Sadaki Asakuma

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.

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.

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.

Determination of Sialyl and Neutral Oligosaccharide Levels in Transition and Mature Milks of Samoan Women, Using Anthranilic Derivatization Followed by Reverse Phase High Performance Liquid Chromatography

An improved analytical method using reverse-phase high performance liquid chromatography following anthranilic acid derivatization for the measurement of each oligosaccharide level in transition (5 to 10 d lactation) and mature (21 to 155 d lactation) milks of sixteen Samoan women is described. The method was applied for the measurement of sialyl as well as neutral oligosaccharide levels. We found that disialyllacto-N-tetraose (DSLNT), sialylacto-N-tetraose c (LSTc), and 6′-sialyllactose (6′-SL) were the most abundant of the sialyl oligosaccharides. In the neutral oligosaccharide fraction, lacto-N-fucopentaose II and III combined (LNFP II+III) were the most dominant, followed by lacto-N-tetraose (LNT) and 3-fucosyllactose (3-FL). 2′-Fucosyllactose (2′-FL) and lacto-N-fucopentaose I (LNFP I) were absent in some and found at low levels in most of the Samoan women. Our study indicates that the milk oligosaccharide composition in Samoan women is similar to that of Japanese women with respect to sialyl but not to neutral oligosaccharides. The differences in neutral oligosaccharides might have a genetic origin.

Purification and Characterization of a Novel Exo-β-1,3-1,6-glucanase from the Fruiting Body of the Edible Mushroom Enoki (Flammulina velutipes)

To elucidate the role of β-glucanases in the cell-wall degradation involved in morphogenesis, an exo-β-1,3-1,6-glucanase (FvBGL1) was purified from fruiting bodies of the edible mushroom Enoki (Flammulina velutipes), and its enzymatic properties were studied. At least three β-glucanases were detected in the crude extract by zymogram assay when 1% laminarin was used as substrate. The molecular mass of FvBGL1 was estimated by SDS–PAGE to be 80 kDa. The optimum pH and temperature for the action of FvBGL1 were 6.1 and 60 °C respectively. FvBGL1 was completely inactivated by 1 mM mercuric ions. FvBGL1 hydrolyzed F. velutipes cell-wall β-glucan as well as β-1,3- and β-1,6-glucans from various sources with glucose as the only reaction product. Transglucosylation was observed when the enzyme acted on laminarinonaose. FvBGL1 can be assumed to degrade F. velutipes cell-wall β-1,3-glucan, but most probably acts more efficiently in concert with other endogenous β-glucan degrading enzymes.

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 microM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4-64.3%) and blastocyst formation (6.5-22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 microM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively). The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 microM Cys is not necessary and TCM199 plus Cys (100 microM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

Characterization of two novel sialyl N-acetyllactosaminyl nucleotides separated from ovine colostrum

The milk/colostrum of some mammalian species is known to contain sugar nucleotides including uridine diphosphate (UDP) oligosaccharides in addition to lactose and milk oligosaccharides, but the detailed structures of these UDP oligosaccharides have not so far been clarified. In this study we isolated two UDP-sialyl N-acetyllactosamines from ovine colostrum and characterized them using 1H-NMR and MALDI-TOFMS spectroscopies. Their structures were found to be Neu5Gc(α2–3)Gal(β1–4)GlcNAcα1-UDP and Neu5Gc(α2–6)Gal(β1–4)GlcNAcα1-UDP.

Effect of time at pasture and herbage intake on profile of volatile organic compounds of dairy cow milk.

Volatile organic compounds (VOCs) in milk were investigated as quantitative markers of herbage intake (HI) at pasture. Eight Holstein cows were fed indoors with concentrate and conserved forages (grass silage, corn silage and hay) (NG), then were divided into three treatments according to the duration of access to pasture: 4 h (G4), 8 h (G8), and 20 h (G20) per day. The HIs were 4.3, 8.6, and 13.0 kg dry matter/day for the G4, G8 and G20 treatments, respectively. Milk from cows was sampled and analyzed VOCs by the steam distillation-extraction method and gas chromatography-mass spectrometry (GC-MS). From the intensity of the GC peak area, the levels of 1-phytene (3,7,11,15-tetramethyl-1-hexadecene) and 2-phytene (3,7,11,15-tetramethyl-2-hexadecene) were lowest in NG treatment and markedly increased with grazing time at pasture. With simple regression analysis on the HI to each diterpenoid, a strong correlation was found between the intensity of 1-phytene in the milk and the HI (r = 0.807, P < 0.001). 1-phytene content in milk could be useful as a quantitative marker of the HI of grazing cows.

Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum

Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.