Ulvans are not equal - Linkage and substitution patterns in ulvan polysaccharides differ with Ulva morphology.

Ulva are hardy green seaweeds that contain the sulfated polysaccharide ulvan and grow in two distinct morphologies: foliose and tubular. The authors hypothesise that ulvan from tubular species are more structurally complex than ulvans from foliose species. Herein, using standardised methods, the glycosyl linkage positions and sulfate ester substitutions of constituent monosaccharides of ulvan isolated from foliose (U. lacinulata and U. stenophylloides) and tubular (U. prolifera and U. ralfsii) species of Ulva were investigated. Comparison of native ulvans with 80 and 100 °C desulfated counterparts indicated that 4-linked rhamnose is predominantly 3-O-sulfated in all four ulvans. Ulvans from the foliose species predominantly contained →3,4)-Rhap-(1→, →4)-GlcAp-(1→ and →4)-IdoAp-(1→, collectively accounting for 67 to 81 mol% of the total linkages. In contrast, these same linkages in ulvans from the tubular species only collectively accounted for 29 to 36 mol%. Instead, ulvan from tubular species contained a combination of →2,3,4)-Rhap-(1→, terminal Rhap-(1→, →4)-GlcAp-(1→, →4)-Xylp-(1→, and/or →4)-Galp-(1→ in high proportions; some of the latter three residues were also likely O-2 sulfated. The results presented here suggest that ulvan from foliose species are predominantly unbranched polysaccharides composed of repeat disaccharides while ulvans from tubular species contain a greater diversity of branch and sulfate substitution locations.

Does harvesting age matter? Changes in structure and rheology of a shear-thickening polysaccharide from Cyathea medullaris as a function of age.

A shear-thickening polysaccharide from the New Zealand Black tree fern (Cyathea medullaris, commonly known as mamaku) extracted from different age fronds (stage 1: young, stage 2: fully grown and stage 3: old) was characterised in terms of structure and rheological properties. Constituent sugar analysis and 1H and 13C NMR revealed a repeating backbone of -4)-ß-D-GlcpA-(1 â†’ 2)-α-D-Manp-(1→, for all mamaku polysaccharide (MP) samples from different age fronds without any alterations in molecular structure. However, the molecular weight (Mw) was reduced with increasing age, from ~4.1 × 106 to ~2.1 × 106 Da from stage 1 to stage 3, respectively. This decrease in Mw (and size) consequently reduced the shear viscosity (ηs-Stage 1 > Î·s-Stage 2 > Î·s-Stage 3). However, the extent of shear-thickening and uniaxial extensional viscosity of MP stage 2 was greater than MP stage 1, which was attributed to a greater intermolecular interaction occurring in the former. Shear-thickening behaviour was not observed in MP stage 3.

Novel 4-O-ß-d-xylopyranosyl-3,6-anhydro-l-galactopyranosyl disaccharide units in a polysaccharide from the red alga Pyrophyllon subtumens.

Thalli of the endemic epiphytic New Zealand red seaweed Pyrophyllon subtumens are known to contain a high level of xylose and a notable amount of arabinose but the extracted polysaccharide has not been characterised. The linkage/substitution of individual sugars within the water-soluble polysaccharide extract and various derivatives were determined by chemical and spectroscopic methods. No 3-linked sugars nor any d-galactose were found, which excluded agar-, carrageenan- or mixed 3-linked/4-linked ß-d-xylan-type polysaccharides found in many other red macroalgae. Instead, the polysaccharide backbone contained predominantly 4-linked ß-d-xylopyranosyl, 4-linked 3,6-anhydro-l-galactopyranosyl and 4-linked l-galactopyranosyl units. Some of each type of sugar were sulfated at various positions. Some xylosyl units were substituted at the 2- or 3-position with l-arabinosyl units. The polysaccharide is complex and likely contains a range of structures. However, partial sequencing was successfully used to recover and identify a novel disaccharide 4-O-d-xylopyranosyl-3,6-anhdydro-l-galactopyranose, which indicates a unique →4)-ß-d-Xylp-(1 â†’ 4)-3,6-anhydro-l-Galp-(1 â†’ repeat unit in the polysaccharide.

