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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-1β and IL-6. This study confirms that ulvan from U. ohnoi has a mild in vitro immunomodulatory effect.

Background Human health depends on a population of microorganisms that inhabit the gut and contribute to homeostasis of the host, including nutrition, immunity and metabolism. Many of the organisms are interactive and mutually dependent, where the end-products for one organism become the fuel for another through substrate and metabolic cross-feeding. To optimise the gut microbiota using diet, the composition and functionality of the gut microbiota, including these interacting networks, must be understood. Microbial composition and functionality is affected by the structure of the energy input, which is primarily dietary fibre for the gut microbiota. The structure of dietary fibre has been reviewed by carbohydrate chemists, but knowledge of how dietary fibre structure affects the gut microbiota is limited. Scope and approach The hierarchical structures of dietary fibre are reviewed, encompassing macrostructure, mesostructure and molecular structure, and how they are affected by food processing and digestion. These factors are considered in relation to their affects on microbial composition and functionality, to provide insight on the interactions between diet, the microbiota, and human health. Key findings and conclusions Food processing and digestion affect food structure, primarily through the removal of some soluble fractions and increased solubilisation of insoluble fractions. The provision of insoluble carbohydrates to the colon appears important for the sustenance of ‘keystone’ species that play a crucial role in stabilising the gut community. Further work is needed at the microbial strain level to understand the impact of increasing fibre solubility. This should be done in studies using well-characterised carbohydrates that consider the impact of food processing and digestion.

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.

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), Smidsrod-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.

Pectic polysaccharides from New Zealand (NZ) spinach (Tetragonia tetragonioides) and karaka berries (Corynocarpus laevigatus) were extracted and analyzed. NZ spinach polysaccharides comprised mostl...

International audience; 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.

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.

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.

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 β-glucan, a constituent of dietary fiber. B. ovatus depolymerized β-glucan and released, but did not utilize, 3-O-β-cellobiosyl-d-glucose (DP3) and 3-O-β-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-β-glucan utilization locus, as well as carbohydrate transporters in S. variabile and H. hathewayi when in batch coculture. Increased transcription of the β-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 β-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.

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.

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.

© 2020 American Chemical Society. 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.

This study investigated the effect of puka gum (PG) (crude and purified) concentration on its emulsification properties in terms of droplet size distribution, zeta potential, rheological and microstructural properties of the emulsions and their visual phase separation. Different concentrations of PG (1–10% w/w) were used to stabilize 15% w/w soybean oil-in-water emulsions. The effect of constant shear rate and time on the apparent viscosity of PG solutions was also tested. PG stabilised-oil-in-water emulsions exhibited monomodal size distributions with average droplet sizes (d32) below 2 μm at 4% w/w PG. The apparent viscosity of these emulsions was dependent on gum concentration exhibiting a Newtonian behaviour at low PG concentrations (≤4% w/w), with shear-thinning at higher concentrations, becoming slightly more pronounced above 100s−1 and especially at 10% w/w gum. The loss modulus (G”) was higher than the storage modulus (G’) at all concentrations indicating that the liquid-like behaviour dominated at all frequencies. The apparent viscosity of PG solutions, showed an irreversible loss of viscosity after shearing over a period of time, presumably resulting from chain breakage and molecular weight reduction. PG droplets were negatively charge with zeta-potential values in the range of −30 – (−45) mV. Furthermore, PG-stabilised emulsions remained stable against phase separation for at least 30 days. Crude and purified PG exhibited no major differences in terms of their emulsifying properties at 4% w/w suggesting that a purification process may not be required for food formulations. Overall, PG can be considered as a promising natural emulsifier for emulsion-based foods and beverage products.

Polysaccharides from feijoa fruit were extracted and analyzed; the composition of these polysaccharides conforms to those typically found in the primary cell walls of eudicotyledons. The two major polysaccharide extracts consisted of mainly pectic polysaccharides and hemicellulosic polysaccharides [xyloglucan (77%) and arabinoxylan (16%)]. A collection of commensal Bacteroides species was screened for growth in culture using these polysaccharide preparations and placed into five categories based on their preference for each substrate. Most of the species tested could utilize the pectic polysaccharides, but growth on the hemicellulose was more limited. Constituent sugar and glycosyl linkage analysis showed that species that grew on the hemicellulose fraction showed differences in their preference for the two polysaccharides in this preparation. Our data demonstrate that the members of the genus Bacteroides show differential hydrolysis of pectic polysaccharides, xyloglucan, and arabinoxylan, which might influence the structure and metabolic activities of the microbiota in the human gut.

A water-soluble mucilage extracted from the leaves of Hoheria populnea was chemically and physically studied. Monosaccharide composition and linkages were determined by high performance anion exchange chromatography (HPAEC), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Lacebark mucilage was composed of rhamnose, galactose, galacturonic acid and glucuronic acid (2:1:2:1). Proton and 13C NMR spectroscopy, and linkage analysis, revealed a predominantly rhamnogalacturonan I-type (RG I-type) structure comprising of a backbone of →4]-α-D-GalpA-[1→2]-α-L-Rhap-[1→. Data indicated the mucilage likely comprises of a polymer containing several structurally discrete domains or possibly more than one discrete polymer. One domain contains a RG I-type backbone with branching at O-3 of GalpA residues to terminal β-D-GlcpA residues, another similarly contains a RG I-type backbone but is branched at O-4 of the Rhap residues to terminal GalpA residues or oligosaccharides containing α-linked 4-Galp and terminal GalpA residues. A possible third domain contains contiguous 2-Rhap residues, some branched at O-3. Hydrated mucilage exhibited pseudoplastic flow behaviour and viscoelastic properties of an entangled biopolymer network. These rheological behaviours were only slightly affected by pH and may prove advantageous in potential end-product applications including oral pharmaceuticals or as a food ingredient.

