Wheat Intercultivar Differences in Susceptibility of Glutenin Protein to Effects of Bug (Eurygaster integriceps) Protease

Preharvest bug damage to wheat can cause significant losses in bread‐making quality. One of the most prevalent forms of bug damage which frequently occurs in most countries of the Middle East, Eastern Europe and North Africa can be attributed to Heteropterous insects, particularly Eurygaster spp. Intercultivar differences in the susceptibility of glutenin to proteolytic degradation by the bug Eurygaster integriceps were investigated using six breadwheat cultivars of Turkish origin. Crude enzyme extract was prepared with distilled water from bug‐damaged wheat. The freeze‐dried extract was blended with sound samples of ground wheat, and the mixture was incubated in distilled water for 30 and 60 min at 37°C and subsequently freeze‐dried. The proteolytic effects of bug damage were determined on large polymeric glutenin. The latter was measured as 50% 1‐propanol insoluble (50PI) glutenin extractable with 50% 1‐propanol in reductant dithiothreitol. The decreases in the amount of 50PI glutenin and the high and low molecular weight subunits were quantified using reversed‐phase HPLC. There was a substantial and progressive decrease in the quantity of 50PI glutenin and its subunits with increasing incubation time. Intercultivar differences were observed that were unrelated to intrinsic levels of proteolytic activity. After 60 min of incubation, the relative decrease in 50PI glutenin compared with control samples ranged from 43% (cv. Ankara) to 65 % (cv. Kirkpinar). Some cultivars (Lancer, Ankara and Gün) with similar levels of intrinsic proteolytic activity showed significantly different responses to bug protease. One cultivar (cv. Kirkpinar) with the lowest proteolytic activity was the most susceptible. High quality breadwheats (cvs. Bezostaya, Lancer, Kiraç and Gün) were generally more resistant to the bug protease, although Ankara, with both intermediate protease activity and breadmaking quality, was the most resistant cultivar. While the 50PI glutenin test was very effective in quantifying the damaging effects of bug protease on wheat protein quality, the nature of the intercultivar differences was unclear.     

Effect of Microbial Transglutaminase on Dough Proteins of Hard and Soft (Triticum aestivium) and Durum (Triticum durum) Wheat Cultivars

Microbial transglutaminase (MTGase), a protein‐glutamine γ‐glutamyl transferase (E.C. 2.3.2.13), catalyzes acyl transfer reactions by introducing a covalent cross‐link between l ‐lysine and l ‐glutamine residues. The use of this enzyme has been proposed as an improver to increase dough strength. The objective of this study was to assess and compare the effect of MTGase on different fractions of dough proteins found in hard, soft, and durum wheat. Three different concentrations of the MTGase (0, 5, and 10U/g of gluten) were tested. Moisture, protein, and dry gluten contents were determined for each concentration in addition to rheological measurements done with the farinograph. Following each treatment, the dough proteins were extracted and analyzed by SE‐HPLC and RP‐HPLC. Soluble polymeric protein, gliadins, albumins, and globulins were quantified in addition to the gliadin subclasses and glutenin subunit types. The combustion procedure was used to determine the amount of insoluble polymeric protein. Differences were observed in susceptibility to MTGase catalysis among the dough proteins of the cultivars studied: the cultivar Cortazar (soft wheat) was the most susceptible. The proteins of this cultivar had a characteristically higher amount of ω and α+β gliadins when compared with the other cultivars. As reported earlier, solubility of high molecular weight glutenin subunits and ω‐gliadins was reduced because of the MTGase treatment. However, all gliadin subclasses, including the γ and α+β gliadins, also participated in cross‐linking. The proteins of the cultivar Altar (durum wheat) were the least susceptible to the effects of MTGase. Albumins and globulins did not show any reduction in solubility, implying that they did not participate in cross‐linking.     

