Binding capacity of a barley beta-D-glucan to the beta-glucan recognition molecule dectin-1

To clarify whether barley beta-glucans exhibit their biological effects via binding to dectin-1, a pivotal receptor for beta-1,3-glucan, the structure of barley beta-glucan E70-S (BBG-70) was unambiguously investigated by NMR spectroscopy and studied for its binding capacity and specificity to dectin-1 by ELISA. NMR spectroscopy confirmed that BBG-70 contains two different linkage glucans, namely, alpha-glucan and beta-glucan, which are not covalently attached to one another. Beta-glucan within BBG-70 is a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose residues but does not contain the continuous 1,3-linkage. Competitive ELISA revealed that highly purified barley beta-glucan E70-S (pBBG-70) inhibits the binding of soluble dectin-1 to sonifilan (SPG), a beta-1,3-glucan, although at a concentration higher than that of SPG and laminarin. It was found that barley beta-glucan can be recognized by dectin-1, implying that barley beta-glucan might, at least in part, exhibit its biological effects via the recognition by dectin-1 of the ligand sugar structure, which may be formed by 1,3-beta- and 1,4-beta-glucosyl linkage.     

Purification and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei with high specific activity and its gene sequence

Production, purification, and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3-1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The K(m) values of purified β-1,3-1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3-1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3-1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.     

Variation in β‐Glucan Fine Structure,Extractability, and Flour Slurry Viscosity in Oats Due to Genotype and Environment

Effects of genotype and environment on (1→3), (1→4)‐β‐d ‐glucan (β‐glucan) extractability, flour slurry viscosity, and β‐glucan polymer fine structure in oats were tested. One environment had a severe negative effect on slurry viscosity as evaluated with a rotational viscometer. Environment also had a strong effect on β‐glucan extractability, whereas genotype had no significant effect. Fine structure of β‐glucan was evaluated from the frequencies of oligosaccharides from lichenase hydrolysis of the β‐glucan polymer. Significant differences in degree of polymerization (DP) fragment frequencies were found associated with both genotype and growth environment. The high‐β‐glucan cultivar HiFi had lower DP3 fragment frequency and higher frequencies of DP4 and DP6 fragments than other cultivars with moderate β‐glucan concentration. Drier environments tended to yield lower DP3 fragment frequencies as well. Drier environments and genotypes with more β‐glucan synthetic potential may have provided cellular environments with more competition for substrate for β‐glucan synthesis, which appeared associated with lower DP3 fragment frequency. In a separate experiment, we found that extractable β‐glucan had higher frequencies of DP3 fragments and lower frequency of DP4 fragments. The observed variations deserve consideration for influence on functional properties, such as viscosity or health benefit potential.     

MALDI-MS and HPLC quantification of oligosaccharides of lichenase-hydrolyzed water-soluble beta-glucan from ten barley varieties

This study is the first to apply matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to both qualitative and quantitative analyses of oligosaccharides of lichenase-hydrolyzed water-soluble beta-glucan from barley. Compared to high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector, MALDI-MS is a rapid technique with high accuracy and sensitivity and could be used to assess primary structural features of water-soluble beta-glucan from different barley varieties.     

Prebiotic properties of alternansucrase maltose-acceptor oligosaccharides

alpha-(1-6) and alpha-(1-3)-linked oligosaccharides were obtained from the reaction between sucrose and maltose, catalyzed by an alternansucrase isolated from Leuconostoc mesenteroides NRRL B-21297 and separated using a Bio-Gel P2 column in six fractions. Fractions 1, 2, and 3 were mainly composed of DP3, DP4, and DP5, respectively. However, fractions 4, 5, and 6 consisted of mixtures from DP5 to DP9, and they are identified here as DP5.7, DP6.7, and DP7.4, respectively. Potential prebiotic properties of these oligosaccharides were tested using pure and mixed cultures. Generally, in pure studies, most of the tested bacteria failed to grow or grew poorly using the DP6.7 and DP7.4 fractions and showed the greatest growth on DP3. Growth of fecal bacteria on the maltose-acceptor products was tested following an in vitro fermentation method. DP3 showed the highest prebiotic effect, followed by DP4 and DP6.7, whereas DP7.4 did not present any prebiotic activity.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley

A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.     