We evaluated eight biorefinery processes targeting the extraction of ulvan from Ulva ohnoi. Using a factorial design the effect of three sequential treatments (aqueous extraction of salt; ethanol extraction of pigments; and Na2C2O4 or HCl (0.05 M) extraction of ulvan) were evaluated based on the yield (% dry weight of biomass) and quality (uronic acid, sulfate, protein and ash content, constituent sugar and molecular weight analysis) of ulvan extracted. The aqueous extraction of salt followed by HCl extraction of ulvan gave higher yields (8.2 ± 1.1% w/w) and purity of ulvan than equivalent Na2C2O4 extracts (4.0 ± 1.0% w/w). The total sugar content of HCl extracts (624–670 μg/mg) was higher than Na2C2O4 extracts (365–426 μg/mg) as determined by constituent sugar with ulvan specific monosaccharides contributing 94.7–96.2% and 70.1–84.0%, respectively. Ulvan extracted from U. ohnoi was 53.1 mol% rhamnose, 27.8 mol% glucuronic acid, 10.1 mol% iduronic acid, and 5.3 mol% xylose with molecular weights ranging from 10.5–312 kDa depending on the biorefinery process employed. Therefore, the extraction of high quality ulvan from U. ohnoi is facilitated by an aqueous pre-treatment and subsequent HCl-extraction of ulvan as part of a cascading biorefinery model delivering salt, ulvan, and a protein enriched residual biomass.

Manuka honey (MH), α-cyclodextrin (C) and a formulation containing these two components (MH + C) were subjected to simulated gastrointestinal digestion followed by fermentation with human faecal microbiota. The honey monosaccharides, glucose and fructose were 9- and 3-fold higher respectively in the digesta of MH + C compared with MH. Methylglyoxal (MGO), characteristic of MH was absent after gastric digestion. The precursor of MGO, 1,3-dihydroxyacetone was found to be at a higher concentration in MH + C, compared with MH, after digestion. The MH + C fermenta were more acidic (pH 4.6, p

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, β-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 β-glucan, pectin, xyloglucan, and arabinoxylan, whereas B. longum subsp. longum utilization was in the order arabinoxylan, arabinan, pectin, and β-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.

The Malvaceae family is a group of flowering plants that include approximately 244 genera, and 4225 species. Grewia mollis, and Hoheria populnea (lacebark), are examples of the Malvaceae family that are used in traditional medicine. For this study polysaccharide samples were extracted from the inner bark of Grewia mollis (unmodified (GG) and destarched grewia gum (GGDS)) and from the leaves of Hoheria populnea (lacebark polysaccharide (LB)). Wound healing properties of grewia gum and lacebark polysaccharides were investigated using 3T3 fibroblast cells cultured in supplemented DMEM. Deposition of collagen using van Gieson's stain, expression of the COL1A1 gene which encodes type I collagen using quantitative PCR, and chemotaxis using a scratch plate assay were analysed following treatment of cells with the test polysaccharides. Quantitative PCR results indicated that all three polysaccharides increased the levels of COL1A1 mRNA, with GG showing the greatest fold change. Histological staining also indicated that the fibroblasts treated with GG deposited more collagen than control cells. Additionally, scratch assay data indicated that simulated cell ‘wounds’ treated with each polysaccharide showed increased wound closure rate over a 36 h period post treatment, with GG exhibiting the greatest effect on wound closure. Analysis of the Malvaceae derived polysaccharides indicates that they could have a positive effect on mechanisms that are integral to wound healing, potentially providing greater scientific understanding behind their use in traditional medicine.

Infants fed breast milk harbor a gut microbiota in which bifidobacteria are generally predominant. The metabolic interactions of bifidobacterial species need investigation because they may offer insight into the colonization of the gut in early life. Bifidobacterium bifidum ATCC 15696 hydrolyzes 2′-O-fucosyl-lactose (2FL; a major fucosylated human milk oligosaccharide) but does not use fucose released into the culture medium. However, fucose is a growth substrate for Bifidobacterium breve 24b, and both strains utilize lactose for growth. The provision of fucose and lactose by B. bifidum (the donor) allowing the growth of B. breve (the beneficiary) conforms to the concept of syntrophy, but both strains will compete for lactose to multiply. To determine the metabolic impact of this syntrophic/competitive relationship on the donor, the transcriptomes of B. bifidum were determined and compared in steady-state monoculture and coculture using transcriptome sequencing (RNA-seq) and reverse transcription-quantitative PCR (RT-qPCR). B. bifidum genes upregulated in coculture included those encoding alpha-l-fucosidase and carbohydrate transporters and those involved in energy production and conversion. B. bifidum abundance was the same in coculture as in monoculture, but B. breve dominated the coculture numerically. Cocultures during steady-state growth in 2FL medium produced mostly acetate with little lactate (acetate:lactate molar ratio, 8:1) compared to that in monobatch cultures containing lactose (2:1), which reflected the maintenance of steady-state cells in log-phase growth. Darwinian competition is an implicit feature of bacterial communities, but syntrophy is a phenomenon putatively based on cooperation. Our results suggest that the regulation of syntrophy, in addition to competition, may shape bacterial communities. IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the infant bowel) using in vitro experimentation with bacterial cultures maintained under controlled growth and environmental conditions. We studied the growth of bifidobacteria whose nutrition centered on the hydrolysis of a human milk oligosaccharide. The results revealed responses relating to metabolism occurring in a Bifidobacterium bifidum strain when it provided nutrients that allowed the growth of Bifidobacterium breve, and so discovered biochemical features of these bifidobacteria in relation to metabolic interaction in the shared environment. These kinds of experiments are essential in developing concepts of bifidobacterial ecology that relate to the development of the gut microbiota in early life.

Summary: 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.

