestic Farm Animals

Human mature milk and colostrum contain 12-13 g/L and 22-24 g/L of milk oligosaccharides respectively, and the structures of least 115 human milk oligosaccharides (HMOs) have been characterized to date. By way of comparison, bovine colostrum collected immediately post partum contains only around 1 g/L of oligosaccharides, and this concentration rapidly decreases after 48 h. It was recently recognized that HMOs have several biological functions, and this study area has become very active, as illustrated by a recent symposium, but it appears that advances in studies on the milk oligosaccharides of domestic farm animals, including cows, have been rather slow compared with those on HMOs. Nevertheless, studies on bovine milk oligosaccharides (BMOs) have progressed recently, especially in regard to structural characterization, with the development of methods termed glycomics. This review is concerned with recent progress in studies on the milk oligosaccharides of domestic farm animals, especially of BMOs and bovine glycoproteins, and it discusses the possibility of industrial utilization in the near future.

Structural determination of three neutral oligosaccharides in bovine (Holstein-Friesian) colostrum, including the novel trisaccharide; GalNAcαl-3Galβ1-4Glc

Two trisaccharides, and a pentasaccharide were obtained from bovine colostrum. Their chemical structures were determined by using methylation and 13C-NMR analyses as follows: GalNac alpha 1-3Gal beta 1-4Glc, Gal alpha-1-3Gal beta 1-4Glc, GaL beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc. GalNAc alpha 1-3Gal beta 1-4Glc, which was identified in this study, is a novel oligosaccharide from natural sources. Gal alpha 1-3Gal beta 1-4Glc and Gal beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc (lacto-N-novopentaose) have been already found in ovine colostrum, and in horse colostrum and marsupial milk, respectively.

Sialyl oligosaccharides of human colostrum: changes in concentration during the first three days of lactation.

Sialyl oligosaccharides of human milk/colostrum are generally believed to be of biological significance, for example with respect to anti-adhesion of pathogenic organism, providing precursors for biosynthesis of brain, and so on. However, the levels of each of the sialyl oligosaccharides in human colostrum have not so far been determined. The present study was designed to determine the concentrations of nine major sialyl oligosaccharides in human colostrum, collected during the first 3 d (days 1–3) from the start of lactation. We found that the concentration of 3′-sialyllactose was significantly higher on day 1 than on day 2 and 3, but the levels of 6′-sialyllactose and sialyllacto-N-tetraose a were higher on day 3 than on day 1. These results are consistent with the view that during the first 3 d of lactation, the concentration of sialyl oligosaccharides in human colostrum change in accordance with the physiological demands of newborn infants.

Chemical characterization of oligosaccharides in chimpanzee, bonobo, gorilla, orangutan, and siamang milk or colostrum

Neutral and acidic oligosaccharides were isolated from the milk or colostrum of four great ape species (chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus)) and one lesser ape species (siamang (Symphalangus syndactylus)), and their chemical structures were characterized by (1)H-NMR spectroscopy. Oligosaccharides containing the type II unit (Gal(beta1-4)GlcNAc) were found exclusively (gorilla and siamang) or predominately (chimpanzee, bonobo, and orangutan) over those containing the type I unit (Gal(beta1-3)GlcNAc). In comparison, type I oligosaccharides predominate over type II oligosaccharides in human milk, whereas nonprimate milk almost always contains only type II oligosaccharides. The milk or colostrum of the great apes contained oligosaccharides bearing both N-glycolylneuraminic acid and N-acetylneuraminic acid, whereas human milk contains only the latter. Great ape milk, like that of humans, contained fucosylated oligosaccharides whereas siamang milk did not. Since these analyses are based on a limited number of individuals, further research on additional samples of great and lesser ape milk is needed to confirm phylogenetic patterns.

CHEMICAL CHARACTERIZATION OF MILK OLIGOSACCHARIDES OF THE BROWN BEAR, URSUS ARCTOS YESOENSIS

Two tri-, two hexa- and two deca-saccharides were isolated from bear milk by chloroform/methanol extraction and gel filtration. The oligosaccharides were characterized, mainly by 1H-NMR, as follows: [structures: see text]. Both trisaccharides were present in bear milk at a higher concentration than lactose.

Changes in the bovine whey proteome during the early lactation period

To investigate time-dependent change in the bovine whey proteome during the early lactation period, a two-dimensional gel-based approach was used in this study. Milk samples were collected from five healthy Friesian-Holstein dairy cows up to 10 days postpartum. Spot patterns of whey proteins varied drastically from immediately after parturition to 48 h, but no significant changes occurred thereafter. Protein identification by mass spectrometry revealed that the ratios of caseins and immunoglobulins drastically decreased during 48 h postpartum, while those of lower molecular mass proteins such as α-lactalbumin and β-lactoglobulin increased. More than 100 spots were detected, being much more abundant in colostral whey than in mature milk whey. Of a total of 25 proteins identified, four, viz. zinc-α-2-glycoprotein, vitamin D-binding protein, immunoglobulin G2 chain C and β2-microglobulin, were detectable only in colostrum. Our results indicate that most of the minor whey proteins in colostrum relate to the passive immunity of newborn calves, but some of them play significant roles in nutritional supplementation of the neonate. The characteristics of whey proteins in transition imply that enhancement of innate immunity becomes more important than protection of the neonate against pathogens via passive immunity after 48 h postpartum.

Sialyllactose occurs as free lactones in ovine colostrum

Three sialyl oligosaccharide fractions were separated from ovine colostrum by gel filtration, anion exchange chromatography and normal-phase HPLC. They were characterized by 1H-NMR spectrometry as follows: Neu5Acalpha2-->3Galbeta1-->4Glc, Neu5Gcalpha2-->6Galbeta1-->4Glc and three forms of Neu5Gcalpha2-->3Galbeta1-->4Glc, namely Neu5Gcalpha2-->3Galbeta1-->4Glc itself, its lactone derivative between the carboxyl group of Neu5Gc and Gal OH-2 and another lactone derivative between the carboxyl group and Gal OH-4. In this study, Neu5Gc-lactose lactones, in their free form, have been isolated for the first time from any natural sources including milk or colostrum.