Publications by authors named "Tadasu Urashima"

74 Publications

Diversification of a fucosyllactose transporter within the genus .

Appl Environ Microbiol 2021 Nov 3:AEM0143721. Epub 2021 Nov 3.

Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO-assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose (FL)), are associated with the formation of a bifidobacteria-rich gut microbial community. Phylogenetic analysis has revealed that FL transporters have diversified into two clades harboring four clusters within the genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO-consumption phenotypes of two bifidobacterial species, subspecies and , which both possess FL binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO-consumption analysis showed that the FL transporter type from subspecies JCM 15439 conferred a novel HMO-uptake pattern that includes the complex fucosylated HMOs (lacto-fucopentaose II and lacto-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that H-antigen or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria to HMO-rich environments. The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breastmilk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated, host-microbe symbiosis. While the co-evolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within . We have shown that the diversification of the solute-binding proteins of the FL-transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allows for bifidobacteria to import different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.
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http://dx.doi.org/10.1128/AEM.01437-21DOI Listing
November 2021

Evolution of milk oligosaccharides: Origin and selectivity of the ratio of milk oligosaccharides to lactose among mammals.

Biochim Biophys Acta Gen Subj 2022 01 16;1866(1):130012. Epub 2021 Sep 16.

School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.

Background: The carbohydrate fraction of mammalian milk is constituted of lactose and oligosaccharides, most of which contain a lactose unit at their reducing ends. Although lactose is the predominant saccharide in the milk of most eutherians, oligosaccharides significantly predominate over lactose in the milk of monotremes and marsupials.

Scope Of Review: This review describes the most likely process by which lactose and milk oligosaccharides were acquired during the evolution of mammals and the mechanisms by which these saccharides are digested and absorbed by the suckling neonates.

Major Conclusions: During the evolution of mammals, c-type lysozyme evolved to α-lactalbumin. This permitted the biosynthesis of lactose by modulating the substrate specificity of β4galactosyltransferase 1, thus enabling the concomitant biosynthesis of milk oligosaccharides through the activities of several glycosyltransferases using lactose as an acceptor. In most eutherian mammals the digestion of lactose to glucose and galactose is achieved through the action of intestinal lactase (β-galactosidase), which is located within the small intestinal brush border. This enzyme, however, is absent in neonatal monotremes and macropod marsupials. It has therefore been proposed that in these species the absorption of milk oligosaccharides is achieved by pinocytosis or endocytosis, after which digestion occurs through the actions of several lysosomal acid glycosidases. This process would enable the milk oligosaccharides of monotremes and marsupials to be utilized as a significant energy source for the suckling neonates.

General Significance: The evolution and significance of milk oligosaccharides is discussed in relation to the evolution of mammals.
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http://dx.doi.org/10.1016/j.bbagen.2021.130012DOI Listing
January 2022

Exploring Potential Bioactive Peptides in Fermented Bactrian Camel's Milk and Mare's Milk Made by Mongolian Nomads.

Foods 2020 Dec 7;9(12). Epub 2020 Dec 7.

Department of Agriculture and Animal Science, Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Nishi, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan.

To date, bioactive proteins and peptides from minor livestock milks and their fermented products have been scarcely reported. In Mongolia, nomads are commonly rearing five livestock animal species (i.e., cow, camel, goat, horse, and sheep) for milking and other purposes. In this study, we analyzed the peptide composition in fermented milks of Bactrian camels () and horses, produced by Mongolian nomads for self-consumption. Peptides from skimmed fermented milks were separated by ultrafiltration and reverse-phase high-performance liquid chromatography. Then, their amino acid sequences were determined by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Consequently, eleven peptides were identified in the fermented camel's milk including four from β-casein (β-CN), three from α-CN, and two from both κ-CN and lactophorin. On the other hand, twenty-four peptides were identified in the fermented mare's milk including nineteen from β-CN, three from α-CN, and one from both κ-CN and α-CN. According to previous reports on the bioactivities of milk-derived peptides, antibacterial and antihypertensive activities were promising in both the fermented camel's milk and mare's milk. In addition, potential antioxidant activity was conjectured in the fermented camel's milk. Further investigations are currently needed to clarify the potential role of immunomodulatory peptides in the two fermented milks.
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http://dx.doi.org/10.3390/foods9121817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762409PMC
December 2020

Factors affecting decreasing viscosity of the culture medium during the stationary growth phase of exopolysaccharide-producing MTCC 25067.

Biosci Microbiota Food Health 2020 20;39(3):160-168. Epub 2020 Mar 20.

Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.