Members of the bacterial genus Bifidobacterium generally dominate the fecal microbiota of infants. The species Bifidobacterium longum is prevalent, but the B. longum subsp. longum and B. longum subsp. infantis strains that are known to colonize the infant bowel are not usually differentiated in microbiota investigations. These subspecies differ in their capacities to metabolize human milk oligosaccharides (HMO) and may have different ecological and symbiotic roles in humans. Quantitative PCR provides a quick analytical method by which to accurately ascertain the abundances of target species in microbiotas and microcosms. However, amplification targets in DNA extracted from samples need to be dependably differential. We evaluated the tuf gene sequence as a molecular target for quantitative PCR measurements of the abundances of B. longum subsp. infantis and B. longum subsp. longum in fecal microbiotas. This approach resulted in the detection of a tuf gene variant (operational taxonomic unit 49 [OTU49]) in Chinese infants that has sequence similarities to both B. longum subsp. infantis and B. longum subsp. longum . We compared the genome sequence and growth and transcriptional characteristics of an OTU49 isolate cultured in HMO medium to those of other B. longum subsp. infantis cultures. We concluded from these studies that OTU49 belongs to B. longum subsp. infantis , that dependable quantitative PCR (qPCR) differentiation between the B. longum subspecies cannot be achieved by targeting tuf gene sequences, and that functional genes involved in carbohydrate metabolism might be better targets because they delineate ecological functions. IMPORTANCE High-throughput DNA sequencing methods and advanced bioinformatics analysis have revealed the composition and biochemical capacities of microbial communities (microbiota and microbiome), including those that inhabit the gut of human infants. However, the microbiology and function of natural ecosystems have received little attention in recent decades, so an appreciation of the dynamics of gut microbiota interactions is lacking. With respect to infants, rapid methodologies, such as quantitative PCR, are needed to determine the prevalences and proportions of different bifidobacterial species in observational and microcosm studies in order to obtain a better understanding of the dynamics of bifidobacterial nutrition and syntrophy, knowledge that might be used to manipulate the microbiota and perhaps ensure the better health of infants.

Pseudomonas syringae pv. actinidiae is the major cause of bacterial canker and is a severe threat to kiwifruit production worldwide. Many aspects of the disease caused by P. syringae pv. actinidiae, such as the pathogenicity-relevant formation of a biofilm composed of extracellular polymeric substances (EPSs), are still unknown. Here, a highly virulent strain of P. syringae pv. actinidiae, NZ V-13, was studied with respect to biofilm formation and architecture using a flow cell system combined with confocal laser scanning microscopy. The biofilm formed by P. syringae pv. actinidiae NZ V-13 was heterogeneous, consisting of a thin cellular base layer 5 μm thick and microcolonies with irregular structures. The major component of the EPSs produced by P. syringae pv. actinidiae NZ V-13 bacteria was isolated and identified to be an exopolysaccharide. Extensive compositional and structural analysis showed that rhamnose, fucose, and glucose were the major constituents, present at a ratio of 5:1.5:2. Experimental evidence that P. syringae pv. actinidiae NZ V-13 produces two polysaccharides, a branched α- d -rhamnan with side chains of terminal α- d -Fuc f and an α- d -1,4-linked glucan, was obtained. The susceptibility of the cells in biofilms to kasugamycin and chlorine dioxide was assessed. About 64 and 73% of P. syringae pv. actinidiae NZ V-13 cells in biofilms were killed when kasugamycin and chlorine dioxide were used at 5 and 10 ppm, respectively. Kasugamycin inhibited the attachment of P. syringae pv. actinidiae NZ V-13 to solid surfaces at concentrations of 80 and 100 ppm. Kasugamycin was bacteriostatic against P. syringae pv. actinidiae NZ V-13 growth in the planktonic mode, with the MIC being 40 to 60 ppm and a bactericidal effect being found at 100 ppm. Here we studied the formation, architecture, and composition of P. syringae pv. actinidiae biofilms as well as used the biofilm as a model to assess the efficacies of bactericidal compounds.

A novel anaerobic pectinolytic bacterium (strain 14T) was isolated from human faeces. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 14T belonged to the family Ruminococcaceae , but was located separately from known clostridial clusters within the taxon. The closest cultured relative of strain 14T was Acetivibrio cellulolyticus (89.7 % sequence similarity). Strain 14T shared ~99 % sequence similarity with cloned 16S rRNA gene sequences from uncultured bacteria derived from the human gut. Cells were Gram-stain-positive, non-motile cocci approximately 0.6 µm in diameter. Strain 14T fermented pectins from citrus peel, apple, and kiwifruit as well as carbohydrates that are constituents of pectins and hemicellulose, such as galacturonic acid, xylose, and arabinose. TEM images of strain 14T, cultured in association with plant tissues, suggested extracellular fibrolytic activity associated with the bacterial cells, forming zones of degradation in the pectin-rich regions of middle lamella. Phylogenetic and phenotypic analysis supported the differentiation of strain 14T as a novel genus in the family Ruminococcaceae . The name Monoglobus pectinilyticus gen. nov., sp. nov. is proposed; the type strain is 14T (JCM 31914T=DSM 104782T).

