Simple Determination of Gluten Protein Types in Wheat Flour by Turbidimetry

A simple method based on turbidimetry has been developed for the quantitative determination of total gliadins, glutenin subunits, and high and low molecular weight (HMW and LMW) subunits of glutenin. The standard procedure includes the subsequent extraction of wheat flour (100 mg) with a salt solution, with 50% 2‐propanol (gliadins), and with 50% propanol under reducing conditions and increased temperature (glutenin subunits). Aliquots of the gliadin and the glutenin extracts are mixed with 2‐propanol to a final concentration of 83%, and the turbidity of the precipitates is measured photometrically at 450 nm and 20°C after 40 min. Another aliquot of the glutenin extract is mixed with acetone to a final concentration of 40% acetone, and precipitated HMW subunits are determined turbidimetrically after 30 min. The sample is then filtered, and an aliquot of the filtrate is mixed with 2‐propanol to a final concentration of 77% to determine the precipitated LMW subunits. Control analyses with reversed‐phase HPLC on C₈ silica gel indicate that the precipitation of the different protein types is quantitative and specific, and studies of 16 different wheat flours demonstrate the strong correlation between quantification by HPLC and turbidimetry. The turbidimetric measurements are reproducible, linear over a wide absorbance range (0.2–1.7), and sufficiently sensitive to analyze 40 μg of protein or 20 mg of flour. The absolute amounts of protein types in flour can be determined by means of calibration curves with protein standards (gliadins, HMW, and LMW subunits). Altogether, the developed method is simple, accurate, sensitive, and specific for the different protein types. The total procedure takes ≈6 hr for the analysis of six flour samples in parallel or ≈4 hr for three samples in overlapping extraction steps. The chemicals used are inexpensive, scarcely toxic, and easy to dispose.     

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.     

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.     

Effects of High and Low Molecular Weight Glutenin Subunits on Rheological Dough Properties and Breadmaking Quality of Wheat

High molecular weight (HMW) or low molecular weight (LMW) subunits of different chemical state (reduced, reoxidized with KBrO₃, or KIO₃) or gliadins were added in 1% amounts to a base flour of the wheat cultivar Rektor and mixed with water. The corresponding doughs were then characterized by microscale extension tests and by microbaking tests and were compared to doughs from the base flour without additives. The maximum resistance of dough was strongly increased by HMW subunits in a reduced state and by HMW subunits reoxidized with KBrO₃. A moderate increase of resistance was caused by HMW subunits reoxidized with KIO₃ and by LMW subunits reoxidized with KBrO₃ or KIO₃. This resistance was strongly lowered by LMW subunits in a reduced state and by gliadins. The extensibility of dough was significantly increased only by gliadins and reduced HMW subunits; HMW subunits reoxidized with KBrO₃ had no effect, and all other fractions had a decreasing effect. In particular, glutenin subunits reoxidized with KIO₃ induced marked decrease of extensibility, resulting in bell‐shaped curve extensigrams, which are typical for plastic properties. The effect of reoxidized mixtures (2:1) of HMW and LMW subunits on maximum resistance depended on the oxidizing agent and on the conditions (reoxidation separated or together); extensibility was generally decreased. Bread volume was increased by addition of HMW subunits (reduced or reoxidized with KBrO₃) and decreased by LMW subunits (reoxidized with KBrO₃ or KIO₃) and by a HMW‐LMW subunit mixture (reoxidized with KBrO₃). The volume was strongly decreased by addition of reduced LMW subunits. A high bread volume was related to higher values for both resistance and extensibility.     

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.     

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.     

Characterization of Glutenin Protein Fractions from Sequential Extraction of Hard Red Spring Wheats of Different Breadmaking Quality