Compared use of HPLC and FZCE for cluster analysis of Triticum spp and for the identification of T. durum adulteration

Wheat quality criteria continually evolve in response to market pressure and consumer preference. Characterization of cereal cultivars for quality and agronomic properties, have widely shown the importance of the protein content to ensure good quality products. The aim of this work is a comparison of reversed-phase high performance liquid chromatography (RP-HPLC) and free zone capillary electrophoresis (FZCE) in the identification of Italian wheat cultivars and detection of durum wheat flour adulteration. Mainly alcohol soluble (gliadins) and water soluble (albumins) proteins were extracted from 14 common wheat cultivars and from 9 durum wheat cultivars. In RP-HPLC chromatograms, wheat albumins and gliadins eluted between 3 and 9 min and between 10 and 42 min, respectively. Even if the chosen chromatographic conditions (reversed phase) did not permit a complete resolution of hydrophilic proteins such as albumins, a good reproducibility was observed for both albumins and gliadins. In FZCE electropherograms, wheat albumins and gliadins migrated between 8 and 14 min and 16-25 min, respectively. A good reproducibility was found for wheat albumins, while the relatively poor reproducibility of gliadin fractions was a consequence of the selected separation conditions aimed to separate in the same run either hydrophilic (albumins) and alcohol-soluble (gliadins) proteins. The principal component analysis (PCA) of HPLC and FZCE data evidenced that both techniques allowed the univocal identification of the great proportion of investigated wheat cultivars. Three peaks were exclusively detected in RP-HPLC chromatograms of common wheat cultivars, while three unique peaks were found in FZCE electropherograms of common wheat cultivars. These peaks were investigated as a basis for detecting and estimating the adulteration of durum wheat flour with flour from common wheat. The direct relationship between the area of the peaks and adulteration level enabled standard curves to be constructed. The standard curves showed that adulteration may be quantified by either RP-HPLC or FZCE.     

Relationship Between Solvent Retention Capacity and Protein Molecular Weight Distribution,Quality Characteristics,and Breadmaking Functionality of Hard Red Spring Wheat Flour

This research aims to investigate the relationship between the solvent retention capacity (SRC) test and quality assessment of hard red spring (HRS) wheat flour samples obtained from 10 HRS cultivars grown at six locations in North Dakota. The SRC values were significantly (P < 0.05) correlated with flour chemical components (protein, gluten, starch, and damaged starch contents, except arabinoxylan); with farinograph parameters (stability [FST], water absorption, peak time [FPT], and quality number); and with breadmaking parameters (baking water absorption [BWA], bread loaf volume [BLV], and symmetry). Differences in locations and cultivars contributed significantly to variation in quality parameters and SRC values. Suitability of SRC parameters for discriminatory analysis of HRS wheat flour is greatly influenced by molecular weight distribution (MWD) of SDS‐unextractable proteins. SRC parameters, except for sucrose SRC, showed significant (P < 0.01) and positive correlations with high‐molecular‐weight (HMW) polymeric proteins in SDS‐unextractable fractions, whereas only lactic acid SRC exhibited significant (P < 0.01) correlations with low‐molecular‐weight polymeric proteins. HMW polymeric proteins also exhibited positive associations with FPT, FST, BWA, and BLV. The discrepant variation in association of SRC parameters with respect to MWD of SDS‐unextractable proteins could improve segregation of HRS wheat flour samples for quality.     

Effects of Suni Bug (Eurygaster spp.) Damage on Milling Properties and Semolina Quality of Durum Wheats (Triticum durum L.)

Economic losses due to suni‐bug (Eurygaster spp. and Aelia spp.) damage are important for the cereal industry in East European and Middle East countries. Samples of five durum wheat cultivars (Diyarbakir, Firat, Ege, Svevo, and Zenith) with zero, medium, and high levels of suni‐bug damage were used to determine the effects of suni‐bug damage on milling properties and semolina quality. As the damage level increased, semolina yields of all cultivars decreased significantly. The loss of semolina yield was greater than decreases in total yield of semolina plus flour, indicating that semolina yields were affected to a higher extent than were flour yields. The ash contents of the semolina samples increased significantly in all cultivars with increasing suni‐bug damage. The falling number values were not correlated with suni‐bug damage level and amylase activities of all samples were quite low. The pasting properties did not differ to a great extent depending on the suni‐bug damage level. Gluten quality of semolina samples substantially deteriorated as suni‐bug damage level increased, as determined by SDS‐sedimentation and mixograph analyses. It was concluded that suni‐bug damage would decrease profits of durum wheat millers substantially by affecting semolina yield and quality.     