Variability in the composition of porcine mucosal heparan sulfates.

Commercial porcine intestinal mucosal heparan sulfate (HS) is a valuable material for research into its biological functions. As it is usually produced as a side-stream of pharmaceutical heparin manufacture, its chemical composition may vary from batch to batch. We analysed the composition and structure of nine batches of HS from the same manufacturer. Statistical analysis of the disaccharide compositions placed these batches in three categories: group A had high GlcNAc and GlcNS, and low GlcN typical of HS; group B had high GlcN and GlcNS, and low GlcNAc; group C had high di- and trisulfated, and low unsulfated and monosulfated disaccharide repeats. These batches could be placed in the same categories based on their 1H NMR spectra and molecular weights. Anticoagulant and growth factor binding activities of these HS batches did not fit within these same groups but were related to the proportions of more highly sulfated disaccharide repeats.

Structural characterization of ulvans extracted from blade (Ulva ohnoi) and filamentous (Ulva tepida and Ulva prolifera) species of cultivated Ulva.

Ulvans from Ulva ohnoi, Ulva tepida and Ulva prolifera were extracted under mild acidic conditions, isolated and their composition and structure determined. The ulvans contained mostly rhamnose (31.6-46.7 mol%) and glucuronic acid (26.6-37.5 mol%), with smaller amounts of xylose (3.4-10.4 mol%) and iduronic acid (3.1-7.6 mol%). In addition, the ulvan samples also contained galactose (4.4-26.0 mol%). Glycosyl linkage analysis showed that ulvan from U. ohnoi contained mostly →4)-GlcpA-(1→ and →3,4)-Rhap-(1→. Preparation of partially methylated alditol acetate standards of idose showed that U. ohnoi contained →4)-IdopA-(1→. In addition to these residues, glycosyl linkage analysis of U. tepida and U. prolifera showed the presence of →2,3,4)-Rhap-(1→, →4)-Xylp-(1→, →2,4)-GlcpA-(1→ and →3,4)-GlcpA-(1→. These two species also contained galactose linkages. These data, together with nuclear magnetic resonance (NMR) spectroscopy indicated that U. ohnoi comprised mostly of type A3S ulvanobiuronic acid repeats [→4)-ß-D-GlcpA-(1→4)-α-L-Rhap3S-(1→], together with smaller amounts of type B3S ulvanobiuronic acid repeats [→4)-α-L-IdopA-(1→4)-α-L-Rhap3S-(1→] and ulvanobiose (U3S [→4)-ß-D-Xylp-(1→4)-α-L-Rhap3S-(1→]). NMR spectra of U. tepida and U. prolifera showed resonances not detected in U. ohnoi, highlighting the complexity of the ulvans from these species. Regardless of the structural diversity of the ulvan samples there was very little antioxidant or inhibitory activity detected on enzymatic processes investigated.

Structure and rheology of pectic polysaccharides from baobab fruit and leaves.

Linkage patterns and relaxation dynamics of baobab (Adansonia digitata) polysaccharides have been investigated by means of linkage analysis and rheometry. The fruit polysaccharide was mostly xylogalacturonan, with co-extracted α-glucan. The leaf polysaccharide consists predominantly of two domains, one branched at O-4 of the →2)-Rhap-(1→ residues and another branched at O-3 of the →4)-GalpA-(1→ backbone to single GlcpA-(1→ residues. Master curves of viscoelasticity of fruit polysaccharides manifested strong pH-dependency. At pH below the dissociation constant of galacturonic acid, dispersions showed liquid-like behaviour. In contrast, at neutral pH, a weak gel network formation was observed that destabilised rapidly under the influence of flow fields. The present work identifies xylogalacturonans from baobab fruit as polysaccharides with unique rheological characteristics that may point to new directions in food and pharmaceutical formulation.

