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Crude β-D-galactosidase from Lactobacillus leichmannii 313 was used to synthesise galacto-oligosaccharides (GOS) from lactose. Using an initial lactose concentration of 170 g/L, the yield of GOS was 52 g/L (i.e. 62% lactose conversion) after 2 h of enzymatic reaction. The highest number of unique individual GOS were generated after 48 h of reaction. ESI-MS and HPAEC analysis of the GOS enriched fractions obtained from 48 h mixtures showed the presence of sugars with degrees of polymerization (DP) ranging from 2 to 6. Further analysis (by NMR spectroscopy) of GOS in fraction 3 (containing ~ 96% w/w GOS) showed non-reducing terminals that were mostly Galp; and reducing ends that were →4)-Glcp, and →6)-Glcp. The most abundant internal linkages in fraction 3 were deduced to be →3)-Galp-(1→, and →6)-Galp-(1→. The predominance of β-D-(1→3, or 6) linkages in GOS fractions suggests that β-D-galactosidase from LL313 is suitable for generating unique GOS from lactose.

Cellulose is a naturally abundant and readily accessible substrate for large scale biorefinery technologies. There has been a significant focus on ionic liquids as alternative solvents for the valo...

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.

Linkage analysis of a xyloglucan from the extracellular medium of suspension cultures of Nicotiana plumbaginifolia showed mostly 4-Glcp and 4,6-Glcp, terminal Xylp and 2-Xylp, and terminal Araf, along with approximately 10% (w/w) O-acetyl groups, equivalent to approximately 0.28 mol acetyl per mol of glycosyl residue. Methylation with methyl trifluoromethanesulfonate under neutral conditions, followed by re-methylation with CD3I under basic conditions, and conversion into partially methylated alditol acetates showed that O-acetyl groups were primarily attached to C-6 of approximately 44% of the 4-Glcp backbone not substituted with Xylp residues and to C-5 of approximately 15% of the terminal Araf residues. These positions of the O-acetyl groups were confirmed by 1H-NMR. Oligosaccharides generated by digestion of native xyloglucan with endo-(1--4)-beta-glucanase were separated by a combination of gel-filtration chromatography and anion-exchange HPLC, and analysed by glycosyl linkage analysis and by electrospray ionisation-mass spectrometry (ESI-MS). The major oligosaccharide subunits were Glc4Xyl2 and Glc5Xyl2, of which 50-60% are substituted with one terminal Araf residue attached to O-2 of a Xylp residue, and a further 20-25% are substituted with two terminal Araf residues attached to O-2 of the Xylp residues. ESI-MS showed that many of the oligosaccharide subunits carried one, two, and, occasionally three O-acetyl groups.

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.

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 (

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

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.

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.

Pectic polysaccharides from New Zealand (NZ) spinach (

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.

The depolymerisation of porcine mucosal heparan sulfate under the action of heparin lyases and analysis by size-exclusion chromatography (SEC) is described. Heparan sulfate treated to enzymic bond scission producing a Δ 4,5 double-bond and quantified by SEC with ultraviolet-visible (UV) spectroscopic detection (230 nm) indicated that the majority of the biopolymer (>85%) was reduced to disaccharides (degree of polymerisation (DP) = 2). However, analysis of the SEC eluant using refractive index (RI), which reflects the mass contribution of the oligosaccharides rather than the molar response of a UV chromophore, indicated that a considerable proportion of the digested HS, up to 43%, was present with DP >2. This was supported by a mass balance analysis. These results contradict the accepted literature where “complete digestion” is routinely reported. Herein we report on the composition and methodology utilised to ascertain the extent of depolymerization and disaccharide composition of this important biopolymer.

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.

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).

Heparan sulfate (HS) is a highly heterogeneous polysaccharide implicated in many important biological processes. Our previous work has demonstrated that a particular affinity-selected HS (referred to henceforth as “HS3”) is capable of enhancing the osteogenic effects of bone morphogenetic protein 2 (BMP2). Here, we gamma-irradiated HS with 26 kGy of ionizing radiation to determine how this affected the structure, composition, and function. Initial structural studies were performed on a commercial preparation of HS as a proof-of-concept. Gamma irradiation of this HS preparation did not significantly alter its structure or composition compared to nonirradiated material, as demonstrated by proton nuclear magnetic resonance spectroscopy, molecular weight analysis using size exclusion chromatography, and disaccharide compositional analysis. When HS3 was gamma irradiated, no significant effect on binding affinity toward BMP2 was observed, based on competitive surface plasmon resonance and differential scanning fluorimetry assays. Furthermore, irradiation did not significantly affect HS3's ability to synergistically enhance the osteogenic effects of BMP2 in vitro; as measured by the relative abundance of osteogenic transcripts in transdifferentiating C2C12 murine myoblasts. Additionally, no significant differences were observed in the levels of alkaline phosphatase (ALP) or calcium deposition in C2C12s treated with BMP2, together with the irradiated, or nonirradiated HS3. Irradiation of HS3 incorporated into collagen type I sponges did not affect its ability to enhance BMP2-mediated ALP expression in C2C12 cells. Our data confirm that gamma irradiation is a cost-effective and viable solution for the sterilization of HS species that allows the retention of its structure and biological function. The work suggests an effective way to incorporate clinically compatible HS species into orthotic implants, scaffolds, and other medical devices for use in the treatment of a range of diseases and disorders. ASTAR (Agency for Sci., Tech. and Research, S’pore)

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.

The role of information technology (IT) is critical in many applications, including accounting. However research into spread sheeting errors indicates financial data emanating from computer applications may contain errors. Auditors review financial data and report, among other things, as to its mathematical accuracy. This study evaluates the reliance trainee auditors place upon IT produced financial data and their application of the concept of mathematical accuracy. Data emanating from a supposedly reliable source was intentionally prepared with mathematical errors and given to participants for evaluation. Despite exposure to concepts such as computer error and audit scepticism, a significant proportion either erroneously concluded the data was accurate and/or failed to appropriately apply the concept of mathematical accuracy. The implications for educators are significant. Basic topics such as data integrity and over-reliance upon IT generated data may need further emphasis during training programs across many disciplines, especially accountancy.

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 .