Immunochemical and Electrophoretic Analysis of the Modification of Wheat Proteins in Extruded Flour Products

Antibodies specific for wheat proteins were used to identify protein fractions modified during extrusion of Hard Red Spring wheat flour (14% protein) under four different combinations of extrusion conditions (18 and 24% feed moisture and 145 and 175°C die temperature). Antibody binding was assessed on immunoblots of proteins extracted from flour and extrudates separated by SDS‐PAGE. Antibodies to high molecular weight glutenin subunits (HMW‐GS) and to B‐group low molecular weight glutenin subunits (LMW‐GS) recognized intact subunits from both flour and extrudates. Antibodies to C‐group LMW‐GS had diminished binding to extruded proteins. Glutenin‐specific antibodies also recognized protein in the extrudates migrating as a smear at molecular weights higher than intact subunits, indicating cross‐linked proteins. Antibodies recognized albumins or globulins in flour but not in extrudates, evidence that these fractions undergo significant modification during extrusion. Acid‐PAGE and antibody reaction of gliadins extracted in 1M urea and in 70% ethanol revealed total loss of cysteine‐containing α, β, γ‐gliadins but no obvious effects on sulfur‐poor ω‐gliadins, suggesting gliadin modification involves replacing intramolecular disulfides with intermolecular disulfide cross‐links. Identifying protein fractions modified during different extrusion conditions may provide new options for tailoring extrusion to achieve specific textural characteristics.     

Effects of Wheat Bug (Eurygaster maura) Protease on Glutenin Proteins

Proteolytic degradation of 50% 1-propanol insoluble (50PI) glutenin of six common wheat cultivars by wheat bug (Eurygaster maura) protease was investigated using reversed-phase HPLC. Wheat at the milk-ripe stage was manually infested with adult bugs. After harvest, bug-damaged kernels were blended (2:1, kernel basis) with undamaged grain of the same cultivar. Samples of ground wheat were incubated in distilled water for different times (0, 30, 60, and 120 min). The incubated whole meal samples were subsequently freeze-dried and stored until analysis. The degree of proteolytic degradation of 50PI glutenin was determined based on the quantity of total glutenin subunits (GS), high molecular weight GS (HMW-GS), and low molecular weight GS (LMW-GS). For ground wheat samples incubated for ≥30 min, 50PI glutenin was substantially degraded as evidenced by a >80% decrease on average in total GS, HMW-GS, and LMW-GS. Some cultivars showed different patterns of glutenin proteolysis as revealed by differences in the ratios of HMW-GS to LMW-GS between sound and bug-damaged samples; a significant decrease in this ratio was found for four cultivars. This evidence, combined with other observations, indicated that there were intercultivar differences in polymeric glutenin resistance to the protease of the wheat bug Eurygaster maura. While the nature of this resistance is unknown, it should be possible to select and develop wheat cultivars with improved tolerance for wheat bug damage. Propanol insoluble glutenin, which corresponds to relatively large glutenin polymers, appears to be an excellent quantitative marker for this purpose.     

Effects of Transglutaminase Enzyme on Fundamental Rheological Properties of Sound and Bug‐Damaged Wheat Flour Doughs

Transglutaminase (TG) catalyzes the formation of nondisulfide covalent crosslinks between peptide‐bound glutaminyl residues and ∊‐amino groups of lysine residues in proteins. Crosslinks among wheat gluten proteins by TG are of particular interest because of their high glutamine content. Depolymerization of wheat gluten proteins by proteolytic enzymes associated with bug damage causes rapid deterioration of dough properties and bread quality. The aim of the present study was to investigate the possibility of using TG to regain gluten strength adversely affected by wheat bug proteases. A heavily bug‐damaged (Eurygaster spp.) wheat flour was blended with sound cv. Augusta or cv. Sharpshooter flours. Dynamic rheological measurements, involving a frequency sweep at a fixed shear stress, were performed after 0, 30, and 60 min of incubation on doughs made from sound or blended flour samples. The complex moduli (G* values) of Augusta and Sharpshooter doughs blended with 10% bug‐damaged flour decreased significantly after 30 min of incubation. These dough samples were extremely soft and sticky and impossible to handle for testing purposes after 60 min of incubation. To test the possibility of using TG to counteract the hydrolyzing effect of bug proteases on gluten proteins, TG was added to the flour blends. The G* values of TG‐treated sound Augusta or Sharpshooter doughs increased significantly after 60 min of incubation. The G* values of the Augusta or Sharpshooter doughs blended with bug‐damaged flour increased significantly rather than decreased after 30 and 60 min of incubation when TG was included in the dough formulation. This indicates that the TG enzyme substantially rebuilds structure of dough hydrolyzed by wheat bug protease enzymes.     

