β-Glucan (Barley; Medium Viscosity)

Traditional enzyme-based methods for measurement of fructan were designed to measure just inulin and branched-type (agave) fructans. The enzymes employed, namely exo-inulinase and endo-inulinase, give incompletely hydrolysis of levan. Levan hydrolysis requires a third enzyme, endo-levanase. This paper describes a method and commercial test kit (Megazyme Fructan Assay Kit) for the determination of all types of fructan (inulin, levan, and branched) in a variety of animal feeds and pet foods. The method has been validated in a single laboratory for analysis of pure inulin, agave fructan, levan, and a range of fructan containing samples. Quantification is based on complete hydrolysis of fructan to fructose and glucose by a mixture of exo-inulinase, endo-inulinase, and endo-levanase, followed by measurement of these sugars using the PAHBAH reducing sugar method which gives the same color response with fructose and glucose. Before hydrolysis of fructan, interfering sucrose and starch in the sample are specifically hydrolyzed and removed by borohydride reduction. The single-laboratory validation (SLV) outlined in this document was performed on commercially available inulin (Raftiline) and agave fructan (Frutafit^©), levan purified from Timothy grass, two grass samples, a sample of legume hay, two animal feeds and two barley flours, one of which (Barley MAX^©) was genetically enriched in fructan through plant breeding. Parameters examined during the validation included working range, target selectivity, recovery, LOD, LOQ, trueness (bias), precision (repeatability and intermediate precision), robustness, and stability. The method is robust, quick, and simple.

Fermentation of β-glucan fractions from barley [average molecular mass (MM), of 243, 172, and 137 kDa] and oats (average MM of 230 and 150 kDa) by the human faecal microbiota was investigated. Fractions were supplemented to pH-controlled anaerobic batch culture fermenters inoculated with human faecal samples from three donors, in triplicate, for each substrate. Microbiota changes were monitored by fluorescent in situ hybridization; groups enumerated were: Bifidobacterium genus, Bacteroides and Prevotella group, Clostridium histolyticum subgroup, Ruminococcus-Eubacterium-Clostridium (REC) cluster, Lactobacillus-Enterococcus group, Atopobium cluster, and clostridial cluster IX. Short-chain fatty acids and lactic acid were measured by HPLC. The C. histolyticum subgroup increased significantly in all vessels and clostridial cluster IX maintained high populations with all fractions. The Bacteroides-Prevotella group increased with all but the 243-kDa barley and 230-kDa oat substrates. In general β-glucans displayed no apparent prebiotic potential. The SCFA profile (51 : 32 : 17; acetate : propionate : butyrate) was considered propionate-rich. In a further study a β-glucan oligosaccharide fraction was produced with a degree of polymerization of 3-4. This fraction was supplemented to small-scale faecal batch cultures and gave significant increases in the Lactobacillus-Enterococcus group; however, the prebiotic potential of this fraction was marginal compared with that of inulin.

A commercially available enzymic method for the quantitative measurement of (1→3),(1→4)-β-glucan has been simplified to allow analysis of up to 10 grain samples in 70 min or of 100–200 samples by a single operator in a day. These improvements have been achieved with no loss in accuracy or precision and with an increase in reliability. The glucose oxidase/peroxidase reagent has been significantly improved to ensure colour stability for periods of up to 1 h after development. Some problems experienced with the original method have been addressed and resolved, and further experiments to demonstrate the quantitative nature of the assay have been designed and performed.

A method developed for the quantification of (1→3)(1→4)-β-D-glucan in barley flour has been modified to allow its use in the measurement of this component in malt, wort, beer and spent grain. For malt samples, free D-glucose was first removed with aqueous ethanol. Quantification of the polymer in wort and beer samples involved precipitation of the β-glucan with ammonium sulphate followed by washing with aqueous ethanol to remove free D-glucose. Spent grain was lyophilised and milled and then analysed by the method developed for malt. In all cases, the β-glucan was depolymerised with lichenase and the resultant β-gluco-oligosaccharides hydrolysed to D-glucose with β-D-glucosidase. The released D-glucose was then specifically determined using glucose oxidase-peroxidase reagent.

