Large-Scale Purification and Characterization of Barley Limit Dextrinase,a Member of the α-Amylase Structural Family

Homogeneous barley limit dextrinase (LD) was isolated on a large scale in a yield of 9 mg/kg of 10-day germinated green malt. This represents a 9,400-fold purification and 29% recovery of the activity in a flour extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The purification protocol consists of precipitation from the extract at 20–70% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DEAE) 650S Fractogel anion-exchange chromatography, and affinity chromatography on β-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mMβ-cyclodextrin and contains a single polypeptide chain of 105 kDa (SDS-PAGE) and pI 4.3. Sequence analysis of tryptic fragments, prepared from 2-vinylpyridinylated LD and purified by RP-HPLC, identified short motifs recognized in β-strand 2, 3, and 5 characteristic of a catalytic (β/α)₈-barrel domain of the α-amylase family of amylolytic enzymes. Barley LD has ≈50 and 85% sequence identity to bacterial pullulanases and rice starch debranching enzyme, respectively. By using ¹H-NMR spectroscopy, LD hydrolyzes specifically α-1,6-glucosidic linkages in pullulan and a branched oligodextrin, 6²-O-α-maltotriosyl-maltotriose, with retention of the α-anomeric configuration. β-Cyclodextrin competitively inhibits the LD activity with K_i of 40 μM, while K_i is 1.9 mM and 2.4 mM for α-cyclodextrin and γ-cyclodextrin, respectively.     

α-Amylolysis of Large Barley Starch Granules

The α-amylolysis of large (volume average 16 μm) barley starch granules was studied by measuring the amount of carbohydrates solubilizing during hydrolysis, and the changes in morphology and molecular structure of the granule residues by scanning electron microscopy, particlesize analysis, size-exclusion chromatography, X-ray diffraction, and differential scanning calorimetry. X-ray diffraction showed that, in the earlier stages of α-amylolysis, both amorphous and crystalline parts of the granules were equally solubilized. More extensive hydrolysis caused a gradual decrease in A-type crystallinity and degradation of the granular structure. Scanning electron microscopy revealed that hydrolysis proceeded through pinholes, and pitted and partially hollow granule residues were formed. The lipid-complexed amylose was less susceptible to α-amylolysis than free amylose and amylopectin. Lipid-complexed amylose started leaching out of the granule residues only after half of the starch had solubilized due to the α-amylase treatment. Even though scanning electron microscopy indicated that there were intact granules left throughout the hydrolysis, the results obtained suggested that α-amylolysis of large barley starch granules proceeded rather evenly among the granules.     

Preparation and Characterization of Films Using Barley and Oat β‐Glucan Extracts

Films for potential food use were prepared from aqueous solutions of β‐glucan extracted from hulled barley, hull‐less barley, and oats. The extracts (75.2–79.3% β‐glucan) also contained proteins, fat, and ash. Glycerol was used as a plasticizer. The films were translucent, smooth, and homogeneous in structure on both sides. Water vapor permeability of films prepared from 4% solutions of β‐glucan extracts were higher than those from 2% solutions, despite similar values for water vapor transmission rate. Mechanical properties were influenced by both β‐glucan source and concentration. The oat β‐glucan films showed higher tensile strength and water solubility, and lower color, opacity, and deformation values than those of barley. Films prepared from hull‐less barley cv. HLB233 remained intact upon immersion in water for 24 hr.     

Glucagel,A Gelling β-Glucan from Barley

A new form of the mixed-linked (1→3),(1→4)-β-d -glucan has been obtained from barley grains using a new extraction and purification process that involves two key steps. A hot-water extraction of the β-glucan from the grain followed by a freeze and thaw of the extract. The commercial product from this process, called Glucagel, forms as a gelatinous or fibrous precipitate, which can be dried. Glucagel has novel functional properties. It forms soft thermoreversible, translucent gels that melt and set at temperatures of ≈60°C.     