Biochemical characterization of a novel thermostable beta-1,3-1,4-glucanase (lichenase) from Paecilomyces thermophila

The purification and characterization of a novel extracellular beta-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila J18 were studied. The strain produced the maximum level of extracellular beta-glucanase (135.6 U mL(-1)) when grown in a medium containing corncob (5%, w/v) at 50 degrees C for 4 days. The crude enzyme solution was purified by 122.5-fold with an apparent homogeneity and a recovery yield of 8.9%. The purified enzyme showed as a single protein band on SDS-PAGE with a molecular mass of 38.6 kDa. The molecular masses were 34.6 kDa and 31692.9 Da when detected by gel filtration and mass spectrometry, respectively, suggesting that it is a monomeric protein. The enzyme was a glycoprotein with a carbohydrate content of 19.0% (w/w). Its N-terminal sequence of 10 amino acid residues was determined as H2N-A(?)GYVSNIVVN. The purified enzyme was optimally active at pH 7.0 and 70 degrees C. It was stable within pH range 4.0-10.0 and up to 65 degrees C, respectively. Substrate specificity studies revealed that the enzyme is a true beta-1,3-1,4-D-glucanase. The K m values determined for barley beta-D-glucan and lichenan were 2.46 and 1.82 mg mL(-1), respectively. The enzyme hydrolyzed barley beta-D-glucan and lichenan to yield bisaccharide, trisaccharide, and tetrasaccharide as the main products. Circular dichroism studies indicated that the protein contains 28% alpha-helix, 24% beta-sheet, and 48% random coil. Circular dichroism spectroscopy is also used to investigate the thermostability of the purified enzyme. This is the first report on the purification and characterization of a beta-1,3-1,4-glucanase from Paecilomyces sp. These properties make the enzyme highly suitable for industrial applications.     

Lectin structure-activity: the story is never over

Advances in plant lectin biochemistry have made great strides during the past decade. Technical advances in biophysical techniques and molecular biology, the availability of synthetic oligosaccharides, and characterization of lectins with unique carbohydrate-binding properties are responsible for these advances. Studies in this laboratory support the view that interesting new discoveries are yet to be made. A new lectin was recently isolated from a fungus (Polyporous squamosus) that recognizes the Neu5Ac alpha2,6 Gal beta1,4 GlcNAc/Glc trisaccharide epitope with high affinity. The lectin does not interact with alpha2,3-linked Neu5Ac or Neu5Ac alpha2,6 GalNAc groups as occur in ovine submaxillary mucin. An unusual lectin with two distinctly different carbohydrate-binding sites is present in tubers of Xanthosoma sagittifolium (L). One species of sites recognizes clusters of oligomannosyl residues. The other type of binding site best accommodates a nonsialylated, triantennary oligosaccharide having LacNAc or Lacto-N-biose (Gal beta1,3GlcNAc) groups at its three nonreducing termini. The banana lectin has also been studied. It recognizes both alpha and beta1,3-linked glucosyl oligosaccharides, generates a precipitin curve with the branched trisaccharide Man alpha1,6[Man alpha1,3]Man, and binds to beta-glucans containing beta1,6-glucosyl end groups.     

