Effect on Dough Functional Properties of Partial Fractionation,Redistribution, and In Situ Deposition of Wheat Flour Gluten Proteins Exposed to Water,Ethanol, and Aqueous Ethanol

The application of the cold‐ethanol laboratory fractionation method to the bulk separation of wheat starch and gluten is accompanied by incidental dissolution, removal, or redeposition of a small part of the functional gliadin protein. The new distribution resulting from process incidental redeposition of soluble components or by purposeful add‐back of soluble and leached components can lead to differences in functionality and more difficult recovery of native properties. To assess this issue, we exposed several wheat flour types to ethanol and water (50–90% v/v) solutions, water, and absolute ethanol at 22°C and –12°C. The exposure was mass conserving (leached components returned to substrate by evaporation of the solvent without separation of phases) or mass depleting (leached components not returned to substrate). The result of the mass‐conserving contact would be flour with altered protein distributions and intermolecular interactions. The result of the mass‐depleting contact would also include altered protein content. Furthermore, the mass‐conserving contact would model an industrial outcome for a cold‐ethanol process in which leached components would be added back from an alcohol solution. The leaching result was monitored by mixography of the flour, nitrogen analysis, and capillary zone electrophoresis of extracts. Although dough rheology was generally like that of the source flour, there were notable differences. The primary change for mass‐conserving contact was an increase in the time to peak resistance and a decrease in the rate of loss of dough resistance following peak resistance. These changes were in direct proportion to the amount of protein mobilized by the solvent. Leaching at 22°C, prevented dough formation for most aqueous ethanol concentrations and greatly reduced gliadin protein content. Minimal changes were noted for solvent contact at –12°C regardless of the ethanol concentration. The data suggested that 1) the conditions applied in cold‐ethanol enrichment of protein from wheat will generally preserve vital wheat gluten functionality, 2) functionality losses can be recovered by returning the solubilized fractions, and 3) the flour to which the gluten is added may require more mixing.     

Mixograph Responses of Gluten and Gluten‐Fortified Flour for Gluten Produced by Cold‐Ethanol or Water Displacement of Starch from Wheat Flour

The total protein of gluten obtained by the cold‐ethanol displacement of starch from developed wheat flour dough matches that made by water displacement, but functional properties revealed by mixing are altered. This report characterizes mixing properties in a 10‐g mixograph for cold‐ethanol‐processed wheat gluten concentrates (CE‐gluten) and those for the water‐process concentrates (W‐gluten). Gluten concentrates were produced at a laboratory scale using batter‐like technology: development with water as a batter, dispersion with the displacement fluid, and screening. The displacing fluid was water for W‐gluten and cold ethanol (≥70% vol, ‐12°C) for CE‐gluten. Both gluten types were freeze‐dried at ‐10°C and then milled. Mixograms were obtained for 1) straight gluten concentrates hydrated to absorptions of 123–234%, or 2) gluten blended with a low protein (9.2% protein) soft wheat flour to obtain up to 16.2% total protein. The mixograms for gluten or gluten‐fortified flour were qualitatively and quantitatively distinguishable. We found differences in the mixogram parameters that would lead to the conclusion of greater stability and strength for CE‐gluten than for W‐Gluten. Differences between the mixograms for these gluten types could be markedly exaggerated by increasing the amount of water to the 167–234% range. Mixograms for evaluation of gluten have not been previously reported in this hydration range. Mixograms for fortification suggest that less CE‐gluten than W‐gluten would be required for the same effect.     

Wheat Proteins Extracted from Flour and Batter with Aqueous Ethanol at Subambient Temperatures

Contact of wheat flour with aqueous ethanol may enrich protein by starch displacement or deplete protein by extraction depending on 1) extraction conditions and 2) the form of the substrate. Extraction at subambient temperatures has not been described for specific gliadins for either dry flour with the protein in native configurations or for wet, developed batter or dough. This limits the ability to interpret technologies such as the cold-ethanol method. Here, we describe specific albumin and gliadin composition of flour extracts by capillary zone electrophoresis CZE in 0–100% (v/v) ethanol from –12 to 22°C. Extraction was reduced for albumin and gliadin protein as the temperature was reduced and the concentration range for extraction narrowed. Extraction dropped precipitously between 0 and –7°C for both albumins and gliadins. Electrophoretically defined gliadins extracted in constant proportion at 22°C and 30–80%(v/v) ethanol, but at lower temperature, the α-gliadins were enriched and β-gliadins depleted in the 30–55% (v/v) range. For extracts from wheat flour batter, depletion of α and β and enrichment of γ relative to the dry flour contact suggested that the electrophoretically slow migrating γ- and ω-proteins are less well incorporated to the dough matrix than electrophoretically fast migrating α and β types.     