Are all ulvans equal? A comparative assessment of the chemical and gelling properties of ulvan from blade and filamentous Ulva.

Green seaweeds of the genus Ulva are rich in the bioactive sulfated polysaccharide ulvan. Herein we characterise ulvan from Ulva species collected from the Bay of Plenty, Aotearoa New Zealand. Using standardised procedures, we quantified, characterised, and compared ulvans from blade (U. australis, U. rigida, U. sp. B, and Ulva sp.) and filamentous (U. flexuosa, U. compressa, U. prolifera, and U. ralfsii) Ulva species. There were distinct differences in composition and structure of ulvans between morphologies. Ulvan isolated from blade species had higher yields (14.0-19.3 %) and iduronic acid content (IdoA = 7-18 mol%), and lower molecular weight (Mw = 190-254 kDa) and storage moduli (G' = 0.1-6.6 Pa) than filamentous species (yield = 7.2-14.6 %; IdoA = 4-7 mol%; Mw = 260-406 kDa; G' = 22.7-74.2 Pa). These results highlight the variability of the physicochemical properties of ulvan from different Ulva sources, and identifies a morphology-based division within the genus Ulva.

A protocol combining breath testing and ex vivo fermentations to study the human gut microbiome.

This protocol describes the application of breath testing and ex vivo fermentations to study the association between breath methane and the composition and functionality of the gut microbiome. The protocol provides a useful systems biology approach for studying the gut microbiome in humans, which combines standardized methods in human breath testing and fecal sampling. The model described is accessible and easy to repeat, but its relative simplicity means that it can deviate from human physiological conditions.

Preferential use of plant glycans for growth by Bacteroides ovatus.

B. ovatus is a member of the human gut microbiota with a broad capability to degrade complex glycans. Here we show that B. ovatus degrades plant polysaccharides in a preferential order, and that glycan structural complexity plays a role in determining the prioritisation of polysaccharide usage.

Sharing a ß-Glucan Meal: Transcriptomic Eavesdropping on a Bacteroides ovatus-Subdoligranulum variabile-Hungatella hathewayi Consortium.

Whole-transcriptome analysis was used to investigate the molecular interplay between three bacterial species that are members of the human gut microbiota. Bacteroides ovatus, Subdoligranulum variabile, and Hungatella hathewayi formed associations in cocultures fed barley ß-glucan, a constituent of dietary fiber. B. ovatus depolymerized ß-glucan and released, but did not utilize, 3-O-ß-cellobiosyl-d-glucose (DP3) and 3-O-ß-cellotriosyl-d-glucose (DP4). These oligosaccharides provided growth substrates for S. variabile and H. hathewayi with a preference for DP4 in the case of the latter species. There was increased transcription of a B. ovatus mixed-linkage-ß-glucan utilization locus, as well as carbohydrate transporters in S. variabile and H. hathewayi when in batch coculture. Increased transcription of the ß-glucan utilization locus did not occur in continuous culture. Evidence for interactions relating to provision of cobalamin, alterations to signaling, and modulation of the "stringent response" (an adaptation to nutrient deprivation) were detected. Overall, we established a bacterial consortium based on barley ß-glucan in vitro, which can be used to investigate aspects of the functional blueprint of the human gut microbiota.IMPORTANCE The microbial community, mostly composed of bacterial species, residing in the human gut degrades and ferments polysaccharides derived from plants (dietary fiber) that would not otherwise be digested. In this way, the collective metabolic actions of community members extract additional energy from the human diet. While the variety of bacteria present in the microbial community is well known, the formation of bacterial consortia, and the consequent interactions that result in the digestion of dietary polysaccharides, has not been studied extensively. The importance of our work was the establishment, under laboratory conditions, of a consortium of gut bacteria that formed around a dietary constituent commonly present in cereals. This enabled the metabolic interplay between the bacterial species to be studied. This kind of knowledge is required to construct an interactive, metabolic blueprint of the microbial community that inhabits the human gut.

