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Arabinoxylan (Wheat Flour; Medium Viscosity)

Arabinoxylan Wheat Flour Medium Viscosity 30 cSt P-WAXYM
Product code: P-WAXYM

3 g

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Content: 3 g
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 10 years under recommended storage conditions
CAS Number: 9040-27-1
Source: Wheat flour
Molecular Weight: 323,000
Purity: ~ 95%
Viscosity: 20-30 cSt
Monosaccharides (%): Arabinose: Xylose = 38: 62
Main Chain Glycosidic Linkage: β-1,4
Substrate For (Enzyme): endo-1,4-β-Xylanase

High purity Arabinoxylan (Wheat Flour; Medium Viscosity) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Recommended substrate for viscometric and reducing-sugar assays of endo-β-D-xylanase activity.

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Megazyme publication
Novel substrates for the automated and manual assay of endo-1,4-β-xylanase.

Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.

endo-1,4-β-Xylanase (EC is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

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Megazyme publication
Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-L-arabinofuranosidase and β-xylosidase.

McCleary, B. V., McKie, V. A., Draga, A., Rooney, E., Mangan, D. & Larkin, J. (2015). Carbohydrate Research, 407, 79-96.

A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 32-α-L-Araf-(1-4)-β-D-xylobiose (A3X), 23-α-L-Araf-(1-4)-β-D-xylotriose (A2XX), 33-α-L-Araf-(1-4)-β-D-xylotriose (A3XX), 22-α-L-Araf-(1-4)-β-D-xylotriose (XA2X), 32-α-L-Araf (1-4)-β-D-xylotriose (XA3X), 23-α-L-Araf-(1-4)-β-D-xylotetraose (XA2XX), 33-α-L-Araf-(1-4)-β-D-xylotetraose (XA3XX), 23 ,33-di-α-L-Araf-(1-4)-β-D-xylotriose (A2+3XX), 23,33-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA2+3XX), 24,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA2+3XXX) and 33,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA3A3XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.

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Formation of Cellulose-Based Composites with Hemicelluloses and Pectins Using Komagataeibacter Fermentation.

Mikkelsen, D., Lopez-Sanchez, P., Wang, D. & Gidley, M. J. (2020). The Plant Cell Wall, 73-87.

Komagataeibacter xylinussynthesizes cellulose in an analogous fashion to plants. Through fermentation of K. xylinus in media containing cell wall polysaccharides from the hemicellulose and/or pectin families, composites with cellulose can be produced. These serve as general models for the assembly, structure, and properties of plant cell walls. By studying structure/property relationships of cellulose composites, the effects of defined hemicellulose and/or pectin polysaccharide structures can be investigated. The macroscopic nature of the composites also allows composite mechanical properties to be characterized. The method for producing cellulose-based composites involves reviving and then culturing K. xylinu in the presence of desired hemicelluloses and/or pectins. Different conditions are required for construction of hemicellulose- and pectin-containing composites. Fermentation results in a floating mat or pellicle of cellulose-based composite that can be recovered, washed, and then studied under hydrated conditions without any need for intermediate drying.

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Structural modifications to water-soluble wheat bran arabinoxylan through milling and extrusion.

Demuth, T., Betschart, J. & Nyström, L. (2020). Carbohydrate Polymers, 240, 116328.

Feruloylated arabinoxylan (AX) is one of the most predominant dietary fiber in cereal grains. In recent decades, soluble AX has gained interest, as a result of its well-established health benefits. Apart from enzymatic degradation during cereal storage, food processing causes AX degradation. These reactions lead to structural modifications and influence both the AX functionalities and its health promoting effects. The aim of this study was to investigate the structural modifications and related property changes of health promoting water-extractable (WE) wheat bran AX through grain milling and extrusion. Multi-detector HPSEC revealed a correlation between Mw, conformational changes and the related viscosity behaviour depending on the processing type. Processing caused molecular degradation of insoluble high Mw AX, which increased the solubility significantly. Moreover, extrusion leaded to a more heterogenic AX fine structure. The detailed characterization of processed dietary fiber may help to facilitate the optimized incorporation of AX in health-promoting foods.

