β-Glucan (Barley; High Viscosity)

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.

Proanthocyanidins (PA) cross-link wheat gluten proteins and dramatically enhance batter viscosity; PA could similarly affect related grains. This study aimed to determine PA effect on viscosity and pasting properties of barley, rye, and oat flours, and the relative contributions of PA interactions with proteins and non-starch polysaccharides (NSP). PA significantly increased batter viscosity, stability, and RVA peak viscosity in rye and barley flours (2.8× and 1.2×, respectively). Interestingly, viscosity peaked distinctively ~75°C in PA-treated rye and barley flours, and their isolated protein-starch systems, indicating prolamins unravelled and complexed with PA during heating. Oat was largely unaffected by PA, likely because of its protein composition. Furthermore, water-soluble rye NSP and arabinoxylans, but not barley β-glucans, significantly increased starch pasting viscosity with PA; oxidative gelation was not a factor. Thus, rye flour viscosity dramatically increased through interactive effects of PA on rye proteins and NSP, which could expand its food applications.

The notion that xyloglucans (XG) play a pivotal role in tethering cellulose microfibrils in the primary cell wall of plants can be traced back to the first molecular model of the cell wall proposed in 1973, which was reinforced in the 1990s by the identification of Xyloglucan Endotransglucosylase/Hydrolase (XTH) enzymes that cleave and reconnect xyloglucan crosslinks in the cell wall. However, this tethered network model has been seriously challenged since 2008 by the identification of the Arabidopsis thaliana xyloglucan-deficient mutant (xxt1 xxt2), which exhibits functional cell walls. Thus, the molecular mechanism underlying the physical integration of cellulose microfibrils into the cell wall remains controversial. To resolve this dilemma, we investigated the cell wall regeneration process using mesophyll protoplasts derived from xxt1 xxt2 mutant leaves. Imaging analysis revealed only a slight difference in the structure of cellulose microfibril network between xxt1 xxt2 and wild-type (WT) protoplasts. Additionally, exogenous xyloglucan application did not alter the cellulose deposition patterns or mechanical stability of xxt1 xxt2 mutant protoplasts. These results indicate that xyloglucan is not essential for the initial assembly of the cellulose network, and the cellulose network formed in the absence of xyloglucan provides sufficient tensile strength to the primary cell wall regenerated from protoplasts.

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.

We present the preparation, morphological analysis, and rheological characterization of ultra-low solid content gels prepared by physically cross-linking TEMPO-oxidized cellulose nanofibrils (TEMPO-CNF) with the soluble plant-cell-wall polysaccharide, mixed-linkage β-glucan (MLG). Of particular note, gel formation was rapidly induced by very small amounts of MLG (e.g. 0.125% w/v) at extremely low TEMPO-CNF concentration (0.05% w/v), which independently were otherwise fluid and thus easily handled. Rheology of these bionanocomposite gel systems as a function of MLG and TEMPO-CNF concentrations revealed that the critical gel concentration of MLG and TEMPO-CNF followed a power-law relation of the concentration of the other component. Surprisingly, these systems also exhibited an additional transition to thick gels at high TEMPO-CNF and MLG concentrations that was visible only at low frequencies. Cryogenic scanning electron microscopy (cryo-SEM) imaging of admixture solutions and gels revealed increased network crowding with increasing MLG amounts. The data are consistent with the hypothesis that non-covalent cellulose-MLG interactions, analogous to those occurring within plant cell walls, drive gel formation. The ability to tune gel physical properties simply by controlling CNF (a promising forest bioproduct) and MLG (a readily available agricultural polysaccharide) fractions at very low solid and polymer content opens new possibilities for material applications in diverse industries.

This study investigated the role of phytate in the Fenton reaction induced oxidative degradation of cereal β-glucan. Viscosity analysis showed that the degradation rate was high in the beginning of oxidation, which fitted to the second order kinetic model. Oat β-glucan contained significant amount of residual phytate and after the residual phytate was removed, faster degradation was shown compared to the original oat β-glucan. Adding the same amount of phytic acid (PA) to the phytate removed β-glucan sample also retarded the degradation but not as efficiently as the residual phytate. Considerable retardation of viscosity loss was shown when the PA to iron ratio was high. The presence of ascorbic acid weakened the retardation effect of phytic acid. Thus, phytate can significantly improve the oxidative stability of β-glucan when the ratio of phytic acid to transition metals and the presence of ascorbic acid are taken into consideration.