Background Collenchyma serves as a mechanical support tissue for many herbaceous plants. Previous work based on solid-state NMR and immunomicroscopy suggested collenchyma cell walls (CWs) may have similar polysaccharide compositions to those commonly found in eudicotyledon parenchyma walls, but no detailed chemical analysis was available. In this study, compositions and structures of cell wall polysaccharides of peripheral collenchyma from celery petioles were investigated. Results This is the first detailed investigation of the cell wall composition of collenchyma from any plant. Celery petioles were found to elongate throughout their length during early growth, but as they matured elongation was increasingly confined to the upper region, until elongation ceased. Mature, fully elongated, petioles were divided into three equal segments, upper, middle and lower, and peripheral collenchyma strands isolated from each. Cell walls (CWs) were prepared from the strands, which also yielded a HEPES buffer soluble fraction. The CWs were sequentially extracted with CDTA, Na2CO3, 1 M KOH and 4 M KOH. Monosaccharide compositions of the CWs showed that pectin was the most abundant polysaccharide [with homogalacturonan (HG) more abundant than rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II)], followed by cellulose, and other polysaccharides, mainly xyloglucans, with smaller amounts of heteroxylans and heteromannans. CWs from different segments had similar compositions, but those from the upper segments had slightly more pectin than those from the lower two segments. Further, the pectin in the CWs of the upper segment had a higher degree of methyl esterification than the other segments. In addition to the anticipated water-soluble pectins, the HEPES-soluble fractions surprisingly contained large amounts of heteroxylans. The CDTA and Na2CO3 fractions were rich in HG and RG-I, the 1 M KOH fraction had abundant heteroxylans, the 4 M KOH fraction was rich in xyloglucan and heteromannans, and cellulose was predominant in the final residue. The structures of the xyloglucans, heteroxylans and heteromannans were deduced from the linkage analysis and were similar to those present in most eudicotyledon parenchyma CWs. Cross polarization with magic angle spinning (CP/MAS) NMR spectroscopy showed no apparent difference in the rigid and semi-rigid polysaccharides in the CWs of the three segments. Single-pulse excitation with magic-angle spinning (SPE/MAS) NMR spectroscopy, which detects highly mobile polysaccharides, showed the presence of arabinan, the detailed structure of which varied among the cell walls from the three segments. Conclusions Celery collenchyma CWs have similar polysaccharide compositions to most eudicotyledon parenchyma CWs. However, celery collenchyma CWs have much higher XG content than celery parenchyma CWs. The degree of methyl esterification of pectin and the structures of the arabinan side chains of RG-I show some variation in the collenchyma CWs from the different segments. Unexpectedly, the HEPES-soluble fraction contained a large amount of heteroxylans. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1046-y) contains supplementary material, which is available to authorized users.

Puka gum (PG), a high molecular weight arabinogalactan-protein (AGP) polysaccharide extracted from the native New Zealand Meryta sinclarii tree formed electrostatic complexes with whey protein isolate (WPI) under specific conditions. The PG and WPI mixtures were slowly acidified using glucono-delta-lactone (GDL), and the progressive change from soluble polymer solution to soluble complexes and to the formation of insoluble complexes (through the association of soluble complexes) were monitored using visual examination, turbidity, particle size (dynamic light scattering) and zeta-potential measurements at total biopolymer concentration C p = 0.3% w/w, protein:polysaccharide ratio Pr:Ps = 2:1, [GDL] = 0.14% w/w, and T = 20 °C. This process was characterised by identifying the phase boundaries, pH c (pH = 5.7 at which soluble complexes of WP and PG were formed) and pH Φ (pH = 4.7 at which complexes associated to form coacervates). The coacervates formed under these conditions appeared as a sticky, viscoelastic and highly extensible material. The effect of ionic strength (0–100 mM NaCl) on complex formation was also studied and a pH c /pH Φ -ionic strength phase diagram was constructed. Coacervation was fully suppressed at ionic strengths above 60 mM NaCl. Rheological measurements of the insoluble coacervate phase at different final coacervation pH (Pr:Ps = 2:1 and 4:1; C p = 3.0% w/w) showed highest complex modulus G * at approx. pH 3.6 for both Pr:Ps = 2:1 and 4:1, i.e. electrical equivalence pH (EEP), where electrostatic interactions are strongest and the overall charge on the insoluble complex is neutral.

A shear-thickening water-soluble polysaccharide was purified from mucilage extracted from the fronds of the New Zealand black tree fern ( Cyathea medullaris or ‘mamaku’ in Māori) and its structure characterised. Constituent sugar analysis by three complementary methods, combined with linkage analysis (of carboxyl reduced samples) and 1 H and 13 C nuclear magnetic resonance spectroscopy (NMR) revealed a glucuronomannan comprising a backbone of 4-linked methylesterified glucopyranosyl uronic acid and 2-linked mannopyranosyl residues, branched at O-3 of 45% and at both O-3 and O-4 of 53% of the mannopyranosyl residues with side chains likely comprising terminal xylopyranosyl, terminal galactopyranosyl, non-methylesterified terminal glucopyranosyl uronic acid and 3-linked glucopyranosyl uronic acid residues. The weight-average molecular weight of the purified polysaccharide was ∼1.9 × 10 6 Da as determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC–MALLS). The distinctive rheological properties of this polysaccharide are discussed in relation to its structure.

Bifidobacteria are dominant members of the microbial community in the intestinal tract of infants, and studies have shown that glycolipids extracted from the cell surface of these bacteria elicit beneficial immune responses. Accordingly, the identification and structural characterization of glycolipids from the cell wall of bifidobacteria is the first step in correlating glycolipid structure with biological activity. Using whole cell MALDI as a screening tool, we herein present for the first time the identification and structural elucidation of the major polar lipids from Bifidobacterium longum subs. infantis. The lipids identified include an unprecedented plasmenyl cyclophosphatidic acid and a mixed acetal glycolipid, with the latter subsequently being isolated and found to suppress the innate immune response.

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.

Lactobacillus ruminis is an inhabitant of human bowels and bovine rumens. None of 10 isolates (three from bovine rumen, seven from human feces) of L. ruminis that were tested could utilize barley β-glucan for growth. Seven of the strains of L. ruminis were, however, able to utilize tetrasaccharides (3- O -β-cellotriosyl- d -glucose [LDP4] or 4- O -β-laminaribiosyl- d -cellobiose [CDP4]) present in β-glucan hydrolysates for growth. The tetrasaccharides were generated by the use of lichenase or cellulase, respectively. To learn more about the utilization of tetrasaccharides by L. ruminis , whole-transcriptome shotgun sequencing (RNA-seq) was tested as a transcriptional screen to detect altered gene expression when an autochthonous human strain (L5) was grown in medium containing CDP4. RNA-seq results were confirmed and extended by reverse transcription-quantitative PCR assays of selected genes in two upregulated operons when cells were grown as batch cultures in medium containing either CDP4 or LDP4. The cellobiose utilization operon had increased transcription, particularly in early growth phase, whereas the chemotaxis/motility operon was upregulated in late growth phase. Phenotypic changes were seen in relation to upregulation of chemotaxis/flagellar operons: flagella were rarely seen by electron microscopy on glucose-grown cells but cells cultured in tetrasaccharide medium were commonly flagellated. Chemotactic movement toward tetrasaccharides was demonstrated in capillary cultures. L. ruminis utilized 3- O -β-cellotriosyl- d -glucose released by β-glucan hydrolysis due to bowel commensal Coprococcus sp., indicating that cross feeding of tetrasaccharide between bacteria could occur. Therefore, the RNA-seq screen and subsequent experiments had utility in revealing foraging attributes of gut commensal Lactobacillus ruminis .