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Barley α‐amylase/subtilisin inhibitor shows inhibitory activity against endogenous xylanase isozyme I of malted barley: A novel protein function.

Sun, J., Xu, F. & Lu, J. (2020). Journal of food Biochemistry, 44(6), e13218.

Mashing process had little influence on the arabinoxylan content in the finished wort. In this paper, a protein with inhibitory activity against the endogenous xylanase isozyme I (X‐I) of malted barley was extracted and purified using a combination of ion‐exchange and size‐exclusion chromatography. The protein was identified as barley α‐amylase/subtilisin inhibitor (BASI). According to the amino acid sequence analysis, BASI was completely different from the previous reported xylanase inhibitors. BASI showed dosage‐dependent inhibitory activity. BASI exhibited a maximum inhibitory activity at 50°C and pH 6.0. BASI inhibited X‐I as a competitive manner.

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A Glycoside Hydrolase Family 62 AL-Arabinofuranosidase from Trichoderma Reesei and Its Applicable Potential during Mashing.

Sun, J., Xu, F. & Lu, J. (2020). Foods, 9(3), 356.

Arabinoxylan is the second most abundant component in the endosperm cell wall of barley and it has been shown to have negative effects on the viscosity and filtration rate of wort and beer. In this study, a glycoside hydrolase (GH) family 62 α-L-arabinofuranosidase (AFase), termed as TrAbf62A, was purified from the culture filtrate of Trichoderma reesei CICC 41495 by a combined chromatographic method. The preferred substrates of the purified TrAbf62A were soluble, highly substituted arabinoxylan oligosaccharides and polymers, similar to the type found in barley grain. TrAbf62A exhibited activity towards oligomeric and polymeric arabinoxylans, as well as colorimetric arabinose-based substrates, thus meeting the criteria to be classified as a type B AFase. TrAbf62A released mainly arabinose and xylose from soluble wheat arabinoxylan, thus indicating a dual lytic enzyme activity. Supplementation of TrAbf62A during mashing, with a loading of 12 mU/g malt, resulted in a 36.3% decrease in arabinoxylan polymer content, a 5.6% reduction in viscosity, and finally, a 22.1% increase in filtration rate. These results revealed that TrAbf62A has a high potential value in improving lautering performance during mashing.

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Quantification of morphochemical changes during in situ enzymatic hydrolysis of individual biomass particles based on autofluorescence imaging.

Kapsokalyvas, D., Loos, J., Boogers, I. A. L. A., Appeldoorn, M. M., Kabel, M. A. & Zandvoort, M. V. (2019). Biopolymers, 111(3), e23347.

Enzymatic hydrolysis of biomass is an established method for producing biofuels. Lignocellulosic biomass such as corn stover is very inhomogeneous material with big variation on conversion rates between individual particles therefore leading to variable recalcitrance results. In this study, we used noninvasive optical microscopy techniques, such as two‐photon microscopy and fluorescence lifetime imaging microscopy, to visualize and analyze morphological and chemical changes of individual corn stover particles pretreated with sulfuric acid during hydrolysis. Morphochemical changes were interpreted based on the fluorescence properties of isolated building blocks of plant cell wall, such as cellulose, hemicellulose, and lignin. Enzymatic hydrolysis resulted in particle size reduction, side wall collapse, decrease of second harmonic signal from cellulose, redshifting of autofluorescence emission, and lifetime decrease attributed to the relative increase of lignin. Based on these observations, tracking compositional change after hydrolysis of individual particles was accomplished. The methodologies developed offer a paradigm for imaging and analyzing enzymatic hydrolysis in vitro and in situ, which could be used for screening enzymes cocktails targeting specific recalcitrant structures or investigating locally enzyme anti‐inhibitory agents.

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Comparison of Japanese and Indian intestinal microbiota shows diet-dependent interaction between bacteria and fungi.