Viscosity of cereal β-glucan during digestion is considered to be a vital factor for its health effects. Thus, studies on solution properties and gelation are essential for understanding the mechanisms of the β-glucan functionality. The aim of this study was to investigate the effect of the dissolution temperature on gelation of cereal β-glucan at low concentrations that are relevant for food products. The rheological properties of oat and barley β-glucans (OBG and BBG) using three dissolution temperatures (37°C, 57°C and 85°C) at low concentration (1.5% and 1%, respectively) were studied for 7 days. Additionally, the β-glucans were oxidised with 70 mM H₂O₂ and 1 mM FeSO₄ × 7H₂O as a catalyst, to evaluate the consequence of oxidative degradation on the gelation properties. The study showed that dissolution at 85°C did not result in gelation. The optimal dissolution temperature for gelation of OBG was 37°C and for gelation of BBG 57°C. At these temperatures, also the oxidised OBG and BBG gelled, although the gel strength was somewhat lower than in the non-oxidised ones. Gelation was suggested to require partial dissolution of β-glucan, which depended on the molar mass and aggregation state of the β-glucan molecule. Therefore, the state of β-glucan in solution and its thermal treatment history may affect its technological and physiological functionality.

Hypothesis: Chitosan and sulfated oat β-glucan are materials suitable to create a prebiotic coating for targeted delivery to gastrointestinal system, using the layer by layer technology. Experiment: Quartz crystal microbalance with dissipation (QCM-D), spectroscopic ellipsometry (SE) and atomic force microscopy (AFM) were used to assess the multilayer formation capacity and characterize the resulting coatings in terms of morphology and material properties such as structure and rigidity. The coating of colloidal materials was proven, specifically on L. acidophilus bacteria as measured by changes in the bacterial suspension zeta potential. Viability of coated cells was shown using plate counting method. The coatings on solid surfaces were examined after exposure to mimics of gastrointestinal fluids and a commercially available β-glucanase. Findings: Successful build-up of multilayers was confirmed with QCM-D and SE. Zeta potential values proved the coating of cells. There was 2 log CFU/mL decrease after coating cells with four alternating layers of chitosan and sulfated β-glucan when compared to viability of uncoated cells. The coatings were partially degraded after exposure to simulated intestinal fluid and restructured as a result of β-glucanase treatment, mimicking enzymes present in the microflora of the human gut, but seemed to resist acidic gastric conditions. Therefore, coatings of chitosan and sulfated β-glucan can potentially be exploited as carriers for probiotics and delicate nutraceuticals.

Oxidation of cereal β-glucans may affect their stability in food products. Generally, polysaccharides oxidise via different pathways leading to chain cleavage or formation of oxidised groups within the polymer chain. In this study, oxidation pathways of oat and barley β-glucans were assessed with different concentrations of hydrogen peroxide (H₂O₂) or ascorbic acid (Asc) with ferrous iron (Fe²⁺) as a catalyst. Degradation of β-glucans was evaluated using high performance size exclusion chromatography and formation of carbonyl groups using carbazole-9-carbonyloxyamine labelling. Furthermore, oxidative degradation of glucosyl residues was studied. Based on the results, the oxidation with Asc mainly resulted in glycosidic bond cleavage. With H₂O₂, both glycosidic bond cleavage and formation of carbonyl groups within the β-glucan chain was found. Moreover, H₂O₂ oxidation led to production of formic acid, which was proposed to result from Ruff degradation where oxidised glucose (gluconic acid) is decarboxylated to form arabinose.

Soluble fiber β-glucan is one of the key dietary materials in healthy food products known for reducing serum cholesterol levels. The micro-structural heterogeneity and micro-rheology of high-viscosity barley β-glucan solutions were investigated by the diffusing wave spectroscopy (DWS) technology. By comparing the mean-square displacement (MSD) of the microspheres imbedded in eight concentrations of β-glucan solutions, we found that the solutions exhibited nearly homogeneous behavior at ≤0.1 %, but the material showed a clear degree of heterogeneity at ≥0.25 %. Micro-rheology investigation revealed that β-glucan solutions displayed nearly perfect viscous behavior at ≤0.1 %, but the property changed into viscoelastic one at ≥0.25 %. The magnitude of high-frequency viscoelastic moduli for the 0.25 % - 0.75 % β-glucan solutions can be characterized by ׀G*׀ α ω3/4, which is the semi-flexible polymer behavior. However, the magnitude of high-frequency viscoelastic moduli (׀G*׀) for the 1.0 % - 1.25 % β-glucan solutions is proportional to ω1/2, which is the flexible polymer behavior. All micro-structural heterogeneity and micro-rheological property shifts occurred in relatively small concentration ranges.