Galacto- and Fructo-oligosaccharides Utilized for Growth by Cocultures of Bifidobacterial Species Characteristic of the Infant Gut.

Bifidobacterial species are common inhabitants of the gut of human infants during the period when milk is a major component of the diet. Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum subspecies longum, and B. longum subspecies infantis have been detected frequently in infant feces, but B. longum subsp. infantis may be disadvantaged numerically in the gut of infants in westernized countries. This may be due to the different durations of breast milk feeding in different countries. Supplementation of the infant diet or replacement of breast milk using formula feeds is common in Western countries. Formula milks often contain galacto- and/or fructo-oligosaccharides (GOS and FOS, respectively) as additives to augment the concentration of oligosaccharides in ruminant milks, but the ability of B. longum subsp. infantis to utilize these potential growth substrates when they are in competition with other bifidobacterial species is unknown. We compared the growth and oligosaccharide utilization of GOS and FOS by bifidobacterial species in pure culture and coculture. Short-chain GOS and FOS (degrees of polymerization [DP] 2 and 3) were favored growth substrates for strains of B. bifidum and B. longum subsp. longum, whereas both B. breve and B. longum subsp. infantis had the ability to utilize both short- and longer-chain GOS and FOS (DP 2 to 6). B. breve was nevertheless numerically dominant over B. longum subsp. infantis in cocultures. This was probably related to the slower use of GOS of DP 3 by B. longum subsp. infantis, indicating that the kinetics of substrate utilization is an important ecological factor in the assemblage of gut communities.IMPORTANCE The kinds of bacteria that form the collection of microbes (the microbiota) in the gut of human infants may influence health and well-being. Knowledge of how the composition of the infant diet influences the assemblage of the bacterial collection is therefore important because dietary interventions may offer opportunities to alter the microbiota with the aim of improving health. Bifidobacterium longum subspecies infantis is a well-known bacterial species, but under modern child-rearing conditions it may be disadvantaged in the gut. Modern formula milks often contain particular oligosaccharide additives that are generally considered to support bifidobacterial growth. However, studies of the ability of various bifidobacterial species to grow together in the presence of these oligosaccharides have not been conducted. These kinds of studies are essential for developing concepts of microbial ecology related to the influence of human nutrition on the development of the gut microbiota.

The molecular weight of ulvan affects the in vitro inflammatory response of a murine macrophage.

Ulvan, a sulfated polysaccharide extracted from the green seaweed genus Ulva, has bioactive properties including an immunomodulating capacity. The immunomodulatory capacity of ulvan from Ulva ohnoi, however, has not been assessed in detail. We depolymerised purified ulvan from U. ohnoi to obtain a range of molecular weight fractions (Mw 7, 9, 13, 21, 209 kDa), which were characterised by constituent sugar analysis, SEC-MALLS, and NMR. Ulvan fractions contained 48.8-54.7 mol% rhamnose, 32.5-35.9 mol% glucuronic acid, 4.5-7.3 mol% iduronic acid, and 3.3-5.6 mol% xylose. 1H and 13C NMR was consistent with hydrolysis occurring at the anomeric centre without further modification to the oligosaccharide structure. The in vitro immunomodulatory effect of ulvan fractions was quantified by measuring levels of inflammatory-mediating signalling molecules released from LPS-stimulated RAW264.7 murine macrophages. All ulvan fractions showed no toxicity on RAW264.7 cells at concentrations below 100 µg mL-1 over 48 h. Secreted interleukin-10 and prostaglandin E2 demonstrated an anti-inflammatory effect by higher molecular weight ulvan fractions at 100 µg mL-1. To a lesser extent, these fractions also enhanced the LPS-induced inflammation through minor increases of IL-1ß and IL-6. This study confirms that ulvan from U. ohnoi has a mild in vitro immunomodulatory effect.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM.

Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 µm long was observed.

Association between the faecal short-chain fatty acid propionate and infant sleep.