Use of Size‐Exclusion High‐Performance Liquid Chromatography for Wheat Quality Prediction in Ethiopia

Wheat quality testing facilities in Ethiopia are limited. The aim of this study was to determine whether size‐exclusion high‐performance liquid chromatography (SE‐HPLC) could be used in breeding programs for quality testing. Thirteen Ethiopian and two South African wheat cultivars were evaluated in two diverse environments for milling and dough characteristics. SE‐HPLC was done on the same samples. Across environments, both SDS‐soluble and SDS‐insoluble polymeric proteins significantly influenced important quality characteristics such as SDS‐sedimentation and mixograph development time. The large monomeric proteins, which are mainly gliadins, had a consistently significantly negative effect on quality. The increase of polymeric protein as opposed to monomeric protein led to improvement of quality characteristics. The SDS‐soluble and SDS‐insoluble polymeric proteins were equally important in quality prediction. The amount of polymeric proteins was significantly higher in the high‐protein environment. Despite a large environmental effect on most fractions, a large ratio of polymeric proteins to monomeric proteins (both SDS‐soluble and SDS‐insoluble) can be a good indicator of baking quality. SE‐HPLC is therefore an option to use in breeding programs in Ethiopia for quality evaluation.     

Effect of Climate Change on Wheat Quality and HMW Glutenin Subunit Composition in the Pannonian Plain

The primary goal of this study is to improve our understanding of the extent of influence of climatic factors in Serbia and high‐molecular‐weight glutenin subunit (HMW‐GS) composition upon wheat end‐use quality. In‐depth analyses were performed on four bread wheat cultivars that are the most common in agricultural practice in Serbia. Total glutenin content showed significant difference between the production years, in opposition to gliadins. Cluster analysis of different percentages of glutenin and gliadin subunit molecular weight ranges (<40,000, 40,000–80,000, 81,000–120,000, and >120,000) indicated that the year of production and the cultivar did not have a significant effect on the percentage ranges for glutenins. However, they had a considerable impact on the percentage ranges for gliadins. Production year and the interaction of year and cultivar had the strongest influences on the percentage of SDS‐unextractable polymeric proteins. A synergistic effect of the HMW‐GS composition and climatic conditions revealed that all eight samples with HMW‐GS composition 2*, 5 + 10, 7 + 9 along with the highest Glu 1 score of 9 (out of a maximum of 10) produced in the year 2011 belonged to two clusters with the best wheat end‐use quality. Furthermore, the climate conditions in 2011 made it possible for the wheat cultivars with HMW‐GS composition –, 2 + 12, 7 + 9 to possess similar qualities as cultivars with HMW‐GS composition 2*, 5 + 10, 7 + 9 produced in 2012.     

Growth Environment Influences Grain Protein Composition and Dough Functional Properties in Three Australian Wheat Cultivars

The objectives of this study were to assess how functional properties of proteins in whole meal wheat (Triticum aestivum L.) flour vary across different growth environments. Grain from three commercial Australian Hard milling wheat cultivars was analyzed from four growth locations in 2008 and from two of the corresponding cultivars and locations in 2009. The protein content of the grain, soluble and insoluble extractable protein fractions, swelling index of glutenin (SIG), glutenin‐to‐gliadin ratio (Glu:Gli), percent unextractable polymeric protein (%UPP), and dough properties including force at maximum resistance (Rmax) and extensibility were measured. Based on analysis of variance of aggregated data for the cultivars, growth locations, and seasons, growth environment factors made significant contributions to variability in the total grain protein, Glu:Gli ratio, %UPP, SIG, Rmax, and extensibility of the wheat flour. Variability of protein content of the soluble and insoluble extractable protein fractions was mostly owing to genotype.     