Galactoglucomannan Oligosaccharides (GGMO) from a molasses byproduct of pine ( Pinus taeda ) fiberboard production

"Temulose" is the trade name for a water-soluble molasses produced on a large scale (300-400 tonnes per year) as a byproduct of the fiberboard industry. The feedstock for Temulose is predominantly a single species of pine ( Pinus taeda ) grown and harvested in stands in southeastern Texas. Because of the method of production, the molasses was predicted to consist of water-soluble hemicelluloses, mainly arabinoxylan-type and galactoglucomannan-type oligosaccharides, plus minor components of lignin, but no detailed structural study had been reported. The structure and composition of the molasses has now been deduced by a combination of MALDI-TOF mass spectrometry, size exclusion chromatography, proton and (13)C NMR techniques, and classic carbohydrate analysis. Limited acid hydrolysis released a series of galactoglucomannan oligosaccharides (GGMO) that were selectively recovered from the acid-labile arabinogalactan by precipitation with ethanol. The precipitate was named "Temulose brown sugar" because of its appearance, and is shown to consist of GGMO with a degree of polymerization (DP) from 4 to 13, with the major component being DP 5-8. The structure of these oligosaccharides is a β-1,4-linked backbone of Man and Glc residues, with occasional α-1,6 branching by single galactosyl units.     

Growth of Trichoderma viride on crude cell wall preparations from barley

Trichoderma viride can utilize crude cell wall preparations from barley starchy endosperm as sole source of carbon and energy. In the process beta-(1-->3)(1-->4)-glucan and arabinoxylan are released. The onset of release of the latter preceded that of glucan, consistent with arabinoxylan being encountered and utilized first. The release of several enzymes was observed during growth of Trichoderma on this substrate: endo-beta-(1-->3)(1-->4)-glucanase, endo-beta-(1-->4)-glucanase, endo-xylanase, arabinofuranosidase, esterase, carboxypeptidase, and "beta-glucan solubilase". It is inferred that each of these activities is necessary for the digestion of this substrate.     

In vitro bile acid binding of flours from oat lines varying in percentage and molecular weight distribution of beta-glucan

Two experimental high beta-glucan oat (Avena sativa) lines (7.64 and 8.05%) and two traditional lines (4.77 and 5.26% beta-glucan) were used to evaluate the effect of beta-glucan quantity and molecular weight on bile acid (BA) binding. The oat flour samples were digested by an in vitro system that simulated human digestion. No significant differences among oat type were found in the overall beta-glucan, starch, and pentosan digestibilities. Considering the standard, cholestyramine, as 100% bound, the relative BA binding for the oat flour samples on a dry matter basis was in the range of 7.5-14.8%, which is higher than the values determined for some other grains and plant materials in the literature. Although the high beta-glucan flours bound a high amount of BA, no significant correlations were found between beta-glucan content in the flours and BA binding. Significant correlations were found between BA binding and insoluble dietary fiber content. Partial hydrolysis with lichenase of the beta-glucan molecules did not affect the BA binding. A summary of all data suggested that BA binding is a multicomponent-dependent process.     

In vivo modeling of beta-glucan degradation in contrasting barley (Hordeum vulgare L.) genotypes

An important determinative of malt quality is the malt beta-glucan content, which in turn depends on the initial barley beta-glucan content as well as the beta-glucan depolymerization by beta-glucanase (EC 3.2.1.73) during malting. Another enzyme, named beta-glucan solubilase, has been suggested to act prior to beta-glucanase; its existence, however, has not been unequivocally proven. We monitored changes in beta-glucan levels and in the development of beta-glucan-degrading enzymes during malting of five lots of contrasting barley genotypes. Two models of in vivo kinetics for beta-glucan degradation were then compared as follows: (i) a biphasic model based on the sequential action of beta-glucan solubilase and beta-glucanase and (ii) a monophasic model assuming that all beta-glucans are depolymerized by beta-glucanase without the previous intervention of another enzyme. Confirmatory regression analysis was used to test the fit of the models to the observed data. Our results show that beta-glucan degradation is mostly monophasic, although some enzyme other than beta-glucanase seems to be required for the early solubilization of a small fraction of insoluble beta-glucans (on average, 7% of total beta-glucans). Furthermore, the genotype-dependent kinetic rate constant (indicating beta-glucan degradability), in addition to beta-glucanase activity, is suggested to play a major role in malting quality.     