Effect of Processing on Functional Properties of Wheat Gluten Prepared by Cold‐Ethanol Displacement of Starch

Functional properties of gluten prepared from wheat flour are altered by separation and drying. Gluten was separated and concentrated by batterlike laboratory methods: development with water, dispersion of the batter with the displacing fluid, and screening to collect the gluten. Two displacing fluids were applied, water or cold ethanol (70% vol or greater, ‐13°C). Both the water‐displaced gluten (W‐gluten) and ethanol‐displaced‐ gluten (CE‐gluten) were freeze‐dried at ‐20°C as a reference. Samples were dried at temperatures up to 100°C using a laboratory, fluidized‐bed drier. Tests of functionality included 1) mixing in a mixograph, 2) mixing in a farinograph, and 3) the baked gluten ball test. Dough‐mixing functionality was assessed for Moro flour (9.2% protein) that was fortified up to 16% total protein with dried gluten. In the mixograph, CE‐gluten (70°C) produced improved dough performance but W‐gluten (70°C) degraded dough performance in proportion to the amount added in fortification. In the microfarinograph, there was a desirable and protein‐proportional increase in stability time for CE‐gluten (70°C) but no effect on stability for W‐gluten (70°C). Baking was evaluated using the baked gluten ball test and the percentage increase in the baked ball volume relative to the unbaked gluten volume (PIBV). PIBV values were as high as 1,310% for freeze‐dried CE‐gluten and as low as 620% for W‐gluten dried at 70°C. PIBV for CE‐gluten was reduced to 77% of the freeze‐dried control by fluid‐bed drying at 70°C. Exposure of CE‐gluten to 100°C air gave a PIBV that was 59% of the reference, but this expansion was still greater than that of W‐gluten dried at 70°C. The highest values of PIBV occurred at the same mixing times as the peak mixograph resistance.     

Wheat Gluten Swelling and Partial Solubility with Potential Impact on Starch-from-Gluten Separation by Ethanol Washing

Swelling of wheat gluten may be a contributing factor in washing or displacement separation of gluten and starch using cold ethanol. To test this hypothesis, dissolution and swelling (settled volume or mass absorption) of a commercial gluten are reported here for the first time as a function of both temperature and ethanol solution concentration. In this test system, instant and substantial volumetric swelling was observed over most of the range of ethanol concentrations but not at 100%, v/v, ethanol. Settled volume reached a maximum of 50–70%, v/v, ethanol, and this was up to 3.5× the volume in absolute ethanol at 22°C and 2× the volume at −15°C. This maximum closely corresponds to the maximum dissolution of whole gluten and prior literature reports of full dissolution of gliadin. The reduction of settled volume at low temperature reflects the possible role of undissolved, gliadin-class proteins in reinforcing the gluten structure and limiting the ultimate swelling. The data suggest gluten-swelling properties as a contributing factor to the success of the cold ethanol, glutenfrom-starch separation process.     

Effect of Morphology of Mechanically Developed Wheat Flour and Water on Starch from Gluten Separation Using Cold Ethanol Displacement

The mechanical development of wheat flour and water creates micro and macro structures in dough or batter that critically influence the ability to separate starch from protein by fluid displacement. This study sought to identify specific structural and rheological features and to relate these to separation as indexed by the separation factor. Structural features, especially protein and starch distributions, were examined using visible light microscopy applied to dough samples that had been exposed to a protein dye. Flour and water samples were developed in a Brabender microfarinograph at conditions (water content and time of development) generally suitable for use of the USDA Western Regional Research Center, cold‐ethanol fluid‐displacement method. No truly homogenous structures were observed. However, distinct segregation of protein and starch were apparent at all conditions. Structural features correlated qualitatively with the success of separation indexed by the overall separation factor (α_p/s) for the separation process. Highly segregated states characterized by large protein bands, clustered starch, and large open spaces were obtained with intermediate development (25 ± 5 min) and were most readily separated (α_p/s = 118 ± 7). Segregated states with relatively thin protein bands (≤10 μm dia) in complex networks entrapping starch were obtained after additional development (≥45 min) and were less completely separable (α_p/s = 32 ± 2). Segregated states with irregularly organized protein in the form of clumps and bands were obtained with minimal development and were partially separable (α_p/s = 65 ± 4). Consistency indicated on the microfarinograph increases monotonically throughout and beyond the period of maximum separability. However, elasticity changes and a high rate of increase in consistency evident in the microfarinogram may reflect changes in the structure that also reduce separability. The study demonstrated the use of the ethanol method to isolate development from displacement phenomena forindependent study.     