Preparation and Functional Properties of Gluten Hydrolysates with Wheat‐Bug (Eurygaster spp.) Protease

Suni‐bug (Eurygaster spp.) enzyme was partially purified from bug‐damaged wheat and used to prepare gluten hydrolysates at 3% and 5% degree of hydrolysis (DH). Functional properties of gluten and gluten hydrolysates were determined at 0.2% (w/v) protein concentration and pH 2–10. Gluten solubility after enzymatic hydrolysis increased significantly (P < 0.05) up to 89.1, 89.6, and 95.0% at pH 7, 8, and 10, respectively. Emulsion stability (ES) of gluten hydrolysates improved at neutral and alkaline pH (P < 0.05) and emulsifying capacity (EC) increased significantly (P < 0.05) except at pH 10. Foaming capacity (FC) values of gluten hydrolysates were significantly higher (P < 0.05) at pH 6, 7, 8; foam stability (FS) values of gluten hydrolysates were significantly higher (P < 0.05) at pH 6 and 7. Enzymatic modification of gluten by wheat‐bug enzyme resulted in hydrolysates with higher antioxidant activity compared to gluten. Significant correlations (P < 0.001) were found between solubility and EC, ES, FC, and FS values of gluten and its hydrolysates with 3% and 5% DH.     

Alkylation of Free Thiol Groups During Extraction: Effects on the Osborne Fractions of Wheat Flour

Wheat proteins are classified according to solubility into the so‐called Osborne fractions. Because wheat flour contains both free thiol and disulfide groups, thiol–disulfide interchange reactions are possible during extraction. Osborne fractionation of 12 different wheat flour samples was performed in the presence of N‐ethylmaleinimide (NEMI) to alkylate free thiol groups and without addition of NEMI (control). The addition of NEMI during extraction tended to decrease the content of gliadins (predominantly α‐gliadins) and caused an increase of the content of glutenins in most flour samples. Thus, alkylation of free thiol groups during extraction led to a decline of the gliadin/glutenin ratio from 2 (control) to approximately 1.5 (NEMI). NEMI and control gliadins were separated by gel‐permeation HPLC into an oligomeric subfraction (high‐molecular‐weight [HMW] gliadins) and two monomeric subfractions. In most flours (8 of 12), the addition of NEMI led to a significant increase of the content of HMW gliadins. HMW gliadins from cultivar Akteur wheat were preparatively isolated from NEMI and control gliadins and characterized by HPLC, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and N‐terminal sequencing. HMW gliadin isolated in the presence of NEMI had a significantly higher content of low‐molecular‐weight glutenin subunits and disulfide‐bound cysteine as well as a lower content of α‐gliadins and disulfide‐bound glutathione compared with the control.     

Baking Quality of Bread Wheat Cultivars Damaged by Nysius simulans

Wheat grain may be attacked by different insect species. Among them, some Heteroptera species (e.g., Aelia spp. and Eurygaster spp.) reduce wheat breadmaking quality; others, such as Nysius simulans , commonly extract water and nutrients from soy plants. The aim of this study was to assess the effect of N. simulans infestation on breadmaking quality of different bread wheat cultivars. Twelve wheat cultivars (damaged and undamaged by N. simulans ) were studied. Infested grain percentage varied between 51 and 78%, depending on cultivar. Protein and gluten quantity and quality were significantly reduced in damaged flours, as shown by gluten index, solvent retention capacity, and SDS sedimentation index. SDS‐PAGE from water‐extractable proteins evidenced an important proteolytic activity in damaged samples. Dough rheological properties showed a reduced dough viscoelasticity in damaged samples. Microbread specific volume changed from 3.26 cm³/g for samples made with undamaged flour to 2.77 cm³/g for bread made with damaged flour. No evidence for modification in starch properties was found. The infestation by N. simulans reduced wheat breadmaking quality in all cultivars studied, as a result of proteolytic activity occurring after dough hydration. Results suggest that the presence of N. simulans should be considered as a factor affecting wheat crops, mainly those located next to soy crop areas, which is the usual host for this insect.     

Biochemical Characterization and Quantification of the Storage Protein (Secalin) Types in Rye Flour