Pareek, S., Kurakawa, T., Das, B., Motooka, D., Nakaya, S., Rongsen-Chandola, T. et al. (2019). NPJ Biofilms and Microbiomes, 5(1), 1-13.

The bacterial species living in the gut mediate many aspects of biological processes such as nutrition and activation of adaptive immunity. In addition, commensal fungi residing in the intestine also influence host health. Although the interaction of bacterium and fungus has been shown, its precise mechanism during colonization of the human intestine remains largely unknown. Here, we show interaction between bacterial and fungal species for utilization of dietary components driving their efficient growth in the intestine. Next generation sequencing of fecal samples from Japanese and Indian adults revealed differential patterns of bacterial and fungal composition. In particular, Indians, who consume more plant polysaccharides than Japanese, harbored increased numbers of Prevotella and CandidaCandida spp. showed strong growth responses to the plant polysaccharide arabinoxylan in vitro. Furthermore, the culture supernatants of Candida spp. grown with arabinoxylan promoted rapid proliferation of Prevotella copri. Arabinose was identified as a potential growth-inducing factor in the Candida culture supernatants. Candida spp. exhibited a growth response to xylose, but not to arabinose, whereas P. copri proliferated in response to both xylose and arabinose. Candida spp., but not P. copri, colonized the intestine of germ-free mice. However, P. copri successfully colonized mouse intestine already harboring Candida. These findings demonstrate a proof of concept that fungal members of gut microbiota can facilitate a colonization of the intestine by their bacterial counterparts, potentially mediated by a dietary metabolite.

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Comparative prebiotic activity of mixtures of cereal grain polysaccharides.

Harris, S., Monteagudo-Mera, A., Kosik, O., Charalampopoulos, D., Shewry, P. & Lovegrove, A. (2019). AMB Express, 9(1), 203.

The main components of the non-starch polysaccharide (NSP) fraction of wheat flour are arabinoxylan (AX) and β-glucan. These are also present in other cereal grains, but their proportions vary with AX being the major component in wheat and rye and β-glucan in barley and oats. Therefore, it was hypothesised that these NSPs could act synergistically when fermented in vitro at the ratios present in the major foods consumed, resulting in increased prebiotic activity. AX and β-glucan were therefore tested in in vitro fermentation studies to assess their prebiotic activity when used individually and/or in different ratios. Short-chain fatty-acids (SCFAs) produced from in vitro fermentation were measured using HPLC and bacterial populations were measured using flow cytometry with fluorescence in situ hybridisation (Flow-FISH). Fermentation of AX alone resulted in a significant bifidogenic activity and increased concentrations of SCFAs, mainly acetate, after 8-24 h of fermentation, however β-glucan alone did not show prebiotic activity. The greatest prebiotic activity, based on concentration of total SCFAs and increases in total bacteria as well as beneficial Bifidobacterium and Clostridium coccoides /Eubacterium groups, was observed when AX and β-glucan were combined at a 3:1 ratio, which corresponds to their ratios in wheat flour which is major source of cereal fibre in the diet. This indicates that the population of bacteria in the human GI tract may be modulated by the composition of the fibre in the diet, to maximise the prebiotic potential.

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High molecular weight mixed-linkage glucan as a mechanical and hydration modulator of bacterial cellulose: Characterization by advanced NMR spectroscopy.

Muñoz-García, J. C., Corbin, K. R., Hussain, H., Gabrielli, V., Koev, T., Iuga, D., Round, A. N., Mikkelsen, D., Gunning, P., Warren, F. J. & Khimyak, Y. Z. (2019). Biomacromolecules, 20(11), 4180-4190.