MTCC 25067 produces a hetero-exopolysaccharide (HePS) when cultured which forms supramolecular networks in the culture medium, increasing the viscosity. In the present study, the viscosity of the bacterial culture reached its maximum at 48 hr of cultivation and then decreased during a stationary growth phase lasting for up to 144 hr. The monosaccharide composition did not change during the stationary growth phase, whereas degradation of HePS molecules was noticeable, leading to partial disintegration of their supramolecular networks. The viscosity values of the HePS purified from the culture and dissolved in a fresh medium indicated little contribution of medium components to the viscosity. Absence of the apparent network structure of the HePS in the surrounding area of bacterial cells was observed during the late growth phase, supporting the idea that the decreases in culture viscosity during the prolonged period of cultivation were caused mainly by reduced interactions between bacterial cells and the intact supramolecular networks as a consequence of decreasing bacterial cell wall integrity and partial degradation of HePS molecules.
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http://dx.doi.org/10.12938/bmfh.2019-051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392921PMC
March 2020

Effects of low-dose milk protein supplementation following low-to-moderate intensity exercise training on muscle mass in healthy older adults: a randomized placebo-controlled trial.

Eur J Nutr 2021 Mar 10;60(2):917-928. Epub 2020 Jun 10.

Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.

Purpose: The purpose of this study was to examine whether long-term ingestion of low-dose milk protein supplementation causes a greater increase in muscle mass and strength of older adults during low-to-moderate intensity exercise training intervention than isocaloric carbohydrate.

Methods: In a randomized, double-blind, and placebo-controlled design, 122 healthy older adults (60-84 year) received either an acidified milk protein drink containing 10 g of milk protein (MILK; n = 61) or an isocaloric placebo drink (PLA; n = 61) daily throughout 6 months of body weight and medicine ball exercise training. Measurements before and after the intervention included body composition, physical performance and blood biochemistry.

Results: Lean body mass significantly increased in the MILK group (+ 0.54 kg, p < 0.001), but did not change in the PLA group (- 0.10 kg, p = 0.534). The increases in the MILK group were significantly greater than in the PLA group (p = 0.004). Fat mass (- 0.77 kg) and plasma uric acid levels (- 0.3 mg/dL) significantly decreased only in the MILK group (p < 0.001), with a significant group difference (p = 0.002 and p < 0.001, respectively). Most of the physical performance tests significantly improved in both groups, but no group differences were found.

Conclusion: We conclude that low-dose milk protein supplementation (10 g of protein/day) combined with low-to-moderate intensity exercise training is associated with increased muscle mass, but not improved physical performance compared to carbohydrate combined with exercise in healthy older adults. This study was registered in the UMIN Clinical Trials Registry (UMIN000032189).
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http://dx.doi.org/10.1007/s00394-020-02302-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900046PMC
March 2021

Chemical structures of oligosaccharides in milks of the American black bear (Ursus americanus americanus) and cheetah (Acinonyx jubatus).

Glycoconj J 2020 02 11;37(1):57-76. Epub 2019 Dec 11.

Smithsonian Environmental Research Center, Smithsonian Institution, Edgewater, MD, 21037, USA.

The milk oligosaccharides were studied for two species of the Carnivora: the American black bear (Ursus americanus, family Ursidae, Caniformia), and the cheetah, (Acinonyx jubatus, family Felidae, Feliformia). Lactose was the most dominant saccharide in cheetah milk, while this was a minor saccharide and milk oligosaccharides predominated over lactose in American black bear milk. The structures of 8 neutral saccharides from American black bear milk were found to be Gal(β1-4)Glc (lactose), Fuc(α1-2)Gal(β1-4)Glc (2'-fucosyllactose), Gal(α1-3)Gal(β1-4)Glc (isoglobotriose), Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (B-tetrasaccharide), Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]Glc (B-pentasaccharide), Fuc(α1-2)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc (difucosyl lacto-N-neotetraose), Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc (monogalactosyl monofucosyl lacto-N-neotetraose) and Gal(α1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (Galili pentasaccharide). Structures of 5 acidic saccharides were also identified in black bear milk: Neu5Ac(α2-3)Gal(β1-4)Glc (3'-sialyllactose), Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)[Fuc(α1-2)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (monosialyl monofucosyl lacto-N-neohexaose), Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3)[Gal(α1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (monosialyl monogalactosyl lacto-N-neohexaose), Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3){Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (monosialyl monogalactosyl monofucosyl lacto-N-neohexaose), and Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-3){Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (monosialyl monogalactosyl difucosyl lacto-N-neohexaose). A notable feature of some of these milk oligosaccharides is the presence of B-antigen (Gal(α1-3)[Fuc(α1-2)]Gal), α-Gal epitope (Gal(α1-3)Gal(β1-4)Glc(NAc)) and Lewis x (Gal(β1-4)[Fuc(α1-3)]GlcNAc) structures within oligosaccharides. By comparison to American black bear milk, cheetah milk had a much smaller array of oligosaccharides. Two cheetah milks contained Gal(α1-3)Gal(β1-4)Glc (isoglobotriose), while another cheetah milk did not, but contained Gal(β1-6)Gal(β1-4)Glc (6'-galactosyllactose) and Gal(β1-3)Gal(β1-4)Glc (3'-galactosyllactose). Two cheetah milks contained Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-neohexaose), and one cheetah milk contained Gal(β1-4)Glc-3'-O-sulfate. Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(β1-4)Glc (disialyllactose) was the only sialyl oligosaccharide identified in cheetah milk. The heterogeneity of milk oligosaccharides was found between both species with respect of the presence/absence of B-antigen and Lewis x. The variety of milk oligosaccharides was much greater in the American black bear than in the cheetah. The ratio of milk oligosaccharides-to-lactose was lower in cheetah (1:1-1:2) than American black bear (21:1) which is likely a reflection of the requirement for a dietary supply of N-acetyl neuraminic acid (sialic acid), in altricial ursids compared to more precocial felids, given the role of these oligosaccharides in the synthesis of brain gangliosides and the polysialic chains on neural cell adhesion.
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http://dx.doi.org/10.1007/s10719-019-09899-7DOI Listing
February 2020