Kernels of the rye cultivars Danko and Halo were milled into white flour and compared with flour of the wheat cultivar Rektor. Flour proteins were extracted stepwise with a salt solution (albumins‐globulins), 60% ethanol (prolamins), and 50% 2‐propanol under reducing conditions (glutelins). The quantification by reversed‐phase HPLC indicated that the extractable proteins of both rye flours consisted of ≈26% albumins‐globulins, 65% prolamins, and 9% glutelins. Compared with wheat flour, rye flours comprised significantly higher proportions of nonstorage proteins (albumins‐globulins) and lower proportions of polymerized storage proteins (glutelins). SDS‐PAGE revealed that the prolamin fractions of rye contained all four storage protein types (HMW, γ‐75k, ω, and γ‐40k secalins), whereas the glutelin fractions contained only HMW and γ‐75k secalins. The quantification of secalin types by RP‐HPLC showed a close relationship between the two cultivars.The γ‐75k secalins contributed nearly half (≈46%) of the total storage proteins, followed by γ‐40k secalins (24%) and ω secalins (17%); HMW secalins (≈7%) were minor components, and 6% of eluted proteins were not identified. The amino acid composition of γ‐40k secalins corresponded to those of γ‐gliadins of wheat, whereas γ‐75k secalins were characterized by higher contents of glutamine and proline. Matrix‐assisted laser desorption/ionization and time of flight mass spectrometry (MALDI‐TOF MS) indicated molecular masses of about 52,000 (γ‐75k) and 32,000 (γ‐40k), respectively. N‐terminal amino acid sequences were homologous with those of wheat γ‐ gliadins except for position 5 (asparagine in γ‐75k and glutamine in γ‐40k secalins) and position 12 (cysteine in γ‐75k secalins). The N‐terminal amino acid sequences of HMW and ω‐secalins were homologous with those of the corresponding protein types of wheat. Gel‐permeation HPLC of prolamin fractions revealed that rye flours contained a significantly higher proportion of ethanol‐soluble oligomeric proteins than wheat flour.     

Correlation of Quality Parameters with the Baking Performance of Wheat Flours

The baking performance of a set of flours from 13 wheat cultivars was determined by means of two different microscale baking tests (10 g of flour each). In the micro‐rapid‐mix test the dough was mixed for a fixed time at a high speed, whereas the microbaking test used mixing to optimum dough consistency in a microfarinograph. Quality parameters such as sedimentation value, crude protein content, dough and gluten extension data, and microfarinograph data were also determined. Finally, quality‐related protein fractions (gliadins, glutenins, SDS‐soluble proteins, and glutenin macropolymer) were quantitated by extraction/HPLC methods with reversed‐phase and gel‐permeation columns. All quality parameters were correlated with the bread volumes of both baking tests. The results demonstrated that the microbaking test (adapted mixing time) was much more closely related to the quality parameters than the micro‐rapid‐mix test (fixed mixing time), which hardly showed any correlation. Among the standard quality parameters, only the crude protein content showed a medium correlation with the bread volume of the microbaking test (r = 0.71), whereas the contents of gliadins (r = 0.80), glutenins (r = 0.76), and glutenin macropolymer (r = 0.80) appeared to be suitable parameters to predict the baking performance of wheat flour. All other quality parameters were not or were only weakly correlated and unsuitable for predicting baking performance.     

Variation and Correlation of Protein Molecular Weight Distribution and Semolina Quality Parameters for Durum Genotypes Grown in North Dakota

This research assessed variation of protein molecular weight distribution (MWD) parameters and their correlations with quality characteristics of semolina samples that were obtained from durum genotypes grown in North Dakota. Sodium dodecyl sulfate buffer extractable and unextractable proteins in semolina were analyzed for MWD by size‐exclusion HPLC with a microbore column. ANOVA indicated that quantitative variations of all the HPLC protein fractions were significantly (P < 0.001) influenced by growing environments. The extractable and unextractable gluten proteins correlated differently with semolina gluten characteristics. Both gluten index and mixograph classification showed positive correlations (P < 0.05) with unextractable polymeric proteins and negative correlations (P < 0.05) with extractable gliadins and polymeric proteins. Quantitative variations of gluten proteins greatly influenced spaghetti cooking characteristics. Specifically, cooked spaghetti firmness (CSF) had high and positive simple linear correlations (P < 0.001) with quantity of gluten proteins in both extractable and unextractable fractions. However, a qualitative MWD parameter, percentage of the extractable gliadins in total protein, had a negative genotypic correlation with CSF (r = –0.81, P < 0.01), whereas percentage of the unextractable polymeric proteins had a positive genotypic correlation (r = 0.75, P < 0.01). Those two MWD parameters also showed significant (P < 0.05) variations for genotypes, indicating that they might be useful for screening durum genotypes for pasta cooking quality.     