Preparative Method for In Vitro Production of Functional Polymers from Glutenin Subunits of Wheat

An in vitro method for preparative‐scale production of artificial glutenin polymers utilizes a controlled environment for the oxidation of glutenin subunits (GS) isolated from wheat flour to achieve high polymerization efficiency. The functionality of in vitro polymers was tested in a 2‐g model dough system and was related to the treatment of the proteins before, during, and after in vitro polymerization. When added as the only polymeric component in a reconstituted model dough (built up from gliadin, water solubles, and starch fractions), in vitro polymers could mimic the behavior of native glutenin, demonstrating properties of dough development and breakdown. Manipulating the high molecular weight (HMW)‐GS to a low molecular weight (LMW)‐GS ratio altered the molecular weight distribution of in vitro polymers. In functional studies using the 2‐g mixograph, simple doughs built up from homopolymers of HMW‐GS were stronger than those using homopolymers of LMW‐GS. These differences may be accounted for, at least in part, by different polymer size distributions. The ability to control the size and composition of glutenin polymers shows the potential of this approach for investigating the effects of glutenin polymer size on dough function and flour end‐use quality.     

Changes in Secondary Protein Structures During Mixing Development of High Absorption (90%) Flour and Water Mixtures

Wheat flour and water mixtures at 90% absorption (dry flour basis) prepared at various mixing times were examined using Fourier transform infrared (FT‐IR) reflectance spectroscopy. Spectra were obtained using a horizontal attenuated total reflection (ATR) trough plate. The apparent amount of protein and starch on the surface of the dough varied with mixing time but this was likely due to the polyphasic nature of the substrate and the changing particle distributions as the batter matrix was developed. Deconvolution of the Amide I band revealed contributions from alpha helical, β‐turn, β‐strand, β‐sheet, and random conformations. The ratio of β‐sheet to nonsheet conformations reached its greatest value about the same time that the mixture was most effectively separated by a laboratory‐scale, cold‐ethanol‐based method but before the peak consistency measured by a microfarinograph.     

Extractability and Molecular Modifications of Gliadin and Glutenin Proteins Withdrawn from Different Stages of a Commercial Ethanol Fuel/Distillers Dried Grains with Solubles Process Using a Wheat Feedstock

Extractability and molecular modifications of gliadin and glutenin proteins withdrawn from different stages of a commercial ethanol fuel/distillers dried grains with solubles (DDGS) process using a wheat feedstock were investigated. Materials were taken postliquefaction (PL), postdistillation (whole stillage), and postdrying (DDGS) during the process and then fractionated to separate the gliadins and the soluble high‐ and low‐molecular‐weight glutenins following a modified Verbruggen extraction method. Each fraction was characterized based on the extraction efficiencies within various aqueous alcohols of propan‐1‐ol, electrophoretic patterns, intrinsic and extrinsic fluorescence, free and total sulfhydryl content, and total disulfide bond levels. Findings indicated significant changes to the composition of extracted proteins and modifications to the protein structure (i.e., surface properties and conformation) throughout the ethanol/DDGS process, beginning with the first step of production (PL, ≈83°C). Overall, processing resulted in a shift toward an unextractable gluten matrix, accompanied by increases in hydrophobicity, disulfide bridging, and excessive protein aggregation.     

Improved Protocols for ELISA Determination of Gliadin in Glucose Syrups

Simple modifications of existing protocols for high‐sensitivity detection of gluten proteins by immunochemical methods allowed rapid and sensitive determination of residual gluten in highly viscous samples of glucose and maltose syrups obtained from processing wheat starch. Dilution of the original syrup to no less than 15–20% in solids allowed retention of gluten proteins in a soluble form so that ELISA determination of gliadin was possible without an extraction step in aqueous ethanol. An ultrafiltration step may be added to concentrate residual gluten proteins in the diluted syrup samples and allow a further increase in sensitivity. The results are relevant for quality assessment of wheat starch derived syrups as raw materials for use in gluten‐free foods for celiac individuals.     