α‐Amylase Inhibitors from Wheat Against Development and Toxigenic Potential of Fusarium verticillioides

Fusarium verticillioides is one of the most important pathogens in maize and is a producer of fumonisin B₁ (FB₁). Although reports of its presence in wheat are scarce, the susceptibility of this cereal to fungus of the same genus motivates interest in investigating compounds present in the grain with inhibitory activity against this species. The aim of this study was to extract α‐amylase inhibitors from wheat and apply them in vitro to evaluate its effect on the development and expression of toxigenic potential of F. verticillioides. The α‐amylase inhibitors, both crude (P₀) and purified (P₁), were applied to in vitro culture containing a pathogen mycelium disc. Mycelial growth of the pathogen, glucosamine content, α‐amylase activity, and production of FB₁ were investigated. All protein extracts of wheat showed the ability to inhibit pathogen growth, especially the extract P₀ from cultivar Quartzo, which resulted in a reduction of glucosamine content (66%) and α‐amylase activity (84%). Furthermore, the protein inhibitors showed antifumonisin effect, reducing by 33 and 47% the mycotoxin production when applied as P₀ and P₁, respectively. These results suggest that α‐amylase inhibitor contributed to resistance against pathogen attack, acting in a diversified manner for each fungal species.     

Low α-Amylase Starch Digestibility of Cooked Sorghum Flours and the Effect of Protein

The comparably low starch digestibility of cooked sorghum flours was studied with reference to normal maize. Four sorghum cultivars that represent different types of endosperm were used. Starch digestibilities of 4% cooked sorghum flour suspensions, measured as reducing sugars liberated following α-amylase digestion, were 15–25% lower than for cooked maize flour, but there were no differences among the cooked pure starches. After the flours were predigested with pepsin to remove some proteins, the starch digestibility of cooked sorghum flours increased 7–14%, while there was only 2% increase in normal maize; however, there was no effect of pepsin treatment on starch digestibility if the flours were first cooked and then digested. After cooking with reducing agent, 100 mM sodium metabisulfite, starch digestibility of sorghum flours increased significantly while no significant effect was observed for maize. Also, starch solubility of sorghum flours at 85 and 100°C was lower than in maize, and sodium metabisulfite increased solubility much more in sorghum than in maize. Differential scanning calorimetry results of the flour residue after α-amylase digestion did not show any peaks over a temperature range of 20–120°C, indicating that sorghum starches had all undergone gelatinization. These findings indicate that the protein in cooked sorghum flour pastes plays an important role in making a slowly digesting starch.     

Release of β‐Glucan from Cell Walls of Starchy Endosperm of Barley

β‐Glucan can be solubilized from barley by warm water, with increasing solubilization as the temperature is increased. Substantially less glucan is extracted if the barley is dehusked using sulfuric acid, particularly if the dehusked barley is denatured. This indicates that enzymes capable of solubilizing glucan are present in barley. Various purified enzymes promote the solubilization of glucan from denatured and dehusked barley. Apart from endo‐β‐(1→3)(1→4)‐glucanase, these enzymes include endo‐xylanases, arabinofuranosidase, xyloacetylesterase, and feruloyl esterase. Ferulic acid and, probably, acetyl groups are esterlinked to arabinoxylan, not β‐glucan, in the cell walls of barley starchy endosperm, so the ability of the esterases, xylanases, and arabinofuranosidase to solubilize glucan indicates the pentosan component of the cell wall can restrict the extraction of glucan.     

β-Glucanase Activity and Molecular Weight of β-Glucans in Barley After Various Treatments

β-Glucanase activity interferes with molecular characterization of mixed-linkage (1→3)(1→4)-β-d -glucans (β-glucans). Reductions in β-glucanase activity were determined after barley cvs. Azhul, Waxbar, and Baronesse were treated with autoclaving (120°C, 45 min), calcium chloride (0.05M, 1 hr), 70% ethanol (80°C, 4 hr), hydrochloric acid (0.1N, 1 hr), oven heating (120 and 140°C, 40 min), sodium hydroxide (0.0025M, 1 hr), and 5% trichloroacetic acid (TCA) (40°C, 1 hr). High-performance size-exclusion chromatography (HPSEC) of α-amylase-treated aqueous extracts was used to demonstrate the effects of treatments on the molecular weights of β-glucans. The HPSEC system included multiple-angle, laser light scattering, refractive index, and fluorescence detectors. β-Glucanase activities, ranging from 52 to 65 U/kg of barley, were reduced by autoclaving (50–75%), hot alcohol (67–76%), oven heating (40–96%), CaCl₂ (75–95%), NaOH (76–89%), and TCA (92–96%). Some malt β-glucanase activity remained after most treatments. HCl and TCA treatments reduced extraction and molecular weights of β-glucans. Weight-average molecular weights (M_w) for β-glucans extracted with water at 23°C were low (most <8 × 10⁵). Base treatment (pH 9) and extraction at 100°C for 2.5 hr resulted in the greatest extraction of β-glucans and highest M_w. As a result, the conditions seem appropriate for measurement of physical characteristics of β-glucans in cereal products.     