Variation in total and soluble beta-glucan content in hulless barley: effects of thermal, physical, and enzymic treatments

Total and soluble beta-glucan content and effects of various treatments of barley grain on extractability and molecular characteristics of soluble beta-glucan were studied. Four types of hulless barley (normal, high amylose, waxy, and zero amylose waxy) from 29 registered and experimental genotypes were analyzed. For each, moisture, protein, amylose, 100 kernel weight, starch, beta-glucan (total and soluble), beta-glucanase activity, and slurry viscosity were determined. Significant differences in total beta-glucan were observed among the groups, with average values of 7. 49%, 6.86%, 6.30%, and 4.38% for high amylose, waxy, zero amylose waxy, and normal barley, respectively. The extractability of beta-glucan in high amylose barley was relatively low (20.6-29.7%) compared to that in normal (29.8-44.3%), zero amylose waxy (34.0-52. 5%), and waxy (36.7-52.7%) barley genotypes. Viscosity of barley flour slurries was affected by the content of soluble beta-glucans, beta-glucanase activity, and molecular weight of beta-glucans. Hydrothermal treatments (autoclaving and steaming) of barley had no effect on extractability of beta-glucans, but prevented enzymic hydrolysis of beta-glucans, and thereby substantially improved their molecular weight. The addition of enzymes (protease and esterase) during extraction and/or physical treatments (sonication) increased extractability of beta-glucans from barley.     

Evaluation of the effect of roasting on the structure of coffee galactomannans using model oligosaccharides

The roasting process induces structural changes in coffee galactomannans. To know more about the reaction pathways that occur during the roasting of coffee, mannosyl and galactomannosyl oligosaccharides, having a degree of polymerization (DP) between 3 and 4, were used as models for galactomannans. These compounds were dry-heated under air atmosphere from room temperature to 200 °C, being maintained at 200 °C for different periods of time. The roasted materials were analyzed by mass spectrometry (ESI-MS, MALDI-MS, and ESI-MSn) and methylation analysis. In the MS spectra were identified several [M+Na]+ ions belonging to a series from a single hexose to 10 hexose residues ([Hex1-10+Na]+). The ions corresponding to their respective mono- and tridehydrated derivatives ([Hex2-10-H2O+Na]+ and [Hex2-10-3H2O+Na]+, respectively) were also identified. ESI-MSn as well as deuterium-labeling and alditol derivatization experiments showed that the tridehydrations occur at the reducing end of the oligosaccharides. The identification of (1→2)- and (1→6)-linked mannose residues and (1→4)-linked glucose residues by methylation analysis allowed the conclusion that transglycosylation and isomerization reactions occur during dry thermal processing.     

Molecular Structure and Some Physicochemical Properties of Buckwheat Starches

The molecular structure and pasting properties of starches from eight buckwheat cultivars were examined. Rapid viscograms showed that buckwheat starches had similar pasting properties among cultivars. The actual amylose content was 16–18%, which was lower than the apparent amylose content (26–27%), due to the high iodine affinity (IA) of amylopectin (2.21–2.48 g/100 g). Amylopectins resembled each other in average chain‐length (23–24) and chain‐length distributions. The long‐chains fraction (LC) was abundant (12–13% by weight) in all the amylopectins, which was consistent with high IA values. The amyloses were also similar among the cultivars in number‐average DP 1,020–1,380 with 3.1–4.3 chains per molecule. The molar‐based distribution indicated that all the amyloses comprised two molecular species differing in molecular size, although the weight‐based distribution showed a single species. A comparison of molecular structures of buckwheat starches to cereal starches indicated buckwheat amylopectins had a larger amount of LC, and their distributions of amylose and short chains of amylopectin on molar basis were similar to those of wheat and barley starches.     