Alterations to the composition of the bowel microbiota (dysbioses) are associated with particular diseases and conditions of humans. There is a need to discover new, indigestible polysaccharides which are selective growth substrates for commensal bowel bacteria. These substrates (prebiotics) could be added to food in intervention studies to correct bowel dysbiosis. A collection of commensal bacteria was screened for growth in culture using a highly-branched xylan produced by New Zealand flax. Two, Bacteroides ovatus ATCC 8483 and Bacteroides xylanisolvens DSM 18836 grew well on this substrate. The utilisation of the xylan was studied chromatographically and by constituent sugar analysis. The two closely related species utilised the xylan in different ways, and differently from their use of wheat arabinoxylan. The growth of Bacteroides species on other plant xylans having differing chemical structures was also investigated. Novel xylans expand the choice of potential prebiotics that could be used to correct bowel dysbioses.

Carbohydrate polymers are industrially and medically important. For instance, a polysaccharide, alginate (from seaweed), is widely used in food, textile and pharmaceutical industries. Certain bacteria also produce alginate through membrane spanning multi-protein complexes. Using Pseudomonas aeruginosa as a model organism, we investigated the biological function of an alginate degrading enzyme, AlgL, in alginate production and biofilm formation. We showed that AlgL negatively impacts alginate production through its enzymatic activity. We also demonstrated that deletion of AlgL does not interfere with polymer length control, epimerization degree or stability of the biosynthesis complex, arguing that AlgL is a free periplasmic protein dispensable for alginate production. This was further supported by our protein-stability and interaction experiments. Interestingly, over-production of AlgL interfered with polymer length control, suggesting that AlgL could be loosely associated with the biosynthesis complex. In addition, chromosomal expression of algL enhanced alginate O-acetylation; both attachment and dispersal stages of the bacterial biofilm lifecycle were sensitive to the level of O-acetylation. Since this modification also protects the pathogen against host defences and enhances other virulence factors, chromosomal expression of algL could be important for the pathogenicity of this organism. Overall, this work improves our understanding of bacterial alginate production and provides new knowledge for alginate production and disease control.

A polysaccharide from the leaves of Sesamum radiatum was extracted by maceration in deionized water followed by ethanol precipitation then chemically and physically characterised. Monosaccharide composition and linkages were determined by high performance anion exchange chromatography (HPAEC), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy respectively. Sesamum gum was composed of glucuronic acid, mannose, galactose, and xylose with trace quantities of glucose, rhamnose and arabinose. Proton and (13)C NMR spectroscopy, and linkage analysis revealed a glucuronomannan based structure comprising a backbone of →4)-β-d-GlcpA-(1→2)-α-d-Manp-(1→ with side-chains of galactose and xylose. Hydrated sesamum gum displayed temperature independent viscoelastic properties with no thermal hysteresis. Intrinsic viscosity was determined to be 3.31 and 4.40dLg(-1) in 0.1M NaCl and deionised water respectively, while the critical concentration was determined to be 0.1% w/v. The characterisation performed in this study will help direct potential applications of this material in foods and pharmaceuticals.

Gums obtained by extraction from the inner bark of stems can be found in association with starch, which must be digested in order to obtain a refined polysaccharide isolate. In the present study, grewia gum obtained from the inner bark of the stems of Grewia mollis was shown to co-exist with starch and the effect of starch digestion on the physicochemical properties of the resultant polysaccharide was evaluated.\ud \ud The gum was extracted by maceration of the inner bark in deionized water and isolated by a combination of filtration, centrifugation and finally precipitation with absolute ethanol to produce the crude grewia gum extract (GG). The presence and content of starch in the gum sample was determined followed by enzymatic digestion of the starch using α-amylase (Termamyl 120L) to give a starch-free extract (GGDS). Physicochemical properties of the extracts such as total carbohydrates, total protein, differential sugar composition, NMR, intrinsic viscosity and rheological behaviour of the samples were evaluated.\ud \ud The GG extract had total carbohydrate content of ∼ 60 % out of which 11.8 % was starch, and a protein content of 2.3 %. Samples also contained galacturonic and glucuronic acid which were highly acetylated. Both samples had a higher proportion of galacturonic acid than glucuronic acid and contained rhamnose, arabinose, galactose, glucose and xylose as neutral sugars in varying proportions. Rheological measurements on 2 %w/w dispersions of the extracts show minor differences between both the original extract and the de-starched material but were influenced by changes in pH.

It is well accepted that our gut bacteria have coevolved with us in relation to our genetics, diet and lifestyle and are integrated metabolically with us to affect our gut health adversely or beneficially. "Who is there" may vary quite widely between individuals, as might "how they do it", but "what they make" may be less variable. Many different individual species of bacteria can perform the same saccharolytic functions and so the availability of substrate (host or diet-derived) along with the degradative enzymes they possess may be key drivers of gut ecology. In this case study, we discuss detailed microbial ecology and metabolism analysis for three individuals following 48 h of in vitro faecal fermentation, using green kiwifruit as the substrate. In parallel, we have analyzed the chemical changes to the kiwifruit carbohydrates present in the fermenta to close the circle on substrate usage/degradative enzymes possessed/microbes present/microbial byproducts produced. In the absence of host carbohydrate, we see that kiwifruit carbohydrates were differentially utilized to drive microbial diversity, yet resulted in similar byproduct production. The starting ecology of each individual influenced the quantitative and qualitative microbial changes; but not necessarily the metabolic byproduct production. Thus, we propose that it is the consistent functional changes that are relevant for assessment of gut health benefits of any food. We recommend that in this era of large scale genotype/-omics studies that hypothesis-driven, bottom-up research is best placed to interpret metagenomic data in parallel with functional, phenotypic data.