Effect of Partial Gelatinization and Lipid Addition on α‐Amylolysis of Barley Starch Granules

The effect of partial gelatinization with and without lipid addition on the granular structure and on α‐amylolysis of large barley starch granules was studied. The extent of hydrolysis was monitored by measuring the amount of soluble carbohydrates and the amount of total and free amylose and lipids in the insoluble residue. Similarly to the α‐amylolysis of native large barley starch granules, lipid‐complexed amylose (LAM) appeared to be more resistant than free amylose and amylopectin. Partial gelatinization changed the hydrolysis pattern of large barley starch granules; the pinholes typical of α‐amylase‐treated large barley starch granules could not be seen. Lipid addition during partial gelatinization decreased the formation of soluble carbohydrates during α‐amylolysis. Also free amylose remained in the granule residues and mostly amylopectin hydrolyzed into soluble carbohydrates. These findings indicate that lysophospholipid (LPL) complexation with amylose occurred either during pretreatment or after hydrolysis, and free amylose was now part of otherwise complexed molecules instead of being separate molecules. Partial gelatinization caused the granules to swell somewhat less during heating 2% starch‐water suspensions up to 90°C, and lipid addition prevented the swelling completely. α‐Amylolysis changed the microstructure of heated suspensions. No typical twisting of the granules was seen, although the extent of swelling appeared to be similar to the reference starch. The granules with added LPL were partly fragmented after hydrolysis.     

Comparisons of β-Glucan Content of Barley and Oat

The cholesterol-lowering effect of cereal grains has been associated with the soluble fiber component of dietary fiber. β-Glucan is the major soluble fiber component of barley (Hordeum vulgare L.) and oat (Avena sativa L.). Much research has been conducted to determine the β-glucan content of barley and oat genotypes from many different countries. However, genotypes of both crops always were grown in separate experiments, making direct comparisons between the two crops difficult. This study compares in the same experiment the β-glucan content of nine barley and 10 oat genotypes grown at two locations in each of two years (i.e., four environments) in North Dakota. Averaged across genotypes, total β-glucan content of barley and oat groat was similar. Soluble β-glucan content of oat groat was greater than barley, and oat groat had a greater ratio of soluble-to-total β-glucan than barley. The soluble β-glucan content and ratio of soluble to total β-glucan content of the “best” barley genotypes were less than that of oat genotypes with the highest levels of these two traits.     

Structure of (1→3)(1→4)‐β‐d‐Glucan in Waxy and Nonwaxy Barley

The endosperm cell walls of barley are composed largely of a (1→3)(1→4)‐β‐d ‐glucan commonly known simply as β‐d ‐glucan (Wood 2001). There has been much research into the characteristics of barley β‐glucan because of the influence of this polysaccharide on performance of barley in malting and subsequent brewing of beer, and in feed value, especially for young chicks (MacGregor and Fincher 1993). The potential for β‐glucan to develop high viscosity is a problem in these uses, but from the perspective of human nutrition, this characteristic may be an advantage. The glycemic response to oat β‐glucan is inversely related to (log)viscosity (Wood et al 1994a) and there is evidence to suggest that the lowering of serum cholesterol levels associated with oat and barley products (Lupton et al 1994; Wood and Beer 1998) is at least in part due to the β‐glucan (Braaten et al 1994) and probably also its capacity to develop viscosity in the gastrointestinal tract (Haskell et al 1992).     