Evolutionary adaptation in fucosyllactose uptake systems supports bifidobacteria-infant symbiosis.

Sci Adv 2019 08 28;5(8):eaaw7696. Epub 2019 Aug 28.

Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan.

The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from subspecies . Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2'-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.
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http://dx.doi.org/10.1126/sciadv.aaw7696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713505PMC
August 2019

Rheological characteristics and supramolecular structure of the exopolysaccharide produced by Lactobacillus fermentum MTCC 25067.

Carbohydr Polym 2019 Aug 27;218:226-233. Epub 2019 Apr 27.

Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan. Electronic address:

Rheological properties and supramolecular structure of the exopolysaccharide (EPS) secreted by Lactobacillus fermentum MTCC 25067 were investigated. The critical concentration representing the lower-limit of the semi-dilute regime was estimated to be 0.71 g/L from the concentration dependence of zero-shear specific viscosity. The storage modulus (G') of a 20 g/L EPS solution was greater than the loss modulus (G″) at 0.1-25 Hz. Approximately linear increases in G' and G″ determined at a frequency of 1 Hz and a strain of 0.01 during cooling from 80 to 25 °C were an indication that the EPS did not undergo thermally-induced cooperative conformational transitions typical of gelling polysaccharides. Atomic force microscopy images revealed that EPS molecules were not completely dissociated into individual molecules in an aqueous solution but remained to form three-dimensional networks. The gel-like dynamic viscoelasticity of the 20 g/L EPS solution was thus attributed to the existence of supramolecular assemblies resulting from significant degrees of intermolecular association of the EPS in the solution.
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http://dx.doi.org/10.1016/j.carbpol.2019.04.076DOI Listing
August 2019

Chemical characterization of the milk oligosaccharides of some Artiodactyla species including giraffe (Giraffa camelopardalis), sitatunga (Tragelaphus spekii), deer (Cervus nippon yesoensis) and water buffalo (Bubalus bubalis).

Glycoconj J 2018 12 22;35(6):561-574. Epub 2018 Nov 22.

Department of Life and Food Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan.

Mammalian milk/colostrum usually contains oligosaccharides along with the predominant disaccharide lactose. It has been found that the number and identity of these milk oligosaccharides varies among mammalian species. Oligosaccharides predominate over lactose in the milk/colostrum of Arctoidea species (Carnivora), whereas lactose predominates over milk oligosaccharides in Artiodactyla including cow, sheep, goat, camel, reindeer and pig. To clarify whether heterogeneity of a variety of milk oligosaccharides is found within other species of Artiodactyla, they were studied in the milk of giraffe, sitatunga, deer and water buffalo. The following oligosaccharides were found: Neu5Ac(α2-3)[GalNAc(β1-4)]Gal(β1-4)Glc (GM tetrasaccharide), and Gal(α1-3)Gal(β1-4)Glc (isoglobotriose) in giraffe milk; Neu5Ac(α2-3)Gal(β1-4)Glc (3'-SL), Neu5Ac(α2-6)Gal(β1-4)Glc (6'-SL), Gal(α1-4)Gal(β1-4)Glc (globotriose) and isoglobotriose in sitatunga colostrum; Gal(β1-3)Gal(β1-4)Glc (3'-GL), Gal(β1-6)Gal(β1-4)Glc (6'-GL), isoglobotriose, Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (lacto-N-neotetraose, LNnT), Gal(β1-4)Glc-3'-O-SO (3'-O-lactose sulphate) in deer milk; 3'-GL, isoglobotriose and Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3',3″-digalactosyllactose, DGL) in water buffalo colostrum. Thus it was shown that the milk oligosaccharides are heterogeneous among these Artiodactyla species.
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http://dx.doi.org/10.1007/s10719-018-9849-0DOI Listing
December 2018

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

Sci Rep 2018 09 18;8(1):13958. Epub 2018 Sep 18.

Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.

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.
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http://dx.doi.org/10.1038/s41598-018-32080-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143587PMC
September 2018

Relationship of a Special Acidified Milk Protein Drink with Cognitive Performance: A Randomized, Double-Blind, Placebo-Controlled, Crossover Study in Healthy Young Adults.

Nutrients 2018 May 8;10(5). Epub 2018 May 8.

Graduate School of Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.

A previous in vivo study with rats suggested that a special milk protein drink manufactured using an acidification procedure to suppress the aggregation of milk proteins was absorbed quickly after feeding. We performed a randomized, double-blind, placebo-controlled, repeated-measure crossover study to investigate the short-term effects on cognitive performance in 29 healthy young adult men after they consumed this drink in the morning. After an overnight fast, subjects were tested for performance in the Uchida⁻Kraepelin serial arithmetic test and the Stroop test as well as for subjective feeling, body temperature, and heart rate variability before and after consumption of either the acidified milk protein drink or an isoenergetic placebo drink. Subjects showed a significant improvement in performance in the Uchida⁻Kraepelin test, the primary outcome measured, when they consumed the acidified milk protein drink compared with the placebo control condition. In addition, consumption of the acidified milk protein drink, compared with the placebo control, was associated with increases in vagally-mediated heart rate variability indices which, from recent theoretical perspectives, may reflect a higher ability to modulate cognitive and behavioral processes. There was no significant difference in subjective feelings and body temperature between the test drink conditions. These data suggest that consumption of the acidified milk protein drink may improve cognitive performance, with possible involvement of physiological systems that regulate cognition and behavior.
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http://dx.doi.org/10.3390/nu10050574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986454PMC
May 2018

Chemical structures of oligosaccharides in milk of the raccoon (Procyon lotor).

Glycoconj J 2018 06 11;35(3):275-286. Epub 2018 Apr 11.

Graduate School of Agriculture, Tohoku University, Sendai, Miyagi, 980-0845, Japan.

In this study on milk saccharides of the raccoon (Procyonidae: Carnivora), free lactose was found to be a minor constituent among a variety of neutral and acidic oligosaccharides, which predominated over lactose. The milk oligosaccharides were isolated from the carbohydrate fractions of each of four samples of raccoon milk and their chemical structures determined by H-NMR and MALDI-TOF mass spectroscopies. The structures of the four neutral milk oligosaccharides were Fuc(α1-2)Gal(β1-4)Glc (2'-fucosyllactose), Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (lacto-N-fucopentaose IV), Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (fucosyl para lacto-N-neohexaose) and Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)[Fuc(α1-2)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (difucosyl lacto-N-neohexaose). No type I oligosaccharides, which contain Gal(β1-3)GlcNAc units, were detected, but type 2 saccharides, which contain Gal(β1-4)GlcNAc units were present. The monosaccharide compositions of two of the acidic oligosaccharides were [Neu5Ac][Hex][HexNAc][deoxy Hex], while those of another two were [Neu5Ac][Hex][HexNAc][deoxy Hex] These acidic oligosaccharides contained α(2-3) or α(2-6) linked Neu5Ac, non reducing α(1-2) linked Fuc, poly N-acetyllactosamine (Gal(β1-4)GlcNAc) and reducing lactose.
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http://dx.doi.org/10.1007/s10719-018-9821-zDOI Listing
June 2018

Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation.

mBio 2017 10 3;8(5). Epub 2017 Oct 3.

Department of Animal Science, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan.

is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced growth. This was impaired in a ATCC 15696 strain harboring a mutation in the gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the α2,6-linked but not to the α2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAcα1-3(Fucα1-2)Galβ] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in -mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates. Adhesion to the host mucosal surface and carbohydrate assimilation are important for bifidobacterium colonization and survival in the host gastrointestinal tract. In this study, we investigated the mechanistic basis for extracellular sialidase (SiaBb2)-mediated adhesion. SiaBb2 cleaved sialyl-human milk oligosaccharides and mucin glycans to produce oligosaccharides that supported growth. Moreover, SiaBb2 enhanced adhesion to mucosal surfaces via specific interactions with the α2,6 linkage of sialyloligosaccharide and blood type A antigen on mucin carbohydrates. These findings provide insight into the bifunctional role of SiaBb2 and the adhesion properties of strains.
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http://dx.doi.org/10.1128/mBio.00928-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626965PMC
October 2017

Identification of sialyl oligosaccharides including an oligosaccharide nucleotide in colostrum of an addax (Addax nasomaculatus) (Subfamily Antelopinae).