To date, the structures of the glycolipids from Lactobacillus plantarum, a commonly used beneficial probiotic, have not been conclusively assigned. Herein, we report for the first time, the full characterisation of the four principal glycolipids of the L. plantarum cell wall using sugar, linkage and FAME analysis, as well as ESI-MS/MS and 1D- and 2D-NMR spectroscopy, and assign the major glycolipids as being: α-D-Glcp-diglyceride, α-D-Galp-(1→2)-α-D-Glcp-diglyceride, β-D-Glcp-(1→6)-α-D-Galp-(1→2)-6-O-acyl-α-D-Glcp-diglyceride and β-D-Glcp-(1→6)-α-D-Galp-(1→2)-α-D-Glcp-diglyceride.

Cell wall polysaccharides are resistant to digestion and absorption in the human small intestine and are considered to be delivered to the colon in a chemically unaltered state. In this paper, pulp from green and gold kiwifruit was subjected to in vitro upper-intestinal tract digestion and the chemical and physical changes to cell wall polysaccharides (dietary fibre) were investigated. Yields of insoluble fibre decreased slightly with simulated digestion while soluble fibre yields increased. Constituent sugar and glycosyl linkage analysis of the soluble and insoluble fibre fractions revealed that the chemical composition and structure of the non-starch polysaccharides remained largely unchanged. However, the degree of methylesterification of galacturonic acid residues present in the pectin-rich soluble fibre fractions of both fruit decreased with treatment; size-exclusion chromatography detected changes in the molecular weight profiles of these fractions. These changes may affect the physicochemical properties and fermentability of kiwifruit dietary fibre in the large intestine.

Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (β-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.

The cell-wall polysaccharide components of gold kiwifruit ( Actinidia chinensis Planch. ‘Hort16A’, ZESPRI ® GOLD) have been investigated for the first time. Alcohol-insoluble residues (AIRs) were prepared from whole, unpeeled gold and green ( Actinidia deliciosa ) kiwifruit and the constituent sugar and glycosyl linkage compositions determined. AIRs from both kiwifruit contained a high proportion of cellulose; the gold kiwifruit contained a higher proportion of hemicellulosic polysaccharides and lower proportion of pectic polysaccharides compared with the green. The gold kiwifruit AIR was partitioned by sequential extraction with water, aqueous CDTA (0.05 M), aqueous Na 2 CO 3 (0.05 M) and aqueous KOH (1 M then 4 M). The glycosyl linkage compositions of each fraction were determined and the types of cell-wall polysaccharides from the gold fruit were found to be similar to those previously reported for the green fruit.

The water-soluble carbohydrates (WSCs) extracted from the underground parts (rhizome) of Arthropodium cirratum (Rengarenga lily extract); third order branches of Cordyline australis (Cabbage tree extract); a seaweed, Undaria pinnatifida (Undaria extract), and exudates from Acacia pycnantha (Acacia extract) were investigated. Extracts of Rengarenga lily, Cabbage tree, Undaria, and Acacia contained 576, 250, 275 and 794 g/kg DM WSCs, respectively. Constituent sugar analysis by gas–liquid chromatography (GLC) showed that extracts of Rengarenga lily and Cabbage tree contained predominantly fructose and glucose (82–95%). The analysis also revealed that Acacia extract contained mainly galactose (78%) and arabinose (22%) while Undaria extract, contained fucose (55%) and galactose (44%). Thin-layer chromatography (TLC) showed that, on the basis of RF values, fructan composition of Rengarenga lily extract and Cabbage tree extract was different. Cabbage tree extract contained 45% (w/w) fructans while Rengarenga lily extract contained 65% (w/w) fructans. High performance size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) showed that the extracts had varying weight average molecular weight due to differences in the average chain length of the major carbohydrates. Data for the amino acid compositions differed considerably depending on the type of extract. Water-soluble carbohydrate extracts prepared from the four plant sources gave a wide range of WSC (250–794 g/kg DM) due to the different proportions of structural material in different species. It is not known how these differences will impact on animal production, if diets are supplemented with the extracts.

The polysaccharides exuded from both Phormium tenax and Phormium cookianum leaves are acidic xylans with molecular weights in the range 1.3×10 6 –1.7×10 6 Da. 1 H and 13 C NMR spectra show that in both cases the backbone comprises 1,4-linked β- d -Xyl p units and their substituted products, including a structural unit that is both 2- O -substituted and 3- O -acetylated. The sidechains contain α- d -GlcA and are terminated by β- d -Xyl p and α- l -Ara f units in relative proportions that differ between plant varieties. The polysaccharides are so highly branched that chain-terminating units account for approximately half of all structural units. These properties place the Phormium polysaccharides in a class which includes brea, sapote and yabo gums.

The non-structural dry matter content of onion bulbs consists principally of fructose, glucose, sucrose and fructans. The objective of this study was to understand the genetic basis for the wide variation observed in the relative amounts of these carbohydrates. Bulb carbohydrate composition was evaluated in progeny from crosses between high dry matter storage onion varieties and sweet, low dry matter varieties. When samples were analysed on a dry weight basis, reducing sugar and fructan content exhibited high negative correlations and bimodal segregation suggestive of the action of a major gene. A polymorphic SSR marker, ACM235, was identified which exhibited strong disequilibrium with bulb fructan content in F(2:3) families from the 'W202A' x 'Texas Grano 438' mapping population evaluated in two environments. This marker was mapped to chromosome 8 in the interspecific population 'Allium cepa x A. roylei'. Mapping in the 'Colossal Grano PVP' x 'Early Longkeeper P12' F2 population showed that a dominant major gene conditioning high-fructan content lay in the same genomic region. QTL analysis of total bulb fructan content in the intraspecific mapping population 'BYG15-23' x 'AC43' using a complete molecular marker map revealed only one significant QTL in the same chromosomal region. This locus, provisionally named Frc, may account for the major phenotypic differences in bulb carbohydrate content between storage and sweet onion varieties.