Disaggregation and reaggregation of gluten proteins by sodium chloride

This study showed that gluten proteins were extracted with distilled water from dough prepared in the presence of NaCl. To elucidate the interrelationship of NaCl and gluten proteins in dough, the extracted proteins were characterized. These proteins were primarily found to be soluble gliadin monomers by N-terminal amino acid sequencing and analytical ultracentrifugation. Extracted proteins were aggregated by the addition of NaCl at concentrations of >10 mM. A decrease in beta-turn structures, which expose tryptophan residues to an aqueous environment in the presence of NaCl, was revealed by Fourier transform infrared analysis and scanning of fluorescence spectra. In addition, cross-linking experiments with disuccinimidyl tartrate showed that a large amount of protein was cross-linked in the dough only in the presence of NaCl. These results suggest that both interactions and distances between proteins were altered by the addition of NaCl.     

Gliadin Immunoreactivity and Dough Rheological Properties of Winter Wheat Genotypes Modified by Thioredoxin

Gliadins are among the most important protein fractions affecting wheat baking quality, but they are also plant allergens and a cause of celiac disease or food intolerance. Therefore, we investigated how gliadin immunoreactivity and dough rheological properties are influenced by thioredoxin, a regulatory disulfide protein that can reduce disulfide bonds, a typical motive in many allergenic proteins. Ten winter wheat genotypes of different qualities were analyzed. Reduction by thioredoxin strongly (>50%) decreased gliadin immunoreactivity as estimated by enzyme‐linked immunosorbent assay with immunoglobulin E (IgE) sera from allergic patients and standard antigliadin antibodies but did not significantly affect dough rheological properties. Most parameters from the Brabender extensigraph were only slightly lower. Simultaneously, the farinograph curve exhibited a drawdown dislocation, possibly due to increased water absorption by modified flour, and dough consistency visibly improved. Results suggest that thioredoxin may be a universal natural beneficial modifier, able to significantly decrease gliadin immunoreactivity (hence its potential allergenicity) without decreasing the unique technological properties of wheat flour.     

Application of a Micro Z‐Arm Mixer to Characterize Mixing Properties and Water Absorption of Wheat Flour

This study applied the use of a new small‐scale apparatus, the micro Z‐arm mixer, which has analogous mixing action to that of the traditional valorigraf and farinograph. A novel methodology has been developed for prediction of water absorption replacing the traditional titration method. The basis of this technique is a common characteristic of wheat flour samples: a reasonably constant slope (20–25.7 BU%) of the relationship between dough resistance and the amount of water present during mixing. Using an average slope value, prediction of water absorption was possible from a single measurement using a simple equation and with a standard error of 1.65%. Applications of the new mixer to cereal research are highlighted, including investigation of the effects of flour protein content and protein composition on mixing properties and water absorption. When protein content and protein composition have been systematically altered by the addition of isolated proteins into the flour, both dough development time (DDT) and water absorption increased when protein content was increased by glutenin addition and decreased when protein content was decreased by starch addition. Gliadin addition decreased DDT; gluten addition slightly increased DDT; glutenin addition significantly increased DDT. Water absorption was not affected by altering the glutenin‐to‐gliadin ratio, but it changed in proportion to the amount of protein added. The effect of HMW‐GS composition on the mixing requirement obtained with the micro Z‐arm mixer and with the 2‐g mixograph was also investigated using a set of single‐, double‐, and triple‐null lines for HMW‐GS coding genes. While subunits coded on the GluD1 locus were most important for determining the mixing requirement in both cases, the sample ranking was different in the two mixing actions. A better differentiation ability of the micro Z‐arm mixer was established for triple‐ and double‐null lines.     