Bacterial cellulose (BC) consists of a complex three-dimensional organization of ultrafine fibers which provide unique material properties such as softness, biocompatibility, and water-retention ability, of key importance for biomedical applications. However, there is a poor understanding of the molecular features modulating the macroscopic properties of BC gels. We have examined chemically pure BC hydrogels and composites with arabinoxylan (BC–AX), xyloglucan (BC–XG), and high molecular weight mixed-linkage glucan (BC–MLG). Atomic force microscopy showed that MLG greatly reduced the mechanical stiffness of BC gels, while XG and AX did not exert a significant effect. A combination of advanced solid-state NMR methods allowed us to characterize the structure of BC ribbons at ultra-high resolution and to monitor local mobility and water interactions. This has enabled us to unravel the effect of AX, XG, and MLG on the short-range order, mobility, and hydration of BC fibers. Results show that BC–XG hydrogels present BC fibrils of increased surface area, which allows BC–XG gels to hold higher amounts of bound water. We report for the first time that the presence of high molecular weight MLG reduces the density of clusters of BC fibrils and dramatically increases water interactions with BC. Our data supports two key molecular features determining the reduced stiffness of BC–MLG hydrogels, that is, (i) the adsorption of MLG on the surface of BC fibrils precluding the formation of a dense network and (ii) the preorganization of bound water by MLG. Hence, we have produced and fully characterized BC–MLG hydrogels with novel properties which could be potentially employed as renewable materials for applications requiring high water retention capacity (e.g. personal hygiene products).

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Microbial enzymatic degradation of tamarind galactoxyloglucan and wheat arabinoxylan by a porcine faecal inoculum.

Feng, G., Flanagan, B. M., Mikkelsen, D., Williams, B. A. & Gidley, M. J. (2019). Bioactive Carbohydrates and Dietary Fibre, 18, 100183.

This study investigated the enzymatic degradation of solubilised tamarind galactoxyloglucan (GXG) and wheat arabinoxylan (AX), as models for abundant polysaccharides in the primary cell walls of fruits/vegetables and cereal grains, respectively. Using a porcine faecal inoculum, it was demonstrated that the microbial consortia produced surface-located endo-β-xyloglucanase and endo-β-xylanase, to enable the degradation of these polymers into oligosaccharides, close to the microbial surface. Subsequently, these oligosaccharides were completely degraded into monosaccharides by exo-degrading enzymes located both within and on the microbial cell wall, while no enzymatic activities were detected in the cytoplasm. This study reveals a competitive utilisation of GXG and AX by a porcine faecal microbiota, and contributes to our understanding of the mechanisms by which fibre polysaccharides are degraded by the gut microbial community.

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A novel thermostable GH10 xylanase with activities on a wide variety of cellulosic substrates from a xylanolytic Bacillus strain exhibiting significant synergy with commercial Celluclast 1.5 L in pretreated corn stover hydrolysis.

Wang, K., Cao, R., Wang, M., Lin, Q., Zhan, R., Xu, H. & Wang, S. (2019). Biotechnology for Biofuels, 12(1), 48.

Background: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. Results: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65°C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25°C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60°C for 60 h, and its half-lives at 65°C and 70°C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-D-cellobioside (pNPC), and p-nitrophenyl-β-D-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. Conclusions: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.

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Partial replacement of meat by sugar cane fibre: cooking characteristics, sensory properties of beef burgers and in vitro fermentation of sugar cane fibre.

Pluschke, A. M., Feng, G., Williams, B. A. & Gidley, M. J. (2019). International Journal of Food Science & Technology, 54(5), 1760-1768.

In this study, sugar cane fibre (SCF) partially replaced meat in beef burger formulations. The effects of SCF on cook yield, dimensional changes, sensory characteristics of beef burgers and in vitro gut fermentation characteristics were evaluated. Replacing beef with 1 to 5% SCF in burgers significantly increased cook yields from 13.8 ± 0.3 to 59.1 ± 0.3% due to its high water‐binding capacity of 5.89 ± 0.08 g g−1 and oil‐binding capacity of 4.68 ± 0.03 g g−1. The inclusion of SCF improved cooking properties whilst improving sensory characteristics. Burgers with 1% SCF had the highest overall acceptability. SCF was steadily fermented with a porcine faecal inoculum for up to 72 h, producing short‐chain fatty acids. The characteristics of high water/oil binding and fermentability suggest that SCF has the potential to provide a range of dietary fibre benefits, and therefore deserves further study.