Isolation and Characterization of High‐Molecular‐Weight (HMW) Gliadins from Wheat Flour

The aim of this study was to isolate high‐molecular‐weight (HMW) gliadins from wheat flour and to characterize the protein components that contribute to HMW gliadins. Wheat flour Akteur was extracted with a modified Osborne procedure, and the fraction soluble in 60% ethanol (total gliadins) was separated by gel‐permeation HPLC, yielding three fractions, GP1–GP3. GP1 (21.5%) consisted of oligomeric HMW gliadins, GP2 (15.2%) of ω5‐gliadins, and GP3 (63.3%) of ω1,2‐, α‐, and γ‐gliadins. Two‐dimensional SDS‐PAGE of HMW gliadins showed that interchain disulfide bonds were present in HMW gliadins. The molecular mass distribution of HMW gliadins determined by gel‐permeation HPLC was in a range from 66,000 to 680,000 with an average degree of polymerization of 13. Reduced HMW gliadins were further separated by preparative reversed‐phase HPLC into four subfractions (RP1, RP2, RP3, and RP4), which were characterized by SDS‐PAGE and semiquantitative N‐terminal sequencing. HMW gliadins of the wheat flour Akteur contained all types of gluten proteins: 48% low‐molecular‐weight glutenin subunits, 18% γ‐gliadins, 13% α‐gliadins, 9% ω1,2‐gliadins, 8% HMW glutenin subunits, and 4% ω5‐gliadins. We postulate that the existence of HMW gliadins can be explained by the presence of terminators, which interrupt the polymerization of glutenin subunits during biosynthesis and lead to polymers of limited size (oligomers) that are still soluble in aqueous ethanol.     

Simplified Dilute Acetic Acid Based Extraction Procedure for Fractionation and Analysis of Wheat Flour Protein by Size Exclusion HPLC and Flow Field‐Flow Fractionation

A simple, highly efficient and reproducible two‐step extraction procedure using dilute acetic acid without (AN) and then with sonication (AS) has been developed for the fractionation of wheat flour protein. Approximately 97% of total protein was extracted from a Canadian hard red spring wheat flour; an additional 1.2% protein could be recovered by further extraction with 1% DDT and 50% 1‐propanol (AR). Size‐exclusion HPLC (SE‐HPLC) and flow field‐flow fractionation (flow FFF) showed that the AN extract, which accounted for most of the total extractable protein (AN + AS + AR), consisted primarily of monomeric protein. The AS extract was composed primarily of polymeric proteins. Flow FFF showed that AN polymeric protein, including that eluting at the SE‐HPLC void volume, showed smaller Stokes diameters than AS polymeric protein. Flow FFF profiles of AS SE‐HPLC subfractions showed that the void volume subfraction contained monomeric and small polymeric protein in addition to large polymeric protein, indicating formation of larger complexes through interaction between some or all of the components. AN and AS extracts, as well as SE‐HPLC and flow FFF fractions thereof, showed a fairly wide range of values among 12 Canadian hard red and white spring wheat cultivars. The proportion of total protein in the AS extract and in the larger sized polymeric protein fractions from SE‐HPLC and flow FFF were highly positively correlated to farinograph mixing time.     

Effect of Dark,Hard, and Vitreous Kernel Content on Protein Molecular Weight Distribution and on Milling and Breadmaking Quality Characteristics for Hard Spring Wheat Samples from Diverse Growing Regions

Kernel vitreousness is an important grading characteristic for segregation of subclasses of hard red spring (HRS) wheat in the United States. This research investigated the protein molecular weight distribution (MWD) and the flour and baking quality characteristics of different HRS wheat market subclasses. The U.S. regional crop quality survey samples obtained from six regions for three consecutive growing years were used for subclass segregation based on the dark, hard, and vitreous (DHV) kernel percentage. Flour milled from HRS wheat with greater percentages of DHV kernel showed higher water absorption capacity for breadmaking. Protein MWD parameters could be related to the association between DHV kernel level and water absorption. Specifically, flour protein fractions rich in gliadins and high‐molecular‐weight polymeric proteins in the SDS‐unextractable fraction were identified to have significant and positive correlations with both DHV kernels and flour water absorption levels. An example further showed the importance of flour water absorption on potential economic incentives that can be gained with having a greater percentage of vitreous kernels. This information could help the flour milling and baking industry to segregate the different subclasses of HRS wheat with varying DHV content for their intended end‐use applications.