Anim Sci J 2018 Jan 7;89(1):167-175. Epub 2017 Sep 7.

Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido, Japan.

Mammalian milk/colostrum usually contains milk oligosaccharides along with the predominant lactose. Although milk oligosaccharides of a variety of Bovidae species including cow, sheep and goat have been characterized, those of the addax, an Antelopinae species of the Bovidae, have not as yet been clarified. In this study, several sialyl oligosaccharides were purified from a sample of addax colostrum and characterized as follows: Neu5Ac(α2-8)Neu5Ac(α2-3)Gal(β1-4)Glc, Neu5Gc(α2-8)Neu5Gc(α2-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-4)Glc, Neu5Ac(α2-6)Gal(β1-4)GlcNAc, Neu5Gc(α2-3)Gal(β1-4)Glc, Neu5Gc(α2-6)Gal(β1-4)Glc, Neu5Gc(α2-6)Gal(β1-4)GlcNAc. In addition, an oligosaccharide nucleotide Neu5Gc(α2-6)Gal(β1-4)GlcNAcα1-UDP was characterized. Molecular species of a variety of sialyl oligosaccharides found in milk and colostrum of these Bovidae were compared.
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http://dx.doi.org/10.1111/asj.12899DOI Listing
January 2018

Complete Genome Sequence of MTCC 25067 (Formerly TDS030603), a Viscous Exopolysaccharide-Producing Strain Isolated from Indian Fermented Milk.

Genome Announc 2017 Mar 30;5(13). Epub 2017 Mar 30.

Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan

MTCC 25067 (formerly TDS030603) is capable of producing a highly viscous slime exopolysaccharide. We report here the complete genome sequence of the strain, which was deciphered by using PacBio single-molecule real-time sequencing technology.
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http://dx.doi.org/10.1128/genomeA.00091-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374239PMC
March 2017

Anchorless cell surface proteins function as laminin-binding adhesins in Lactobacillus rhamnosus FSMM22.

FEMS Microbiol Lett 2017 03;364(6)

Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

Anchorless cell surface proteins (CSPs) were extracted with 1 M lithium chloride solution from Lactobacillus rhamnosus FSMM22. Loss of the anchorless CSPs resulted in a 2-fold decrease in FSMM22 cells bound to a constitutive extracellular matrix glycoprotein, laminin, in vitro. DNA-binding protein HU, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and 30S ribosomal protein S19 (RpsS) were identified by mass spectrometry in the extract as laminin-binding adhesins. Among the four proteins, RpsS was immunohistochemically confirmed to exist on the cell surface. Our findings strongly suggest that anchorless CSPs can enhance bacterial adhesion to the host.
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http://dx.doi.org/10.1093/femsle/fnx056DOI Listing
March 2017

In vitro safety assessments and antimicrobial activities of Lactobacillus rhamnosus strains isolated from a fermented mare's milk.

Anim Sci J 2017 Mar 1;88(3):517-525. Epub 2016 Aug 1.

Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.

Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4-8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α-chymotrypsin and β-glucosidase were detected, but those of α-galactosidase, β-glucuronidase and N-acetyl-β-glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell-free culture supernatant inhibited growths of Salmonella Typhimurium LT-2, Shigella sonnei, Listeria monocytogenes, and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain.
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http://dx.doi.org/10.1111/asj.12668DOI Listing
March 2017

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

Glycoconj J 2016 10 20;33(5):789-96. Epub 2016 May 20.

Intensive Grazing Research Team, National Agricultural Research Center for Hokkaido Region, Sapporo, Hokkaido, 062-8555, Japan.

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 (1)H-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.
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http://dx.doi.org/10.1007/s10719-016-9672-4DOI Listing
October 2016

Chemical characterization of milk oligosaccharides of the tiger quoll (Dasyurus maculatus), a marsupial.

Glycoconj J 2016 10 20;33(5):797-807. Epub 2016 May 20.

School of Molecular Biosciences, The University of Sydney, NSW, Sydney, 2006, Australia.

