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.