Purification and Partial Characterization of an Endoxylanase Inhibitor from Barley

Hordeum vulgare L. xylanase inhibitor (HVXI), an endoxylanase inhibitor with a protein structure, was purified to homogeneity from barley (Hordeum vulgare L.). HVXI is a nonglycosylated monomeric protein, with a molecular weight of ≈40,000 and a pI ≥ 9.3. Although it inhibits different endoxylanases to a varying degree, the activities of an α‐L‐arabinofuranosidase and a β‐d ‐xylosidase were not inhibited. Apparently, HVXI occurs in two molecular forms. These characteristics and the N‐terminal sequences of the composing polypeptides show that HVXI is homologous with Triticum aestivum L. xylanase inhibitor I, an endoxylanase inhibitor from wheat flour.     

Development of a Field Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of α-Amylase in Preharvest-Sprouted Wheat

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed for detection of α-amylase in preharvest sprouted wheat and adapted to rapid field-use formats requiring 15–20 min to perform. Polyclonal and monoclonal antibodies were prepared to detect a mixture of high and low pI isozymes of α-amylase and high pI isozymes only. All antibodies detected α-amylase on immunoblots of either a crude wheat extract or of purified enzyme, but only the polyclonal antibodies functioned in a sandwich ELISA. Depending on the antibody combination, the tube ELISA detected either the high and low pI isozymes of α-amylase or the high pI isozymes only with a detection limit of ≈0.5–1.0 ng/mL of amylase. Wheats with falling numbers (FN) of <350 sec could be discriminated from sound wheats, with decreasing FN producing increasing assay color. Using 130 wheat grain samples, ELISA absorbances for detection of both high and low pI isozymes and of high pI isozymes only were highly positively correlated with amylase enzyme activity and negatively correlated with FN. The correlations were similar for detection of both isozyme families and for detection of high pI isozymes only. Analyses of three sets of wheat samples from different environments demonstrated that the relationship between ELISA absorbance and FN had little dependence on wheat cultivar. The precision of sample analysis using the field ELISA was similar to the precision of FN test apparatus.     

Characterization of β‐ d‐glucosidase extracted from soil fractions

One way to study the state in which stabilized extracellular enzymes persist and are active in the soil is by extraction from the soil, with subsequent fractionation of enzyme–organomineral complexes and characterization of such complexes. In order to investigate the location and characteristics of soil β‐glucosidase, three soil fractions were obtained both from real (undisturbed) soil aggregates and from structural (dispersed in water and physically disrupted) aggregates using two different granulometric procedures. The β‐glucosidase activity of the fraction was then assayed. When the aggregates were dispersed, more than 73% of activity was in the soil microaggregates with diameters of less than 50 μm (SF₅₀). These aggregates were associated with strongly humified organic matter. Solutions of diluted pyrophosphate at neutral pH liberated active β‐glucosidase from all fractions, although the efficacy of extraction varied according to the type of fraction. The SF₅₀ fraction and aggregates of 2000–100 μm obtained by sieving (SF₂₀₀₀) showed the greatest β‐glucosidase activity (34.5 and 36.0%, respectively). Micro‐ and ultrafiltration of SF₅₀ extracts increased the total β‐glucosidase activity, whereas these procedures, applied to the RF₂₀₀₀ fraction, decreased it. Humus–β‐glucosidase complexes in the SF₅₀ fraction, between 0.45 μm and 10⁵ nominal molecular weight limit ( nmwl ) (SF₅₀II) and < 10⁵nmwl (SF₅₀III) showed an optimum pH at 5.4, and in the SF₅₀I fraction (> 0.45 μm) the optimum was 4.0. The stability of β‐glucosidase in the aggregates of the smallest size SF₅₀II and SF₅₀III decreased at acid pHs. The presence of two enzymes (or two forms of the same enzyme) catalysing the same reaction with different values of Michaelis constant and maximum velocity was observed in all but one of the β‐glucosidase complexes extracted and partially purified from the SF₅₀ aggregates.     