Milk oligosaccharides were separated from the carbohydrate fraction of milk of the tiger quoll a species of marsupial that is closely related to the eastern quoll, Dasyurus viverrinus. They were characterized by (1)H - nuclear magnetic resonance spectroscopy and matrix - assisted laser desorption/ionization time-of-flight mass spectrometry. The following oligosaccharides were identified; Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Neu5Ac(α2-3) Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc with an α(2-3)Neu5Ac linked to β(1-4)Gal residue of either branch of Gal(β1-4)GlcNAc(β1-6) units, and Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc with a β(1-3) linked Gal and an α(2-3) linked Neu5Ac. In addition, larger oligosaccharides were characterized as follows; Gal(β1-3){Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)GlcNAc(β1-6)}Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3){Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)GlcNAc(β1-6)}Gal(β1-4)Glc and their α(2-3) linked Neu5Ac derivatives.
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http://dx.doi.org/10.1007/s10719-016-9675-1DOI Listing
October 2016

Effects of prebiotic oligosaccharides consumption on the growth and expression profile of cell surface-associated proteins of a potential probiotic Lactobacillus rhamnosus FSMM15.

Biosci Microbiota Food Health 2016 6;35(1):41-9. Epub 2015 Oct 6.

Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

To investigate carbohydrate preference of a potential probiotic, Lactobacillus rhamnosus FSMM15, six prebiotics, including two milk-derived prebiotics, galactooligosaccharides and lacto-N-biose I, and four plant-origin prebiotics, beet oligosaccharide syrup, difructose anhydride III, fructooligosaccharides, and raffinose, were examined. The strain utilized the milk-derived prebiotics at similar levels to glucose but did not utilize the plant-origin ones in the same manner, reflecting their genetic background, which allows them to adapt to dairy ecological niches. These prebiotics had little influence on the expression pattern of cell surface-associated proteins in the strain; however, an ATP-binding cassette transporter substrate-binding protein and a glyceraldehyde-3-phosphate dehydrogenase were suggested to be upregulated in response to carbon starvation stress.
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http://dx.doi.org/10.12938/bmfh.2015-014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735032PMC
February 2016

Chemical characterization of milk oligosaccharides of the common wombat (Vombatus ursinus).

Anim Sci J 2016 Sep 26;87(9):1167-77. Epub 2015 Nov 26.

Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido, Japan.

Previous structural characterizations of marsupial milk oligosaccharides have been performed in the tammar wallaby, red kangaroo, koala, common brushtail possum and the eastern quoll. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of wombat milk carbohydrate were characterized in this study. Neutral and acidic oligosaccharides were isolated from the carbohydrate fractions of two samples of milk of the common wombat and characterized by (1) H-nuclear magnetic resonance spectroscopy. The structures of six neutral saccharides were found to be Gal(β1-4)Glc (lactose), Gal(β1-3)Gal(β1-4)Glc (3'-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3',3"-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I) and Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novooctaose), while those of six acidic saccharides were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc. (sialyl 3'-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3',3"-digalactosyllactose), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc,, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc. In addition, small amounts of sulfated oligosaccharides but no oligosaccharides containing Neu5Gc or α(2-6) linked Neu5Ac were detected.
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http://dx.doi.org/10.1111/asj.12566DOI Listing
September 2016

Chemical characterization of milk oligosaccharides of the eastern quoll (Dasyurus viverrinus).

Glycoconj J 2015 Aug 6;32(6):361-70. Epub 2015 Jun 6.

Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture & Vererinary Medicine, Obihiro, Hokkaido, 080-8555, Japan,

Structural characterizations of marsupial milk oligosaccharides have been performed in four species to date: the tammar wallaby (Macropus eugenii), the red kangaroo (Macropus rufus), the koala (Phascolarctos cinereus) and the common brushtail possum (Trichosurus vulpecula). To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, the oligosaccharides in the carbohydrate fraction of eastern quoll milk were characterized in this study. Neutral and acidic oligosaccharides were separated and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3'-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3",3'-digalactosyllactose), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II), Gal(β1-3)[Gal(β1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose III) and Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novooctaose). The structures of the acidic oligosaccharides detected are Neu5Ac(α2-3)Gal(β1-4)Glc (3'-sialyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a), Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), Neu5Ac(α2-3) Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, and Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc with an α(2-3) Neu5Ac linked to β(1-4)Gal residue of either branch of Gal(β1-4)GlcNAc(β1-6) units. The most predominant oligosaccharides in the carbohydrate fraction of mid-lactation milk were found to be lacto-N-novopentaose I and lacto-N-novooctaose, i.e., branched oligosaccharides that contain N-acetylglucosamine. The predominance of these branched oligosaccharides, rather than of a series of linear β(1-3) linked galacto oligosaccharides, appears to be the main feature of the eastern quoll milk oligosaccharides that differentiates them from those of the tammar wallaby and the brushtail possum.
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http://dx.doi.org/10.1007/s10719-015-9600-zDOI Listing
August 2015

4-O-Acetyl-sialic acid (Neu4,5Ac2) in acidic milk oligosaccharides of the platypus (Ornithorhynchus anatinus) and its evolutionary significance.