The gut microbiota harvests energy from indigestible plant polysaccharides, forming short-chain fatty acids (SCFAs) that are absorbed from the bowel. SCFAs provide energy-presumably after easily digested food components have been absorbed from the small intestine. Infant night waking is believed by many parents to be due to hunger. Our objective was to determine whether faecal SCFAs are associated with longer uninterrupted sleep in infants. Infants (n = 57) provided faecal samples for determining SCFAs (7 months of age), and questionnaire data for determining infant sleep (7 and 8 months). Linear regression determined associations between SCFAs-faecal acetate, propionate and butyrate-and sleep. For each 1% higher propionate at 7 months of age, the longest night sleep was 6 (95% CI: 1, 10) minutes longer at both 7 and 8 months. A higher proportion of total faecal SCFA as propionate was associated with longer uninterrupted infant sleep.

Substrate Use Prioritization by a Coculture of Five Species of Gut Bacteria Fed Mixtures of Arabinoxylan, Xyloglucan, ß-Glucan, and Pectin.

Dietary fiber provides growth substrates for bacterial species that belong to the colonic microbiota of humans. The microbiota degrades and ferments substrates, producing characteristic short-chain fatty acid profiles. Dietary fiber contains plant cell wall-associated polysaccharides (hemicelluloses and pectins) that are chemically diverse in composition and structure. Thus, depending on plant sources, dietary fiber daily presents the microbiota with mixtures of plant polysaccharides of various types and complexity. We studied the extent and preferential order in which mixtures of plant polysaccharides (arabinoxylan, xyloglucan, ß-glucan, and pectin) were utilized by a coculture of five bacterial species (Bacteroides ovatus, Bifidobacterium longum subspecies longum, Megasphaera elsdenii, Ruminococcus gnavus, and Veillonella parvula). These species are members of the human gut microbiota and have the biochemical capacity, collectively, to degrade and ferment the polysaccharides and produce short-chain fatty acids (SCFAs). B. ovatus utilized glycans in the order ß-glucan, pectin, xyloglucan, and arabinoxylan, whereas B. longum subsp. longum utilization was in the order arabinoxylan, arabinan, pectin, and ß-glucan. Propionate, as a proportion of total SCFAs, was augmented when polysaccharide mixtures contained galactan, resulting in greater succinate production by B. ovatus and conversion of succinate to propionate by V. parvula Overall, we derived a synthetic ecological community that carries out SCFA production by the common pathways used by bacterial species for this purpose. Systems like this might be used to predict changes to the emergent properties of the gut ecosystem when diet is altered, with the aim of beneficially affecting human physiology.IMPORTANCE This study addresses the question as to how bacterial species, characteristic of the human gut microbiota, collectively utilize mixtures of plant polysaccharides such as are found in dietary fiber. Five bacterial species with the capacity to degrade polymers and/or produce acidic fermentation products detectable in human feces were used in the experiments. The bacteria showed preferential use of certain polysaccharides over others for growth, and this influenced their fermentation output qualitatively. These kinds of studies are essential in developing concepts of how the gut microbial community shares habitat resources, directly and indirectly, when presented with mixtures of polysaccharides that are found in human diets. The concepts are required in planning dietary interventions that might correct imbalances in the functioning of the human microbiota so as to support measures to reduce metabolic conditions such as obesity.

Utilization of Complex Pectic Polysaccharides from New Zealand Plants (Tetragonia tetragonioides and Corynocarpus laevigatus) by Gut Bacteroides Species.

Pectic polysaccharides from New Zealand (NZ) spinach (Tetragonia tetragonioides) and karaka berries (Corynocarpus laevigatus) were extracted and analyzed. NZ spinach polysaccharides comprised mostly homogalacturonan (64.4%) and rhamnogalacturonan I (5.8%), with side chains of arabinan (8.1%), galactan (2.2%), and type II arabinogalactan (7.1%); karaka berry polysaccharides comprised homogalacturonan (21.8%) and rhamnogalacturonan I (10.0%), with greater proportions of side chains (arabinan, 15.6%; galactan, 23.8%; and type II arabinogalactan, 19.3%). Screening of gut commensal Bacteroides showed that six were able to grow on the NZ spinach extract, while five were able to grow on the karaka berry extract. Analysis of the polysaccharides remaining after fermentation, by size-exclusion chromatography and constituent sugar analysis, showed that the Bacteroides species that grew on these two substrates showed preferences for the different pectic polysaccharide types. Our data suggest that, to completely degrade and utilize the complex pectin structures found in plants, members of Bacteroides and other bowel bacteria work as metabolic consortia.