A gum that exudes from the wounded trunk of the New Zealand native tree Meryta sinclairii has been isolated. The gum was completely precipitated by the β-glucosyl Yariv reagent and was thus determined to be an arabinogalactan-protein (AGP). It contained >95% w/w carbohydrate and only 2% w/w protein with a high level of hydroxyproline. SEC-MALLS showed that the gum had a weight-average molecular weight of 4.45×10 6 Da compared with 6.02×10 5 Da for gum arabic. Constituent sugar and linkage analyses were consistent with polymers comprised of a highly branched backbone of 1,3-linked galactopyranosyl (Gal p ) residues, with side-chains made up of arabinofuranose- (Ara f ) containing oligosaccharides, terminated variously by rhamnopyranosyl (Rha p ), arabinopyranosyl (Ara p ), Gal p and glucuronopyranosyl (Glc p A) residues. Analysis by one-dimensional and two-dimensional 1 H and 13 C NMR experiments confirmed the linkage analyses. The structure of the gum is discussed in comparison with the structure of gum arabic and other AGPs.

The molecular mechanisms of alginate polymerization/modification/secretion by a proposed envelope-spanning multiprotein complex are unknown. Here, bacterial two-hybrid assays and pulldown experiments showed that the catalytic subunit Alg8 directly interacts with the proposed copolymerase Alg44 while embedded in the cytoplasmic membrane. Alg44 additionally interacts with the lipoprotein AlgK bridging the periplasmic space. Site-specific mutagenesis of Alg44 showed that protein-protein interactions and stability were independent of conserved amino acid residues R17 and R21, which are involved in c-di-GMP binding, the N-terminal PilZ domain, and the C-terminal 26 amino acids. Site-specific mutagenesis was employed to investigate the c-di-GMP-mediated activation of alginate polymerization by the PilZAlg44 domain and Alg8. Activation was found to be different from the proposed activation mechanism for cellulose synthesis. The interactive role of Alg8, Alg44, AlgG (epimerase), and AlgX (acetyltransferase) on alginate polymerization and modification was studied by using site-specific deletion mutants, inactive variants, and overproduction of subunits. The compositions, molecular masses, and material properties of resulting novel alginates were analyzed. The molecular mass was reduced by epimerization, while it was increased by acetylation. Interestingly, when overproduced, Alg44, AlgG, and the nonepimerizing variant AlgG(D324A) increased the degree of acetylation, while epimerization was enhanced by AlgX and its nonacetylating variant AlgX(S269A). Biofilm architecture analysis showed that acetyl groups promoted cell aggregation while nonacetylated polymannuronate alginate promoted stigmergy. Overall, this study sheds new light on the arrangement of the multiprotein complex involved in alginate production. Furthermore, the activation mechanism and the interplay between polymerization and modification of alginate were elucidated., IMPORTANCE This study provides new insights into the molecular mechanisms of the synthesis of the unique polysaccharide, alginate, which not only is an important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa but also has, due to its material properties, many applications in medicine and industry. Unraveling the assembly and composition of the alginate-synthesizing and envelope-spanning multiprotein complex will be of tremendous significance for the scientific community. We identified a protein-protein interaction network inside the multiprotein complex and studied its relevance with respect to alginate polymerization/modification as well as the c-di-GMP-mediated activation mechanism. A relationship between alginate polymerization and modification was shown. Due to the role of alginate in pathogenesis as well as its unique material properties harnessed in numerous applications, results obtained in this study will aid the design and development of inhibitory drugs as well as the commercial bacterial production of tailor-made alginates.

At about 20-year intervals, since at least the 1860s, there have been accounts of the accumulation of large quantities of mucilage in the waters off the northern coast of the South Island of New Zealand. On a few occasions these events have been associated with harmful effects, such as the mass mortality of marine fauna and the impediment of fishing activities, though previous attempts to identify the causative organism(s) or the environmental conditions which promote its development have been unsuccessful. In January 2000, there was a moderate re-occurrence of mucilage accumulation in Tasman Bay that led to the identification of the primary origin of this material as the planktonic, thecate, dinoflagellate Gonyaulax hyalina (Ostenfeld et Schmidt). Macro-aggregate accumulation was occurring in the water column when dinoflagellate cell numbers were relatively low. Experimental work on cultures of this organism showed that the dinoflagellate produced transparent exo-polymers (TEPs) composed primarily of galactose and glucose monomers (44.8 and 29.6 mol%, respectively). Analysis of natural and cultured mucilage showed this had a high inorganic content (>60% (w/w)). A substantial proportion (40% (w/w)) of this inorganic fraction was composed of calcium that apparently played an important role in stabilizing the gel. Phosphorus was also enriched in the mucilage. It is hypothesized that the high inorganic N/P ratio observed in the water column at the time of mucilage formation was an effect rather than a cause of this event, which resulted in the scavenging of particulate material from the water column and enrichment of sediments and bottom waters with organic material and phosphorus.