Gluten proteins from two cultivars of hard red spring (HRS) wheat with good and poor breadmaking quality were fractionated into 13 fractions by sequential extraction with dilute hydrochloric acid. Each subfraction was characterized by multistacking (MS) SDS‐PAGE under nonreducing conditions, followed by imaging densitometry. The glutenin polymers from the origins of MS‐SDS‐PAGE were analyzed by SDSP‐PAGE under reducing conditions to determine the composition of high and low molecular weight subunits. The results showed that fractions differed significantly in glutenin‐to‐gliadin ratios and in the size distribution of glutenin polymers. The earlier precipitated fractions were composed of more gliadins but fewer glutenin polymers. However, the glutenin polymers gradually increased in their relative quantities with the residue having the largest glutenin‐to‐gliadin ratio. The size distribution of glutenin polymers differed significantly from early precipitated to later fractions. The relative quantities of glutenin aggregates at the 4% origins increased significantly. The ratio of high molecular weight (HMW) to low molecular weight (LMW) glutenin subunits increased significantly from early to intermediate fractions. Between the two cultivars, significant differences were found in the ratio of HMW to LMW glutenin subunits and quantity of SDS insoluble glutenin polymers in the residue fraction with the better breadmaking quality cultivar ND706 having a greater ratio than the cultivar Sharp. It was concluded that the size distribution of glutenin polymers played an important role in determining the differences in breadmaking quality between the good and poor HRS wheat cultivars.     

Quantitative Determination of Gluten Protein Types in Wheat Flour by Reversed-Phase High-Performance Liquid Chromatography

A combined extraction-HPLC procedure was developed on a microscale to determine the amounts of the different gluten protein types (ω5-, ω1,2-, α- and γ-gliadins; high molecular weight [HMW] and low molecular weight [LMW] glutenin subunits) in wheat flour. After preextraction of albumins and globulins from flour (100 mg) with a salt solution (2 × 1.0 mL), extraction of gliadins was achieved with 60% aqueous ethanol (3 × 0.5 mL). Subsequently, the glutenin subunits were extracted under nitrogen and at 60°C with 50% aqueous 1-propanol containing Tris-HCl (0.05 mol/L, pH 7.5), urea (2 mol/L) and dithioerythritol (1%). The separation and quantitative determination of gliadins and glutenin subunits was then performed by reversed-phase HPLC on C₈ silica gel at 50°C using a gradient of increasing acetonitrile concentration in the presence of 0.1% trifluoroacetic acid. The flow rate was 1.0 mL/min, and the detection wavelength was 210 nm. Temperature and flow rate were modified for the quantitation of single underivatized HMW subunits. To determine the absolute amounts of protein types, different protein standards (gliadin, LMW and HMW subunits, bovine serum albumin) with known protein contents were compared to HPLC absorbance areas. The calibration curves were almost identical and linear over a broad range (20–220 μg). This extraction-HPLC procedure allows an accurate, reproducible, sensitive, and relatively fast quantitative determination of all gluten protein types in wheat flour, and can be applied to quality evaluation of cereals as raw materials or in processed products.     

Effect of the Molecular Weight Distribution of Glutenin Protein from an Extra‐Strong Wheat Flour on Rheological and Breadmaking Properties Through Reconstitution Studies

Ten glutenin fractions were separated by sequential extraction of wheat gluten protein with dilute hydrochloric acid from defatted glutenin‐rich wheat gluten of the Canadian hard red spring wheat (HRSW) cultivar Glenlea. The molecular weight distribution (MWD) of 10 different soluble glutenin fractions was examined by multistacking SDS‐PAGE under nonreduced conditions. Also, the subunit composition of the different glutenin fractions was determined by SDS‐PAGE under reduced conditions. The MWD of the fractions (especially HMW glutenins) varied from fraction to fraction. From early to later fractions, the MWD shifted from low to high. The early extracted fractions contained more LMW glutenin subunits (LMW‐GS) and less HMW glutenin subunits (HMW‐GS). The later extracted fractions and the residue fraction contained much more HMW‐GS (2*, 5, and 7 subunits) than the early extracted fractions. The trend in the amounts of 2*, 5, and 7 subunits in each fraction from low to high matched the extraction solvent sequence containing from lower to higher levels of HCl. The influence of glutenin protein fractions from the extra‐strong mixing cultivar, Glenlea, on the breadmaking quality of the weak HRSW, McVey, was assessed by enriching (by 1%) the McVey base flour with isolated glutenin protein fractions from Glenlea. The mixograph peak development times and loaf volumes of enriched flour were measured in an optimized baking test. The results indicated that the higher content in Glenlea glutenin of HMW‐GS with higher molecular weight, such as 2*, 5, and 7, seem to be the critical factor responsible for the strong mixing properties of Glenlea. Our results confirmed that subunit 7 occurred in the highest quantity of all the HMW‐GS. Therefore, it seems that the greater the content of larger molecular weight glutenin subunits, the larger the glutenin polymers and the stronger the flour.     