Polysaccharides from New Zealand Native Plants: A Review of Their Structure, Properties, and Potential Applications.

Water-soluble, non-starch polysaccharides from plants are used commercially in a wide range of food and non-food applications. The increasing range of applications for natural polysaccharides means that there is growing demand for plant-derived polysaccharides with different functionalities. The geographical isolation of New Zealand and its unique flora presents opportunities to discover new polysaccharides with novel properties for a range of applications. This review brings together data published since the year 2000 on the composition and structure of exudate gums, mucilages, and storage polysaccharides extracted from New Zealand endemic land plants. The structures and properties of these polysaccharides are compared with the structures of similar polysaccharides from other plants. The current commercial use of these polysaccharides is reviewed and their potential for further exploitation discussed.

Molecular, rheological and physicochemical characterisation of puka gum, an arabinogalactan-protein extracted from the Meryta sinclairii tree.

A water-soluble polysaccharide (type II arabinogalactan-protein) extracted from the gum exudate of the native New Zealand puka tree (Meryta sinclairii), was characterised for its molecular, rheological and physicochemical properties. In 0.1 M NaCl, the weight average molecular weight (Mw) of puka gum is 5.9 × 106 Da with an RMS radius of 56 nm and z-average hydrodynamic radius of 79 nm. The intrinsic viscosity of the polysaccharide is 57 ml/g with a coil overlap concentration 15% w/w. Together, the shape factor, p, of 0.70 (exponent of RMS radius vs. hydrodynamic radius), Smidsrød-Haug's stiffness parameter B of 0.031 and Mark-Houwink exponent α of 0.375 indicate that the polysaccharide adopts a spherical conformation in solution, similar to gum arabic. The pKa is 1.8. The polysaccharide exhibits a Newtonian to shear-thinning behaviour from 0.2 to 25% w/w. Viscosity of the polysaccharide (1 s-1) decreases with decreasing concentration, increasing temperature, ionic strength, and at acidic pH.

Genomic insights from Monoglobus pectinilyticus: a pectin-degrading specialist bacterium in the human colon.

Pectin is abundant in modern day diets, as it comprises the middle lamellae and one-third of the dry carbohydrate weight of fruit and vegetable cell walls. Currently there is no specialized model organism for studying pectin fermentation in the human colon, as our collective understanding is informed by versatile glycan-degrading bacteria rather than by specialist pectin degraders. Here we show that the genome of Monoglobus pectinilyticus possesses a highly specialized glycobiome for pectin degradation, unique amongst Firmicutes known to be in the human gut. Its genome encodes a simple set of metabolic pathways relevant to pectin sugar utilization, and its predicted glycobiome comprises an unusual distribution of carbohydrate-active enzymes (CAZymes) with numerous extracellular methyl/acetyl esterases and pectate lyases. We predict the M. pectinilyticus degradative process is facilitated by cell-surface S-layer homology (SLH) domain-containing proteins, which proteomics analysis shows are differentially expressed in response to pectin. Some of these abundant cell surface proteins of M. pectinilyticus share unique modular organizations rarely observed in human gut bacteria, featuring pectin-specific CAZyme domains and the cell wall-anchoring SLH motifs. We observed M. pectinilyticus degrades various pectins, RG-I, and galactan to produce polysaccharide degradation products (PDPs) which are presumably shared with other inhabitants of the human gut microbiome (HGM). This strain occupies a new ecological niche for a primary degrader specialized in foraging a habitually consumed plant glycan, thereby enriching our understanding of the diverse community profile of the HGM.