A set of fungal polysaccharide samples was characterised by elemental analysis and FTIR spectroscopy and compared with reference chitins, chitosans and β-D-glucans. The nitrogen to carbon (N/C) values and FTIR spectra were used to compare the samples based on their composition. It was found that the N/C ratio correlates well with deacetylation degree (DD) of chitosans and chitin/glucan ratio R_chit of fungal chitin – β-D-glucan complexes with the exception of some samples having significant nitrogen and/or carbon admixtures. FTIR spectroscopy was indicative for the N-acetylation of chitins (chitosans) as well as for the chitin (chitosan) contribution to fungal polysaccharide preparations. Multivariate analyses of the FTIR data (HCA, PCA) discriminated samples and reference materials into several clusters depending on their similarity. Chitosan lactates, chitosan – β-D-glucans and chitin – β-D-glucans of high and low amounts of chitin were successfully discriminated from the reference polysaccharides and from each other. The proposed procedures based on the N/C ratio and multivariate analyses of FTIR spectra may be used in screening fungal polysaccharide preparations.

Purified and semi-purified polysaccharides characteristic of cereals were fermented in vitro with a pig faecal inoculum, using the cumulative gas production technique, to examine the kinetics and end-products of fermentation after 48 h. It was shown that arabinoxylan and mixed linkage (1,3;1,4) β-glucan were rapidly fermented if soluble, while less soluble substrates (insoluble arabinoxylan, maize and wheat starch granules, and bacterial cellulose) were more slowly fermented. Relevant monosaccharides were fermented at very similar rates to soluble polymeric arabinoxylan and β-glucan, showing that depolymerisation was not a limiting step, in contrast to some previous studies. Bacterial cellulose is shown to be a useful model substrate for fermentation of plant cellulose which is difficult to obtain without harsh chemical treatments. Fermentation end-products were related to kinetics, with slow carbohydrate fermentation resulting in increased protein fermentation. Ratios of short-chain fatty acid products were similar for all arabinoxylan and β-glucan substrates.

Several recombinant fungal enzymes (endoglucanase and pectinase) were studied for their interactions with α-expansin in cell wall extension and polysaccharide degradation. Both Cel12A and Cel5A were able to hydrolyze cellulose CMC-Na and mixed-linkage β-glucan. In contrast to Cel5A, Cel12A could also hydrolyze xyloglucan and induce wall extension of cucumber hypocotyls in an in vitro assay. Combining α-expansin, even at high concentrations, with Cel12A did not enhance the maximum/final wall extension rate induced by Cel12A alone. These results strongly suggest that modification/degradation of the xyloglucan molecule/network is the key for cell wall extension, and α-expansin and Cel12A may share the same acting site in the substrate. Pectinase (Pel1, a pectin lyase) enhanced α-expansin-induced wall extension in a concentration-dependent manner, suggesting that the pectin network may normally regulate accessibility of expansin to the xyloglucan–cellulose complex. α-Expansin enhanced Cel12A's hydrolytic activity on cellulose CMC-Na but not on xyloglucan and β-glucan. Expansin did not affect Cel5A's hydrolytic activity. Interestingly, expansin also enhanced Pel1's activity on degrading high esterified pectin. A potential explanation for why expansin could synergistically interact with only certain enzymes on specific polysaccharides is discussed. Additional results also suggested that cell wall swelling may not be a significant event during the action of expansin and hydrolases.

The production of β-glucanase in a mycoparasitic fungus, Coniothyrium minitans, was investigated using a wild type strain 2134. Through ultraviolet (UV) irradiation of strain 2134, four mutants, M11-3B2, A7-3D, A8-1 and A10-4, exhibiting enhanced β-glucanase activities were isolated. Strains A8-1 and A10-4 were constitutive mutants that expressed barley β-glucan hydrolysing activity in the absence of a supplemental inducer (β-glucan substrate). Supernatant from A8-1 and A10-4 cultures grown in potato dextrose broth (PDB), the medium without β-glucan, had maximum levels of β-glucanase activity on average 10 times greater than the wild type strain 2134. M11-3B2 had low levels of constitutive β-glucanase expression and enhanced laminarin hydrolysing activity when grown in presence of β-glucan-rich substrate.