Immunocytochemical Localization of Gluten Proteins Uncovers Structural Organization of Glutenin Macropolymer

The content and composition of the disulfide‐bonded glutenin macropolymer has been shown to influence dough properties, although its structural organization is poorly characterized. The structure of the glutenin macropolymer in dough was studied using an immunolocalization transmission electron microscopy (TEM) technique by localizing gliadins, low molecular weight glutenin subunits (LMW‐GS), and high molecular weight glutenin subunits (HMW‐GS) in sections of dough using antibody probes selective for each of the three classes of gluten polypeptides. Distinct differences in the distribution patterns of gliadins, LMW‐GS, and HMW‐GS were observed, which suggests that proteins have different roles in the structural organization of the gluten matrix. On the basis of the observed distribution of the proteins in dough, it is speculated that gliadins, which are randomly distributed as individual particles, fill space within the glutenin macropolymer; LMW‐GS, which are present as clusters, are speculated to form aggregated branch structures; and HMW‐GS, which are present as chains, are speculated to form a network from which the LMW‐GS branches are formed. Changes in the distribution of gliadins, LMW‐GS, and HMW‐GS in dough during mixing were also noted. Such an arrangement supports previous biochemical evidence which has established that gliadins, LMW‐GS, and HMW‐GS have specific roles in the structural organization of the glutenin macropolymer in doughs.     

Effect of Multiple Substitution of Glutenin and Gliadin Proteins on Flour Quality of Canada Prairie Spring Wheat

The effect of genetic substitution of two to four glutenin and gliadin subunits from a Canada Prairie Spring (CPS) cv. Biggar BSR into Alpha 16, another CPS wheat line, was studied for rheological and baking quality. Results from double substitution showed that the presence of a gliadin component from Biggar BSR (BGGL) and low molecular weight glutenin subunit 45 (LMW 45) contributed to improved dough strength characteristics. Presence of BGGL in combination with high molecular weight glutenin subunit 1 (HMW 1) or 17+18 (HMW 17+18) also showed improved dough strength over control Alpha lines. When three or four protein subunits were substituted, even though improved quality performance was observed, it was associated with the negative effect of lowered flour water absorptions in spite of similar protein contents. The study confirms that LMW glutenins, as well as gliadins, play an important role along with HMW glutenins in wheat flour quality. CPS wheat lines with improved dough strength properties can be selected from the double substitution lines with the combination of BGGL/LMW 45 and BGGL/HMW 1.     

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.     

Biochemical Studies of Proteins in Nondeveloped,Partially Developed,and Developed Doughs

Nondeveloped, partially developed with shear and extensional deformations, and developed doughs represent different stages of dough development. To understand the relationship between gluten proteins and dough rheology, this study used disulfide‐sulfhydryl analyses, gel filtration chromatography, SDS‐PAGE, acid polyacrylamide gel electrophoresis (A‐PAGE), and densitometry to examine proteins in the four types of doughs mentioned. Free sulfhydryl content was the lowest in native flour and nondeveloped dough, and the highest in partially developed doughs, while a reverse trend was observed for disulfide content. For each flour sample, the protein elution profile from gel filtration chromatography shifted with the level of dough development. With respect to the smallest sized molecules, native flour had the most, followed by nondeveloped, partially developed, and then developed doughs. SDS‐PAGE and A‐PAGE exhibited similar protein patterns among the same chromatographed protein fractions of each native flour and its different doughs. Densitometric data showed that the amount of high molecular weight (HMW) glutenins increased and the amounts of low molecular weight (LMW) glutenins, gliadins, and albumins/globulins decreased with progressive stages of dough development. In conjunction with previously published results, indications are that the increase in the size and the amount of HMW glutenins is related to the strength of dough and the amount of protein matrix present in the dough.     