Summary Starch granules isolated from leaves and seeds of Lolium temulentum L. were roughly spherical with an irregular perimeter having mean diameters of 1.7 and 4.1 μm respectively. On a dry mass basis, 47 % of the seed and maximally 4 % of the leaf were starch. Starch structures were deduced from a combination of data obtained using acid-hydrolysis/Smith degradation, glycosyl-linkage analysis/GC-MS, iodine complex formation, isoamylase digestion, high performance size exclusion chromatography and high performance anion exchange chromatography/pulsed amperometry. The mean degree of polymerisation (DP) of seed starch was 4800 (Mr = 778 kDa) and consisted entirely of a highly branched, amylopectin-like polymer with an average chain length of DP = 46 ± 6 and an average of 104 chains per molecule. Leaf starch was 98.8 % by mass of a highly-branched, amylopectin-like polymer of DP = 5850 (Mr 947 kDa) containing on average, 136 chains of mean DP = 43 ± 6. The remaining 1.2 % of the leaf starch was isoamylase-resistant, of high Mr (5850 kDa) and was designated amylose. Both starches were unusual in that the chain lengths were longer by a factor of 2, than is normal for amylopectins and also because they contained little or no long 1,4-linked chains, as determined by isoamylase digestion. HPAEC of isoamylase fragments from leaf starch revealed a bi-modal distribution of branch chain lengths which was not evident in the seed starch. With the exceptions of this bi-modality and the trace of amylose in the leaf starch, the structure of the starches from the two organs were very similar. Seed starch exhibited an anomalous reaction with iodine. The chromogen was deep blue with a λmax = 608 nm, indicative of the presence of long amylose chains. On digestion with isoamylase, all of the mass of starch was converted to low molecular mass with an iodine λmax shifted to 567 nm. Hence, short 1,4-linked glucan chains which were contiguous through 1,6-linkages formed blue chromogens and can give the appearance of amylose.

A fructan polymerase activity was partially purified and concentrated by sequential acid and salt precipitation from extracts of excised, illuminated leaves of timothy grass (Phleum pratense). The polymerase catalysed the de novo synthesis of oligo-and polyfructan from sucrose as sole substrate at near-physiological rates (0.5-0.9 mg g -1 fresh mass h -1 ; 0.9-1.5 nkat g -1 ). Rates of in vitro polymerisation were high, at up to 4.1 mg cm -3 h -1 (7.1 nkat cm -3 ) of total products of degree of polymerization greater than 2 (DP > 2). The trisaccharides 1-kestose and 6-kestose together with oligosaccharides of up to DP ≤ c. 10 were synthesized in under 2 h at 30°C. In longer incubations, ethanol-precipitable polymers of DP = c. 10-35 (1.6-5.7 kDa) were detected by anion-exchange chromatography and pulsed amperometry. When this polymeric product was used as a primer and re-incubated with fresh enzyme and sucrose, abundant polymers of up to DP = 50 (8.1 kDa) were formed. The structure of the polymeric enzyme product was compared with native fructan from timothy leaves and with standard inulin, using glycosyl-linkage analysis followed by identification of partially methylated alditol acetate derivatives by GC-MS. The deduced structure was a linear (unbranched) 2,6-linked fructose chain terminated with glucose and fructose. The linkage structures of the native and enzyme-generated polymers were identical, increasing confidence in the physiological relevance of the activity. After ultracentrifugation of tissue homogenates at 265 000 g ar , the polymerase remained in the supernatant, demonstrating no tight association with particulate components. The polymerizing reaction was dependent on enzyme concentration, requiring at least 3 g fresh mass equivalent cm 3 (c. 2.7 nkat cm 3 ) for the efficient in vitro generation of fructans of DP > 3. In common with other trisaccharide-synthesizing and oligofructan-glycosylating enzymes from grasses, the polymerase reaction exhibited both a maximal velocity at pH 5.0-5.5 and a low affinity for sucrose. The polymerization reaction did not saturate fully even at 1.5 M sucrose, and the concentration causing half maximal velocity (apparent K m ) was c. 560 mM. The preparation contained substantial invertase activity (1.8 mg sucrose g -1 fresh mass h -1 = 1.5 nkat g -1 fresh mass) with a K m for sucrose hydrolysis of 5 mM. A single peak of polymerase activity with an M r of 51 kDa was recovered from size-exclusion chromatography (SEC). Invertases of M r 51 and 110 kDa were identified in the preparation. The 110-kDa invertase isoform exhibited no polymerase activity, but synthesized trisaccharide (mainly 1-kestose) from sucrose. The 51-kDa isoform co-eluted with the polymerase. The trisaccharide fraction produced by this isoform contained abundant 1-and 6-kestose. After SEC, the purification of the polymerase was 41-fold relative to the original tissue homogenate. The properties of enzymatic polymerization of fructan are discussed with respect to the physiology of accumulation in grass leaves and other systems.

The rheological properties of three xyloglucans (XGs) from the extracellular medium of suspension cultured Nicotiana plumbaginifolia cells, apple pomace and tamarind seeds, with different structural features and molecular weights have been studied. The molecular weight (weight average) of the Nicotiana , apple pomace and tamarind seed XGs determined by multi-angle laser light scattering were 129, 219 and 833 kDa, respectively. Tamarind seed XG had the highest viscosity and Nicotiana XG had the lowest viscosity, with that of apple pomace XG intermediate. The viscosity of apple pomace XG at 5% w/v was almost equivalent to that of tamarind seed XG at 2% w/v, but their behaviour at high shear rates differed; both XGs were non-Newtonian in their rheological properties, but that from tamarind seeds showed more pronounced shear-thinning. The viscosity of Nicotiana XG at 5% w/v was almost equivalent to tamarind seed XG at 0.5% w/v, displaying Newtonian behaviour. Modification of the molecular weight of the XGs and their degree of branching revealed that differences in viscosity between the molecules, and their shear-field behaviour, was due primarily to differences in molecular weight. Removal of fucose residues from apple pomace XG decreased the viscosity of solutions from 8 to 4 mPa·s, whereas removal of both fucose and galactose from apple pomace XG, resulted in precipitation from solution. Deacetylation of Nicotiana XG also resulted in precipitation from solution.

SUMMARY Chlorarachniophytes are amoeboid/flagellate eukaryotes that harbor reduced green algal endosymbionts. The carbohydrate stores of chlorarachniophyte algae have been investigated using methylation analysis to determine monosaccharide composition. An appreciable quantity of long chain β-1,3 glucan occurs in these algae. Immunogold electron microscopy using an antibody specific for (β-1,3 glucans localized (β-1,3 glucans within a vacuole in the host cell cytoplasm. The results suggest that photosynthate produced by the endosymbiont is stored by the host. Implications of the data for endosymbiosis are discussed.