Viscosity and Solubility of β‐Glucan Extracted Under In Vitro Conditions from Barley β‐Glucan‐Fortified Bread and Evaluation of Loaf Characteristics

Fortifying bread with β‐glucan has been shown to reduce bread quality and the associated health benefits of barley β‐glucan. Fortification of bread using β‐glucan concentrates that are less soluble during bread preparation steps has not been investigated. The effects of β‐glucan concentration and gluten addition on the physicochemical properties of bread and β‐glucan solubility and viscosity were investigated using a less soluble β‐glucan concentrate, as were the effects of baking temperature and prior β‐glucan solubilization. Fortification of bread with β‐glucan decreased loaf volume and height (P ≤ 0.05) and increased firmness (P ≤ 0.05). Gluten addition to bread at the highest β‐glucan level increased height and volume (P ≤ 0.05) to values exceeding those for the control and decreased firmness (P ≤ 0.05). β‐Glucan addition increased (P ≤ 0.05) extract viscosity, as did gluten addition to the bread with the highest β‐glucan level. Baking at low temperature decreased (P ≤ 0.05) β‐glucan viscosity and solubility, as did solubilizing it prior to dough formulation. Utilization of β‐glucan that is less soluble during bread preparation may hold the key to effectively fortifying bread with β‐glucan without compromising its health benefits, although more research is required.     

Development of an Orange‐Flavored Barley β‐Glucan Beverage

Barley β‐glucan concentrate shows great potential as a functional food ingredient, but few product applications exist. The objectives of this study were to formulate a functional beverage utilizing barley β‐glucan concentrate, and to make a sensory evaluation of beverage quality in comparison to pectin beverages and to assess shelf stability over 12 weeks. Three beverage treatments containing 0.3, 0.5, and 0.7% (w/w) barley β‐glucan were developed in triplicate. Trained panelists found peely‐ and fruity‐orange aroma and sweetness intensity to be similar (P > 0.05) for all beverages tested. Beverage sourness intensity differed among beverages (P ≤ 0.05). Panelists evaluated beverages containing 0.3% hydrocolloid as similar (P > 0.05), whereas beverages with 0.5 and 0.7% β‐glucan were more viscous (P ≤ 0.05) than those with pectin at these levels. Acceptability of beverages was similar according to the consumer panel. Shelf stability studies showed no microbial growth and stable pH for all beverages over 12 weeks. Colorimeter values for most beverages decreased (P ≤ 0.05) during the first week of storage, mostly stabilizing thereafter. With an increase in concentration, β‐glucan beverages became lighter in color (P ≤ 0.05) and cloudier, but these attributes for pectin beverages were not affected (P > 0.05). β‐Glucan beverages exhibited cloud loss during the first three weeks of storage. β‐Glucan can therefore be successfully utilized in the production of a functional beverage acceptable to consumers.     

Molecular Characterization of Barley β-Glucans by Size-Exclusion Chromatography with Multiple-Angle Laser Light Scattering and Other Detectors

Molecular characteristics were determined for mixed-linkage (1→3) (1→4)-β-d -glucans (β-glucans) extracted from Azhul, Crystal, Waxbar, and Prowashonupana barleys. β-Glucans in extracts (with or without α-amylase, protease, hemicellulase, or xylanase treatment) were separated from other components by high-performance size-exclusion chromatography and detected with multiple-angle laser light scattering, refractive index, and fluorometry following postrefractive index treatment with Calcofluor. Pretreatment of barley with 70% ethanol (80°C, 4 hr) reduced β-glucanase activity by ~20%. Hot-alcohol treatment also reduced β-glucan extraction at 23 and 65°C by 42 and 14%, respectively. Molecular weights of β-glucans in the first water extract were generally higher than in succeeding water and alkali extracts. Weight average molecular weights ranged from 0.44 × 10⁶ to 2.34 × 10⁶ g/mol after α-amylase treatment to remove interfering starch. Interference due to pentosans was not demonstrated using enzyme treatments.     

Waxy Starch Barley Genotypes with Reduced β-Glucan Contents

Random inbred lines were produced from a cross between the genotypes Chalky Glenn and Waxy Hector, and two-row lines were classified as waxy or nonwaxy by an iodine staining test. Mean nitrogen and β-glucan contents of the waxy types were higher than those of the nonwaxy types but, in contrast to previous data, mean milling energies of the two groups were not significantly different. Waxy lines with low milling energy had much lower β-glucan levels than those with high milling energy, and they also demonstrated much more extensive cell wall modification during malting. From a trial grown the following season, the waxy types with low milling energy were again identified and had levels of β-glucan content similar to those of nonwaxy types. β-Glucan contents and, particularly, milling energies showed good agreement between seasons. It is suggested that, although waxy starch is usually associated with high β-glucan content, a genetic factor from Chalky Glenn that confers low levels of β-glucan can express in a waxy background.