Mixed linkage β-glucane and pentosanes (mainly arabinoxylanes) are the major endosperm cell-wall polysaccharides of barley and wheat respectively. These polysaccharides, although minor components of the whole grain, significantly affect the industrial utilization of these cereals. The modification of barley corns during malting requires the dissolution of the β-glucan in the cell-wall of the starch endosperm. High β-glucane concentration in wort and beer effect the rate of filtration and can also lead to precipitate or gel formation in the final product. In a similar manner, pentosane is thought to cause filtration problems with wheat starch hydrolysates by increasing viscosity and by producing gelatinous precipitate which blocks filters. Ironically, it is this same viscosity building and water binding capacity which is considered to render pentosanes of considerable value in dough development and bread storage (anti-staling functions). In the current paper, some aspects of the beneficial and detrimental effects of pentosans and β-glucan in the industrial utilization of wheat and barley are discussed. More specifically, enzymic methods for the preparation, analysis and identification of these polysaccharides and for the removal of their functional properties, are described in detail.

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in values are determined for each sample allowing the direct measurement of β-glucan in malt samples. α-Amylase does not interfere with the assay. The method is suitable for the routine analysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0-1 for barley flour samples containing 3-8 and 4-6% (w/w) β-glucan content.

Background: Lignocellulose biomass is the most abundant and renewable material in nature. The objectives of this study were to characterize two endoglucanases TrepCel3 and TrepCel4, and determine the effect of the combination of them (1.2 mg TrepCel3, 0.8 mg TrepCel4) on in vitro rumen fermentation characteristics. In this study, three nature lignocellulosic substrates (rice straw, RS; wheat straw, WS; leymus chinensis, LC) were evaluated for their in vitro digestibility, gas, NH₃-N and volatile fatty acid (VFA) production, and microbial protein (MCP) synthesis by adding enzymatic combination. Methods: Two endoglucanases’ genes were successfully expressed in Escherichia coli (E. coli) BL21 (DE3), and enzymatic characteristics were further characterized. The combination of TrepCel3 and TrepCel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability. Results: The maximum enzymatic activity of TrepCel3 was determined at pH 5.0 and 40°C, while TrepCel4 was at pH 6.0 and 50°C. They were stable over the temperature range of 30 to 60°C, and active within the pH range of 4.0 to 9.0. The TrepCel3 and TrepCel4 had the highest activity in lichenan 436.9 ± 8.30 and 377.6 ± 6.80 U/mg, respectively. The combination of TrepCel3 and TrepCel4 exhibited the highest efficiency at the ratio of 60:40. Compared to maximum hydrolysis of TrepCel3 or TrepCel4 separately, this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4% for RS, 236.7% for wheat straw WS, 222.4% for LC and 131.1% for sugar beet pulp (SBP). Supplemental this combination enhanced the dry matter digestion (DMD), gas, NH₃-N and VFA production, and MCP synthesis during in vitro rumen fermentation. Conclusions: The TrepCel3 and TrepCel4 exhibited the synergistic relationship (60:40) and significantly increased the saccharification yield of lignocellulosic substrates. The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates. This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants. If possible, in the future, experiments in vivo should be carried out to fully evaluate its effect.