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Extracellular depolymerisation triggers fermentation of tamarind xyloglucan and wheat arabinoxylan by a porcine faecal inoculum.

Feng, G., Flanagan, B. M., Williams, B. A., Mikkelsen, D., Yu, W. & Gidley, M. J. (2018). Carbohydrate Polymers, 201, 575-582.

Arabinoxylan (AX) and xyloglucan (XG) are important components of primary cell walls of cereal grains and vegetables/fruits, respectively. Despite the established health benefits of these non-starch polysaccharides, the mechanisms of their utilisation by the gut microbiota are poorly understood. In this study, the mechanisms of solubilised wheat AX and tamarind XG degradation were investigated under in vitro fermentation conditions using a porcine faecal inoculum. Through structural analysis of the polymers, we demonstrate that depolymerisation by microbial surface accessible endo-degrading enzymes occurs prior to active fermentation of AX or XG. Breakdown products are released into the medium and potentially utilised cooperatively by other microbes. Acetate and propionate are the main fermentation products and are produced concurrently with polysaccharide depletion. Butyrate, however, is produced more slowly consistent with it being a secondary metabolite.

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Effect of butyrate and fermentation products on epithelial integrity in a mucus-secreting human colon cell line.

Nielsen, D. S. G., Jensen, B. B., Theil, P. K., Nielsen, T. S., Knudsen, K. E. B. & Purup, S. (2018). Journal of Functional Foods, 40, 9-17.

Butyrate produced by microbial fermentation of dietary fibres beneficially affect the colonic epithelium. Here we enhanced butyrate production through in vitro incubations of different sources of arabinoxylan (AX) with butyrogenic bacteria (Butyrivibrio fibrisolvens, Eubacterium rectale) to explore their potential synbiotic effects on the intestinal epithelial barrier function. B. fibrisolvens incubated with AX produced the highest butyrate levels (15 mM). Sodium-butyrate (1–10 mM) and fermentation products (5% v/v) from B. fibrisolvens increased the barrier function in a human colonic epithelial cell line. This was associated with regulation of different genes involved in epithelial integrity, mucus secretion, apoptosis, oxidative stress, and butyrate transport. In conclusion, butyrate in concentrations that can be achieved by dietary intervention in vivo enhanced the epithelial barrier function in vitro. B. fibrisolvens might be a potential new probiotic for increasing colonic butyrate production in humans, specifically in synbiotic combination with AX, a common DF component of food cereals.

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Binding selectivity of dietary polyphenols to different plant cell wall components: quantification and mechanism.

Phan, A. D. T., Flanagan, B. M., D'Arcy, B. R. & Gidley, M. J. (2017). Food Chemistry, 233, 216-227.

Selected polyphenols exhibited binding selectivity to different cellulose-based composites and apple cell walls. For catechin, cellulose is the dominant binding component, whereas hemicelluloses (xyloglucan and arabinoxylan) apparently did not contribute to polyphenol adsorption in the presence of cellulose. In contrast, ferulic acid and cyanidin-3-glucoside bound to cellulose-based composites and apple cell walls with different affinities, showing that both electrostatic interactions and plant cell wall microstructure were important. Negatively-charged pectin-containing cell walls exhibited the most extensive binding of positively-charged cyanidin-3-glucoside, and bound negatively-charged ferulic acid least effectively. Langmuir binding isotherms predicted the maximum amount of adsorbed polyphenols to be in the range of 30–150% plant cell wall mass. NMR and CLSM analysis support the interactions between polyphenols and plant cell walls and show that although polyphenols are associated with plant cell walls under hydrated conditions, they are not immobilised on polymer surfaces.

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Effect of Water-Extractable Arabinoxylans from Wheat Aleurone and Bran on Lipid Peroxidation and Factors Influencing their Antioxidant Capacity.

Malunga, L. N., Izydorczyk, M. & Beta, T. (2017). Bioactive Carbohydrates and Dietary Fibre, 10, 20-26.