Physicochemical and Rheological Characterization of Wheat Flour Dough

Rheological and structural behavior of dough prepared with two Argentinean flours (FI and FII) of different dough extensibilities were studied. Flours were analyzed by composition and rheological assays. Structural properties of dough prepared at different mixing times were analyzed by scanning electron microscopy, free sulfhydryls quantification, and yield of different protein fractions, as well as their protein surface hydrophobicity. Size of high molecular weight glutelin soluble aggregates was analyzed through multistacking gel electrophoresis. Dynamic viscoelasticity of dough was also studied. Flours FI and FII presented similar physicochemical properties but different rheological properties. Structural properties of both flour components were different. Starch from FI flour generated a more viscous paste than that of FII. FI presented a higher glutenin‐to‐gliadin ratio and a higher content of free sulfhydryls than FII. The resulting dough of FI showed a high development time and was more stable than FII. FI contained a high proportion of soluble HMW glutenins and formed dough with a more depolymerized insoluble protein residue containing a lower amount of gliadin in its matrix than FII. FI also formed a more elastic and stable dough with higher development time than FII. The specific structural characteristic of FI turns this flour into suitable raw material for the preparation of different bakery products in which elasticity of dough would be an important functional property.     

Substitution of Concentrated Ethanol for Water in the Laboratory Washing Fractionation of Protein and Starch from Hydrated Wheat Flour

An unprecedented, ethanol-based, washing process was used at a laboratory scale to produce both concentrated protein and starch fractions from hydrated wheat flour. In this multistep process, flour was first hydrated and mixed to a batter and then chilled and rested. The cold batter was then mixed and washed in chilled and concentrated ethanol using a modified device that normally applies the water-based Martin process. Control of the separation was affected by each of these steps. For instance, the hydration of the flour, the time of mixing, the temperature of the wash, the ethanol concentration, and the time of washing were influential. The method produced a gluten concentrate similar in yield and protein content to that reported for a pilot-scale Martin process but without the need for added salt. Notably, ethanol washing resulted in nonsticky, partially disintegrated curds that dried easily, whereas water washing resulted in a sticky, glutinous, cohesive mass that dried slowly. The process has commercial potential to reduce water and energy use, reduce wastewater generation and environmental impact, and improve product recovery. The process also has the potential to reduce the capital complexity of the drying step and create convenient opportunities for protein subfractionation.     

Effects of Nitrogen and Sulfur Fertilizer on Protein Composition,Mixing Requirements,and Dough Strength of Four Wheat Cultivars

Two field trials using four New Zealand wheat cultivars were undertaken to observe the effects of nitrogen and sulfur fertilization on protein composition, mixing requirements, and dough strength and to compare the results with that observed with a single cultivar, Otane. The results confirmed that adequate sulfur fertilization was necessary to ensure lower dough mixing requirements. The existence of a nexus between mixing requirements and dough strength was confirmed and genotype has significant effects on it. Variation in the content of HMW‐GS in the protein corresponded to changes in dough mixing requirement of Otane. Across the four cultivars, dough mixing requirements (mechanical dough development work input and mixograph development time) and dough strength (Extensigraph resistance to extension) depended on different aspects of protein composition. As the content of polymeric proteins increased, MDD work input increased, but mixograph development time decreased, while the effect on Rmax was small. Rmax, however, was more affected by either the content of small monomerics in the flour or the ratio between HMW‐GS peak area to total gliadin peak area. The ratio of MDD work input to Rmax was largely explained by the gliadin content of the flour. Thus, depending on the genetic background, it should be possible to adjust dough mixing requirements by modifying overall HMW‐GS, LMW‐GS, or gliadin content while maintaining dough strength.     

Farinograph Responses for Wheat Flour Dough Fortified with Wheat Gluten Produced by Cold‐Ethanol or Water Displacement of Starch

The objective of this research was to identify and define mixing characteristics of gluten‐fortified flours attributable to differences in the method for producing the gluten. In these studies, a wheat gluten concentrate (W‐gluten) was produced using a conventional process model. This model applied physical water displacement of starch (dispersion and screening steps), freeze‐drying, and milling. W‐gluten was the reference or “vital” gluten in this report. An experimental W‐concentrate was produced using a new process model. The new model applied coldethanol (CE) displacement of starch (dispersion and screening steps), freeze‐drying, and milling. Freeze‐drying was used to eliminate thermal denaturation and thereby focus on functional changes due only to the separation method. The dry gluten concentrates were blended with a weak, low‐protein (9.2%), soft wheat flour and developed with water in a microfarinograph. We found that both water and cold‐ethanol processed gluten successfully increased the stability (St) and improved mixing tolerance index (MTI) to create in the blended flour the appearance of a breadbaking flour. Notably, in the tested range of 9–15% protein, the St for CE‐gluten was always higher then the St for W‐gluten. Furthermore, the marginal increase in St (slope of the linear St vs. protein concentration) for the CE‐gluten was ≈57% greater than that for the W‐gluten. The slope of the MTI vs. protein data was lower for the CE‐gluten by 24%. Flour fortified with CE‐gluten exhibited higher water absorption (up to 1.8% units at 13.5% P) than flour fortified with W‐gluten.     