Improving the redispersion and recycling of dried cellulose nanofibrils (CNFs) without compromising their nanoscopic dimensions and inherent mechanical properties are essential for their large-scale applications. Herein, mixed-linkage (1,3;1,4)-β-d-glucan (MLG) was studied as a rehydration medium for the redispersion and recycling of dried CNFs, benefiting from the intrinsic affinity of MLG to both cellulose and water molecules as inspired from plant cell wall. MLG from barley with a lower molar ratio of cellotriosyl to cellotetraosyl units was found homogeneously coated on CNFs, facilitating rehydration of the network of individualized CNFs. The addition of barley MLG did not impair the mechanical properties of the CNF/MLG composites as compared to neat CNFs nanopaper. With the addition of 10 wt% barley MLG, dry CNF/MLG composite film was successfully redispersed in water and recycled with well-maintained mechanical properties, while lichenan from Icelandic moss, cationic starch, and xyloglucan could not help the redispersion of dried CNFs.

The lignocellulosic biomass comprises three main components: cellulose, hemicellulose, and lignin. Degradation and conversion of these three components are attractive to biotechnology. This study aimed to prospect fungal lignocellulolytic enzymes with potential industrial applications, produced through a temporal analysis using Hymenaea courbaril and Tamarindus indica seeds as carbon sources. α-L-arabinofuranosidase, acetyl xylan esterase, endo-1,5-α-L-arabinanase, β-D-galactosidase, β-D-glucosidase, β-glucanase, β-D-xylosidase, cellobiohydrolase, endoglucanase, lichenase, mannanase, polygalacturonase, endo-1,4-β-xylanase, and xyloglucanase activities were determined. The enzymes were produced for eight filamentous fungi: Aspergillus fumigatus, Trametes hirsuta, Lasiodiplodia sp., two strains of Trichoderma longibrachiatum, Neocosmospora perseae, Fusarium sp. and Thermothelomyces thermophilus. The best producers concerning enzymatic activity were T. thermophilus and T. longibrachiatum. The optimal conditions for enzyme production were the media supplemented with tamarind seeds, under agitation, for 72 h. This analysis was essential to demonstrate that cultivation conditions, static and under agitation, exert strong influences on the production of several enzymes produced by different fungi. The kind of sugarcane, pretreatment used, microorganisms, and carbon sources proved limiting sugar profile factors.

Transglycanases are enzymes that remodel the primary cell wall in plants, potentially loosening and/or strengthening it. Xyloglucan endotransglucosylase (XET; EC 2.4.1.207), ubiquitous in land plants, is a homo-transglucanase activity (donor, xyloglucan; acceptor, xyloglucan) exhibited by XTH (xyloglucan endotransglucosylase/hydrolase) proteins. By contrast, hetero-trans-β-glucanase (HTG) is the only known enzyme that is preferentially a hetero-transglucanase. Its two main hetero-transglucanase activities are MLG : xyloglucan endotransglucosylase (MXE) and cellulose : xyloglucan endotransglucosylase (CXE). HTG is highly acidic and found only in the evolutionarily isolated genus of fern-allies, Equisetum. We now report genes for three new highly acidic HTG-related XTHs in E. fluviatile (EfXTH-A, EfXTH-H and EfXTH-I). We expressed them heterologously in Pichia and tested the encoded proteins’ enzymic activities to determine whether their acidity and/or their Equisetum-specific sequences might confer high hetero-transglucanase activity. Untransformed Pichia was found to secrete MLG-degrading enzyme(s), which had to be removed for reliable MXE assays. All three acidic EfXTHs exhibited very predominantly XET activity, although low but measurable hetero-transglucanase activities (MXE and CXE) were also detected in EfXTH-H and EfXTH-I. We conclude that the extremely high hetero-transglucanase activities of Equisetum HTG are not emulated by similarly acidic Equisetum XTHs that share up to 55.5% sequence identity with HTG.

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.