Glycobiology 2015 Jun 18;25(6):683-97. Epub 2015 Jan 18.

Smithsonian Environmental Research Center, Smithsonian Institution, Edgewater, MD 21037, USA

Monotremes (echidnas and platypus) retain an ancestral form of reproduction: egg-laying followed by secretion of milk onto skin and hair in a mammary patch, in the absence of nipples. Offspring are highly immature at hatching and depend on oligosaccharide-rich milk for many months. The primary saccharide in long-beaked echidna milk is an acidic trisaccharide Neu4,5Ac2(α2-3)Gal(β1-4)Glc (4-O-acetyl 3'-sialyllactose), but acidic oligosaccharides have not been characterized in platypus milk. In this study, acidic oligosaccharides purified from the carbohydrate fraction of platypus milk were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. All identified structures, except Neu5Ac(α2-3)Gal(β1-4)Glc (3'-sialyllactose) contained Neu4,5Ac2 (4-O-acetyl-sialic acid). These include the trisaccharide 4-O-acetyl 3'-sialyllactose, the pentasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-tetraose d) and the hexasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-fucopentaose III). At least seven different octa- to deca-oligosaccharides each contained a lacto-N-neohexaose core (LNnH) and one or two Neu4,5Ac2 and one to three fucose residues. We conclude that platypus milk contains a diverse (≥ 20) array of neutral and acidic oligosaccharides based primarily on lactose, lacto-N-neotetraose (LNnT) and LNnH structural cores and shares with echidna milk the unique feature that all identified acidic oligosaccharides (other than 3'-sialyllactose) contain the 4-O-acetyl-sialic acid moiety. We propose that 4-O-acetylation of sialic acid moieties protects acidic milk oligosaccharides secreted onto integumental surfaces from bacterial hydrolysis via steric interference with bacterial sialidases. This may be of evolutionary significance since taxa ancestral to monotremes and other mammals are thought to have secreted milk, or a milk-like fluid containing oligosaccharides, onto skin surfaces.
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http://dx.doi.org/10.1093/glycob/cwv010DOI Listing
June 2015

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

Biosci Biotechnol Biochem 2015 29;79(2):271-9. Epub 2014 Oct 29.

a Department of Animal Science, School of Veterinary Medicine , Kitasato University , Towada, Japan.

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.
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http://dx.doi.org/10.1080/09168451.2014.972325DOI Listing
December 2015

Erratum to: Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula).

Glycoconj J 2014 Jul 12. Epub 2014 Jul 12.

Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan,

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http://dx.doi.org/10.1007/s10719-014-9537-7DOI Listing
July 2014

Enhancement of Exopolysaccharide Production of Lactobacillus fermentum TDS030603 by Modifying Culture Conditions.

Biosci Microbiota Food Health 2014 29;33(2):85-90. Epub 2014 Apr 29.

Department of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

To optimize culture conditions that enhance production of a highly viscous exopolysaccharide of Lactobacillus fermentum TDS030603, a chemically defined medium was examined. The best yield was found to be 199 ± 23 mg/l when 48-hr cultivation was microaerobically performed at 30°C in the chemically defined medium supplemented with 5% glucose and 1% ammonium citrate without pH control. In response to the optimized exopolysaccharide production, the mRNA expression levels of epsB, epsE, and epsG elevated significantly. Our results indicated that the optimal C/N ratio and/or microaerobic condition can alter the expression levels of several exopolysaccharide biosynthesis-related genes promoting the exopolysaccharide production yield.
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http://dx.doi.org/10.12938/bmfh.33.85DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4081186PMC
July 2014

Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula).

Glycoconj J 2014 Jul 7;31(5):387-99. Epub 2014 Jun 7.

Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan,

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by (1)H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3'-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3", 3'-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3'-galactosyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)(-3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)(-3-O-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulphate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulphate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)(-3-O-sulphate)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.
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http://dx.doi.org/10.1007/s10719-014-9533-yDOI Listing
July 2014

Can an ancestral condition for milk oligosaccharides be determined? Evidence from the Tasmanian echidna (Tachyglossus aculeatus setosus).

Glycobiology 2014 Sep 7;24(9):826-39. Epub 2014 May 7.

Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.