Reduced-Oxidized Glutathione Status as a Potential Index of Oxidative Stress in Mature Cereal Grain

The purpose of this study was to examine the reduced and oxidized glutathione status of selected cereal grains as a potential index of balance between oxidative stress and antioxidant systems, and the contribution of reduced glutathione to the total antioxidant status in cereal grain extracts. Wheat cultivars Almari and Henika, barley cultivars Gregor and Mobek, rye cultivar Dañkowskie Złote, oat cultivar Sławko, and buckwheat cultivar Kora were used. Total antioxidant status (TAS) was measured by the ABTS (2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate)) method. Contents of total phenolic compounds were also determined. Reduced (GSH) and oxidized glutathione (GSSG) (γ-glutamyl-cysteinyl-glycine) were assayed using the spectrofluorimetric method, and results were confirmed by the enzyme recycling method. Correlation coefficient for the GSH/GSSG ratio was r = 0.79. Correlation between TAS and the total phenolic compound content was r = 0.81. Correlation between GSH/GSSG ratio and TAS values was r= 0.46, depending on the extraction system used. The GSH/GSSG ratio may indicate a hierarchy among different cultivars and variance of cereal grains against damage caused by reactive oxygen species. For the main water-soluble antioxidants, our data indicate a potential hierarchy of resistance in investigated cereals against oxidative stress (buckwheat > wheat > barley ≈ rye > oat). This hierarchy was confirmed by the ability of investigated cereal extracts to scavenge superoxide anion radicals in vitro. The reduced-oxidized glutathione status in different cereal grains can be applied as a potential index of balance between oxidative stress and antioxidant systems.     

Influence of corn size distribution on the diastatic power of malted barley and its impact on other malt quality parameters

Detailed studies were carried out on the influence of corn size distribution on the values obtained for diastatic power (DP) of commercially malted barley. Malted barley was screened using a screening box, and the DP activities of the different corns retained on the different compartments of the screening box were determined. The malt samples retained on the 2.8 mm screen had the highest DP activity, whereas the small corns (相似文献   

Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 degrees C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 degrees C. The k(cat) values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s(-1), respectively, whereas the k(cat) values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s(-1), respectively. The K(M) values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.     

Grain Yield,Quality, and Nutrient Concentrations of Feed,Food, and Malt Barley

Barley (Hordeum vulgare L.) is a cereal grown for animal feed, human consumption, and malting. Nutrient concentrations are important as they provide information regarding the dietary values of barley consumed by animals or human beings. In addition, grain nutrient removal may be useful for refining fertilizer recommendations. A study was conducted in 2015 and 2016 investigating the cultivar effects on grain yield, quality, and grain nutrient concentrations and removal under irrigated conditions for two-row barley cultivars. Adjunct and feed cultivars produced the highest yields compared with the all-malt and food cultivars. Specific quality and nutrient values were greater than or equal to in the food cultivar compared to the malt or feed cultivars. Variations in nutrient concentrations were measured among the adjunct and all-malt cultivars, which could potentially affect the malting and brewing qualities. Grain yield, quality, nutrient concentrations and nutrient removal varied among cultivars grown under identical environmental conditions, which may influence end-use.     

Normal phase high-performance liquid chromatography method for the determination of tocopherols and tocotrienols in cereals

The eight vitamers of vitamin E (alpha-, beta-, gamma-, and delta-tocopherols and -tocotrienols) have different antioxidant and biological activities and have different distributions in foods. Some cereals, especially oat, rye, and barley, are good sources of tocotrienols. A fast procedure for the determination of tocopherols and tocotrienols (tocols) in cereal foods was developed. It involves sample saponification and extraction followed by normal phase high-performance liquid chromatography (HPLC). The results have been compared with those found by direct extraction without saponification. The method is sensitive and selective enough to be tested on a wide variety of cereal samples. The highest tocol levels were found in soft wheat and barley ( approximately 75 mg/kg of dry weight). beta-Tocotrienol is the main vitamer found in hulled and dehulled wheats (from 33 to 43 mg/kg of dry weight), gamma-tocopherol predominates in maize (45 mg/kg of dry weight) ), and alpha-tocotrienol predominates in oat and barley (56 and 40 mg/kg of dry weight, respectively).