Wheat (Triticum aestivum L) and barley (Hordeum vulgare L) are two important cereals cultivated worldwide. The effect of aleurone cell wall structure on water diffusion and distribution within wheat and barley grains was evaluated at different relative humidity levels. Time domain nuclear magnetic resonance was used to measure the transverse relaxation time T₂ of grains. Two water states were distinguished within grains, namely W₁ (lower mobility) and W₂ (higher mobility). Grains with thicker aleurone layer cell walls had a higher W₂ The water-absorption and desorption rates were mainly determined by the thickness of the aleurone cell walls and decreased with increasing cell wall thickness. The higher W₂ values observed in grains with thicker aleurone cell walls with the a water content of 2.0% (w/w) and 12.1% (w/w) were probably related to a higher water motion within the grains, in response to higher porosity. Arabinoxylan and (1,3)(1,4)-β-glucan alternating multilayer films were prepared, each film had 20 layers, one layer was approximately 1 µm thick. The film was used as a model to simulate the aleurone layers. These results show that cell walls of the aleurone layer regulate the diffusion and distribution of water within grains.

Background: The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked D-xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity. Results: The expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila, Sporotrichum thermophile) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated TtXyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4-O-methyl-D-glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, TtXyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of TtXyn30A to cleave linear parts of xylan and UXOS as well. Conclusions: Hereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, TtXyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits.

Lignocellulosic materials are abundant, renewable and are emerging as valuable substrates for many industrial applications such as the production of second-generation biofuels, green chemicals and pharmaceuticals. However, the recalcitrance and the complexity of cell wall polysaccharides require multiple enzymes for their complete conversion to oligo- and monosaccharides. The endoglucanases from GH45 family are a small and relatively poorly studied group of enzymes with potential industrial application. The present study reports cloning, heterologous expression and functional characterization of two GH45 endoglucanases from mesophilic fungi Gloeophyllum trabeum (GtGH45) and thermophilic fungi Myceliophthora thermophila (MtGH45), which belong to subfamilies GH45C and GH45A, respectively. Both enzymes have optimal pH 5.0 and melting temperatures (Tm) of 66.0°C and 80.9°C, respectively, as estimated from circular dichroism experiments. The recombinant proteins also exhibited different mode of action when incubated with oligosaccharides ranging from cellotriose to cellohexaose, generating mainly cellobiose and cellotriose (MtGH45) or glucose and cellobiose (GtGH45). The MtGH45 did not show activity against oligosaccharides smaller than cellopentaose while the enzyme GtGH45 was able to depolymerize cellotriose, however with lower efficiency when compared to larger oligosaccharides. Furthermore, both GHs45 were stable up to 70°C for 24 h and useful to enhance initial glucan hydrolysis rates during saccharification of sugarcane pith by a mixture of cellulolytic enzymes. Recombinant GHs45 from diverging subfamilies stand out for differences in substrate specificity appearing as new tools for preparation of enzyme cocktails used in cellulose hydrolysis.

Chinese Nong-flavor (NF) daqu has been enriched with plenty of active enzymes by man-made environment for thousand years. Based on our previous metatranscriptomics, an endo-β-glucanase gene (NFEg16A), which showed high expression level in NF daqu, was directly obtained and expressed in Escherichia coli BL21 (DE3). NFEg16A shared the highest sequence identity of 87% with endo-1,3-1,4-β-glucanase from Paecilomyces thermophile. It was optimally active at pH 6.5 and 60°C and highly stable (>75% residual activity) at pH 3-8 and temperature 30-90°C. The activity of NFEg16A was strongly inhibited by 10 mM Fe³⁺ and Hg²⁺. Compared with endoglucanases with high similarities, NFEg16A was more stable at 70°C and had higher half-lives of 3.4 h and 1.4 h at 80°C and 90°C, respectively. Its specific activity was 85.3 U/mg on barley β-glucan. Moreover, NFEg16A could efficiently hydrolyze substrate at high concentration of 15 mg/mL, and released glucose and cellobiose as its main end-products. Therefore, this work to some extent verified the important role of NFEg16A in NF daqu, and it would stimulate the acquisition of more enzymes from NF daqu to improve the baijiu quality in future. High thermostability of NFEg16A could also strengthen its potential applications in feed industry.