The monotreme pattern of egg-incubation followed by extended lactation represents the ancestral mammalian reproductive condition, suggesting that monotreme milk may include saccharides of an ancestral type. Saccharides were characterized from milk of the Tasmanian echidna Tachyglossus aculeatus setosus. Oligosaccharides in pooled milk from late lactation were purified by gel filtration and high-performance liquid chromatography using a porous graphitized carbon column and characterized by (1)H NMR spectroscopy; oligosaccharides in smaller samples from early and mid-lactation were separated by ultra-performance liquid chromatography and characterized by negative electrospray ionization mass spectrometry (ESI-MS) and tandem collision mass spectroscopy (MS/MS) product ion patterns. Eight saccharides were identified by (1)H NMR: lactose, 2'-fucosyllactose, difucosyllactose (DFL), B-tetrasaccharide, B-pentasaccharide, lacto-N-fucopentaose III (LNFP3), 4-O-acetyl-3'-sialyllactose [Neu4,5Ac(α2-3)Gal(β1-4)Glc] and 4-O-acetyl-3'-sialyl-3-fucosyllactose [Neu4,5Ac(α2-3)Gal(β1-4)[Fuc(α1-3)]Glc]. Six of these (all except DFL and LNFP3) were present in early and mid-lactation per ESI-MS, although some at trace levels. Four additional oligosaccharides examined by ESI-MS and MS/MS are proposed to be 3'-sialyllactose [Neu5Ac(α2-3)Gal(β1-4)Glc], di-O-acetyl-3'-sialyllactose [Neu4,5,UAc3(α2-3)Gal(β1-4)Glc where U = 7, 8 or 9], 4-O-acetyl-3'-sialyllactose sulfate [Neu4,5Ac(α2-3)Gal(β1-4)GlcS, where position of the sulfate (S) is unknown] and an unidentified 800 Da oligosaccharide containing a 4-O-acetyl-3'-sialyllactose core. 4-O-acetyl-3'-sialyllactose was the predominant saccharide at all lactation stages. 4-O-Acetylation is known to protect sialyllactose from bacterial sialidases and may be critical to prevent microbial degradation on the mammary areolae and/or in the hatchling digestive tract so that sialyllactose can be available for enterocyte uptake. The ability to defend against microbial invasion was probably of great functional importance in the early evolution of milk saccharides.
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http://dx.doi.org/10.1093/glycob/cwu041DOI Listing
September 2014

Distinct substrate specificities of three glycoside hydrolase family 42 β-galactosidases from Bifidobacterium longum subsp. infantis ATCC 15697.

Glycobiology 2014 Feb 23;24(2):208-16. Epub 2013 Nov 23.

Enzyme and Protein Chemistry, Department of Systems Biology.

Glycoside hydrolase family 42 (GH42) includes β-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different β-d-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of β-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite -1 to be key specificity determinants. Bga42A thus prefers the β1-3-galactosidic linkage from human milk and other β1-3- and β1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Galβ1-4Galβ1-4Glc) as well as 4-galactobiose (Galβ1-4Gal) and 4-galactotriose (Galβ1-4Galβ1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the β-galactoside-rich gut niche and emphasize the importance and diversity of β-galactoside metabolism in probiotic bifidobacteria.
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http://dx.doi.org/10.1093/glycob/cwt104DOI Listing
February 2014

Chemical characterization of milk oligosaccharides of the koala (Phascolarctos cinereus).

Glycoconj J 2013 Nov 4;30(8):801-11. Epub 2013 Jul 4.

Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture & Veterinary Medicine, Obihiro, Hokkaido, 080-8555, Japan,

Previous structural characterizations of marsupial milk oligosaccharides had been performed in only two macropod species, the tammar wallaby and the red kangaroo. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of the koala milk carbohydrate fraction were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of milk of the koala, a non-macropod marsupial, and characterized by (1)H-nuclear magnetic resonance spectroscopy. The structures of the neutral saccharides were found to be Gal(β1-4)Glc (lactose), Gal(β1-3)Gal(β1-4)Glc (3'-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3',3″-digalactosyllactose), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I) and Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl lacto-N-novopentaose I), while those of the acidic saccharides were Neu5Ac(α2-3)Gal(β1-4)Glc (3'-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Gal (sialyl 3'-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), and Neu5Ac(α2-3)Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl sialyl lacto-N-novopentaose a). The neutral oligosaccharides, other than fucosyl lacto-N-novopentaose I, a novel hexasaccharide, had been found in milk of the tammar wallaby, a macropod marsupial, while the acidic oligosaccharides, other than fucosyl sialyl lacto-N-novopentaose a had been identified in milk carbohydrate of the red kangaroo. The presence of fucosyl oligosaccharides is a significant feature of koala milk, in which it differs from milk of the tammar wallaby and the red kangaroo.
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http://dx.doi.org/10.1007/s10719-013-9484-8DOI Listing
November 2013
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