Dietary lipid hydroperoxides (LOOH) are implicated in the rise of colon cancers. We investigated the effect of water-extractable arabinoxylans (WEAX) on lipid peroxidation under simulated gastric conditions. Water-extractable fractions containing mostly arabinoxylans were isolated from wheat aleurone and wheat bran and fractionated by stepwise precipitation with (NH4)2SO4 at 50 and 75% saturation. LOOH concentration (121.2 ± 3.8 µM/g) of grilled chicken breast muscle increased by 30%; however, it remained unaltered or decreased (≤35%) depending on type and concentration of WEAX fractions. Antioxidant capacity of WEAX fractions based on DPPH, ABTS and ORAC was 28.2 – 147.9, 91.2 – 355.3, and 185.9 – 527.5 µM TE/g, respectively. The content of ferulic acid residues in WEAX fractions (R = 0.99) and relative proportions of monosubstituted xylose residues (R = 0.80) influenced the antioxidant capacity. Consumption of diets rich in feruloylated WEAX may offer protection against oxidative damage in the gastrointestinal tract.

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Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.

van Munster, J. M., Thomas, B., Riese, M., Davis, A. L., Gray, C. J., Archer, D. B. & Flitsch, S. L. (2017). Scientific Reports, 7.

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

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Multi-scale characterisation of deuterated cellulose composite hydrogels reveals evidence for different interaction mechanisms with arabinoxylan, mixed-linkage glucan and xyloglucan.

Martínez-Sanz, M., Mikkelsen, D., Flanagan, B. M., Gidley, M. J. & Gilbert, E. P. (2017). Polymer, 124, 1-11.

The interactions of cellulose with other major plant cell wall polysaccharides - arabinoxylan (AX), xyloglucan (XG) and mixed linkage glucans (MLG) - have been investigated by characterising the architecture of composite deuterated cellulose hydrogels by means of SAXS and SANS, combined with XRD, NMR and microscopy. The results indicate that cellulose-AX interactions, limited to the ribbons' surface, take place via a non-specific adsorption mechanism. In contrast, XG and MLG interact specifically with cellulose, forming two different fractions: (i) interfibrillar domains interacting with the cellulose microfibrils and (ii) surface domains, responsible for the cross-linking of ribbons. XG co-crystallises with cellulose, promoting the formation of Iβ-richer microfibrils and forming intercalated amorphous regions. On the other hand, MLG interacts with cellulose forming a paracrystalline coating layer. This structural role of XG and MLG in preventing microfibril aggregation may help explain their key function in the cell expansion process of growing plant tissues.

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Removal of lignin from straw spent pulping liquor using synthetic cationic and biobased flocculants.

Piazza, G. J., Lora, J. H., Wayman, L. I. & Garcia, R. A. (2017). Separation and Purification Technology, 188, 348-357.

The spent pulping liquor (SPL) obtained from straw processed by soda (alkaline) pulping contains dissolved non-sulfonated lignin. The lignin can be separated from SPL using acid or cationic flocculants which are potentially hazardous to the environment. In this study, the performance of the biobased protein flocculant hemoglobin (HEM) with and without added calcium chloride was compared with that of a high charge density synthetic cationic flocculant, poly (diallyldimethylammonium chlorides) (pDADMAC). Turbidity measurements gave overly broad concentration ranges for optimal lignin removal which may be related to the mechanism of flocculation which requires several major steps: Conversion of dissolved lignin to particulate lignin, formation of particulate lignin flocs, and subsequent sedimentation of these flocs. An optimal flocculant concentration or concentration range was estimated using the three methods: average Zeta potential corresponding to the lowest turbidity range; a novel method using the maximum percent pellet mass calculated using the dried masses of the pellet and supernatant after centrifugation; measurement of supernatant lignin using spectroscopy. HEM light absorption interfered with spectroscopic lignin determination, and a method for correcting the measurements was devised. This study shows that HEM is an effective flocculant for nonsulfonated lignin in SPL. The HEM-lignin complex is a potential high protein component of animal feed.

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