Nitric oxide (NO) is a critical signaling molecule involved in the regulation of a wide variety of physiological processes across every domain of life. In most aerobic and facultative anaerobic bacteria, heme-nitric oxide/oxygen binding (H-NOX) proteins selectively sense NO and inhibit the activity of a histidine kinase (HK) located on the same operon. This NO-dependent inhibition of the cognate HK alters the phosphorylation of the downstream response regulators. In the marine bacterium Saccharophagus degradans (Sde), in addition to a typical H-NOX (Sde 3804)/HK (Sde 3803) pair, an orphan H-NOX (Sde 3557) with no associated signaling protein has been identified distant from the H-NOX/HK pair in the genome. The characterization reported here elucidates the function of both H-NOX proteins. Sde 3557 exhibits a weaker binding affinity with the kinase, yet both Sde 3804 and Sde 3557 are functional H-NOXs with proper gas binding properties and kinase inhibition activity. Additionally, Sde 3557 has an NO dissociation rate that is significantly slower than that of Sde 3804, which may confer prolonged kinase inhibition in vivo. While it is still unclear whether Sde 3557 has another signaling partner or shares the histidine kinase with Sde 3804, Sde 3557 is the only orphan H-NOX characterized to date. S. degradan is likely using a dual-H-NOX system to fine-tune the downstream response of NO signaling.

Climate change threatens to release abundant carbon that is sequestered at high latitudes, but the constraints on microbial metabolisms that mediate the release of methane and carbon dioxide are poorly understood. The role of viruses, which are known to affect microbial dynamics, metabolism and biogeochemistry in the oceans, remains largely unexplored in soil. Here, we aimed to investigate how viruses influence microbial ecology and carbon metabolism in peatland soils along a permafrost thaw gradient in Sweden. We recovered 1,907 viral populations (genomes and large genome fragments) from 197 bulk soil and size-fractionated metagenomes, 58% of which were detected in metatranscriptomes and presumed to be active. In silico predictions linked 35% of the viruses to microbial host populations, highlighting likely viral predators of key carbon-cycling microorganisms, including methanogens and methanotrophs. Lineage-specific virus/host ratios varied, suggesting that viral infection dynamics may differentially impact microbial responses to a changing climate. Virus-encoded glycoside hydrolases, including an endomannanase with confirmed functional activity, indicated that viruses influence complex carbon degradation and that viral abundances were significant predictors of methane dynamics. These findings suggest that viruses may impact ecosystem function in climate-critical, terrestrial habitats and identify multiple potential viral contributions to soil carbon cycling.

Brewer’s spent grain (BSG) constitutes various valuable carbohydrates that may contribute to a healthy diet. These components may be obtained from BSG via hydrothermal treatment (HT), a procedure for dissolving water-inextricable carbohydrates. The objective of this study was to investigate HT as an environmentally friendly technology for extracting high-molecular-weight fiber with proven beneficial effects on human health. Cellulose, β-glucan, and arabinoxylan (AX) served as model substances and were subjected to auto-hydrolysis at different temperatures and reaction times. The results were evaluated in terms of structural and chemical characteristics. When the treatment temperature was increased, the original weight-average molar mass of AX (370 kDa) and β-glucan (248 kDa) decreased gradually (<10 kDa), and the molar mass distribution narrowed. Further investigations focused on the heat-induced formation and elimination of monosaccharides and undesirable by-products. The concentrations of by-products were successfully described by kinetic models that can be used to optimize the hydrolysis process.

The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine.

Mixed-linkage glucan : xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land-plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-D-glucan (MLG), with radioactive oligosaccharides of xyloglucan as acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography, HPLC and gel-permeation chromatography. Equisetum HTG consistently cleaved the MLG at the third consecutive β-( 1→4)-bond following (towards the reducing terminus) a β-( 1→3)-bond. It then formed a β-( 1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its XTH subfamily membership. Using size-homogeneous barley MLG as donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.