Formation of Homopolymers and Heteropolymers Between Wheat Flour and Several Protein Sources by Transglutaminase‐Catalyzed Cross‐Linking

The effect of different protein sources (soy flour, lupin flour, egg albumin, gelatin powder, protein‐rich beer yeast flour) on wheat dough functionality was tested by determining gluten index, texture properties, and thermomechanical parameters. Transglutaminase (TG) was also added to improve the dough functionality by forming cross‐links. The presence of protein sources had a significant effect on the gluten index, with the exception of lupin flour. Gelatin and the presence of TG resulted in significant single effects on the texture properties of the wheat‐protein dough. All the protein sources significantly modified the mixing characteristics of the dough or the thermal behavior. Capillary electrophoresis studies of the water‐soluble, salt‐soluble, and glutenin proteins indicated that interactions were mainly within proteins, thus homologous polymers. Scanning electron microscopy studies of the doughs made from blends of wheat and protein sources doughs supported the formation of heterologous structures in the wheat‐lupin blends. The combination of TG and lupin would be a promising method to be used on the treatment of insect‐damaged or weak flours, to increase the gluten strength.     

Effects of Wheat Protein Fractions on Flour Tortilla Quality

Commercial wheat protein fractions (10) were evaluated during processing for quality of tortillas prepared using pastry, tortilla, and bread flours. Protein fractions that separately modify dough resistance and extensibility were evaluated in tortillas to determine whether the proteins could increase diameter, opacity, and shelf stability. Tortillas were prepared using laboratory‐scale, commercial equipment with fixed processing parameters. Dough and tortilla properties were evaluated using analytical methods, a texture analyzer, and subjective methods. Tortillas were stored in plastic bags at 22°C for up to 20 days. Adjustments in water absorption and level of reducing agent were made to normalize differences in functionality of 3% added proteins on dough properties. Tortilla weight, moisture, pH, opacity, and specific volume were not affected by added proteins, except for glutenin and vital wheat gluten treatments, which had decreased opacity in tortillas prepared from pastry flour. Increased insoluble polymeric protein content corresponded to decreased tortilla diameter and improved shelf stability. Treatments yielding tortillas with improved shelf stability and similar tortilla properties were produced when commercially processed vital wheat gluten products, FP600, FP6000, FP5000, or gliadin were added to pastry or tortilla flour. These wheat protein fractions improved processing and tortilla quality of wheat flours, especially pastry flour, by modifying protein content and quality.     

Synergistic and Additive Effects of Three High Molecular Weight Glutenin Subunit Loci. I. Effects on Wheat Dough Rheology

The high molecular weight glutenin subunits (HMW‐GS) play an important role in governing the functional properties of wheat dough. To understand the role of HMW‐GS in defining the basic and applied rheological parameters and end‐use quality of wheat dough, it is essential to conduct a systematic study where the effect of different HMW‐GS are determined. This study focuses on the effect of HMW‐GS on basic rheological properties. Eight wheat lines derived from cvs. Olympic and Gabo were used in this study. One line contained HMW‐GS coded by all three loci, three lines were each null at one of the loci, three lines were null at two of the loci and one line null at all three loci. The flour protein level of all samples was adjusted to a constant 9% by adding starch. In another set of experiments, in addition to the flour protein content being held at 9%, the glutenin‐to‐gliadin ratio was maintained at 0.62 by adding gliadin. Rheological properties such as elongational, dynamic, and shear viscometric properties were determined. The presence of Glu‐D1 subunits (5+10) made a significantly larger contribution to dough properties than those encoded by Glu‐B1 (17+18), while subunit 1, encoded by Glu‐A1, made the least contribution to functionality. Results also confirmed that HMW‐GS contributed to strength and stability of dough.