Relationship Between the Qualitative and Quantitative Compositions of Gluten Protein Types and Technological Properties of Synthetic Hexaploid Wheat Derived from Triticum durum and Aegilops tauschii

The contribution of the diploid wheat species Aegilops tauschii (Coss.) Schmall to the technological properties of bread wheat (Triticum aestivum L.) was previously studied by the investigation of synthetic hexaploids derived from tetraploid durum wheat (T. turgidum L.) and three diploid Ae. tauschii lines. The results indicated that bread volume, gluten index, SDS‐sedimentation volume, and maximum resistance of gluten were significantly influenced by the Ae. tauschii lines. To determine the relationship between technological properties and qualitative and quantitative compositions of gluten proteins, the flours of parental and synthetic lines were extracted using a modified Osborne fractionation. Gliadin and glutenin fractions were then characterized by reversed‐phase (RP) HPLC on C₈ silica gel. The HPLC patterns revealed typical differences between synthetic and parental lines. The gliadin patterns of three synthetic lines and the glutenin patterns of two synthetic lines were more similar to that of the diploid Ae. tauschii parents involved in the hybrids. In the glutenin pattern of one synthetic line, characteristics from both Ae. tauschii and the durum wheat parents were observed. The amount of total gliadin and gliadin types of the synthetic lines was mostly intermediate between those of the durum and Ae. tauschii parents. The amounts of total glutenin and glutenin types (HMW and LMW subunits) of the synthetic lines were generally higher than those of the parental lines, and the ratio of gliadins to glutenins was significantly decreased. High positive correlations were found between the amount of total glutenins, HMW, and LMW subunits and bread volume, maximum resistance and extension area of gluten, and SDS‐sedimentation volume. The ratio of gliadins to glutenin subunits had a strong negative influence on these properties. The protein content of the flours and the amount of total gluten proteins were not correlated with any of the technological properties. Results on the relationship between biochemical characteristics and the breadmaking properties indicated that wheat prebreeding would benefit from studies on protein types and quantification in the choice of parents. In addition, the potential of the diploid Ae. tauschii for improvement of breadmaking quality should be further exploited.     

Role of Gluten and Its Components in Determining Durum Semolina Dough Viscoelastic Properties

Gluten was isolated from three durum wheat cultivars with a range in strength. Gluten was further fractionated to yield gliadin, glutenin and high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits (GS). The gluten and various fractions were used to enrich a base semolina. Enriched dough samples were prepared at a fixed protein content using a 2‐g micromixograph. Mixing strength increased with addition of gluten. Dynamic and creep compliance responses of doughs enriched with added gluten ranked in order according to the strength of the gluten source. Gliadin addition to dough resulted in weaker mixing curves. Gliadin was unable to form a network structure, having essentially no effect on dough compliance, but it did demonstrate its contribution to the viscous nature of dough (increased tan δ). Source of the gliadin made no difference in response of moduli or compliance. Addition of glutenin to the base semolina increased the overall dough strength properties. Glutenin source did influence both dynamic and compliance results, indicating there were qualitative differences in glutenin among the three cultivars. Enrichment with both HMW‐GS and LMW‐GS increased overall dough strength. Source of HMW‐GS did not affect compliance results; source of LMW‐GS, however, did have an effect. The LMW‐2 proteins strengthened dough to a greater extent than did LMW‐1. Mechanisms responsible for dough viscoelastic properties are described in terms of reversible physical cross‐links.     

Flour Protein Composition and Functional Properties of Transgenic Rye Lines Expressing HMW Subunit Genes of Wheat

Flours from nonsprouted (ns) kernels and dried sprouted (s) kernels of transgenic rye expressing HMW glutenin subunits (HMW‐GS) 1Dy10 (L10) or 1Dx5+1Dy10 (L5+10) from wheat were compared with flours from the corresponding wildtype rye (Lwt). The crude protein content of nonsprouted flours ranged from 9.2% (Lwt) to 10.4% (L5+10) and was lowered by ≈1% due to sprouting. Flour proteins were separated into albumins/globulins, prolamins, and glutelin subunits by a modified Osborne fractionation and into SDS‐soluble and insoluble fractions. Portions of the prolamin fractions were reduced in the same manner as glutelins. The different fractions were then characterized and quantified by RP‐HPLC on C₈ silica gel. The proportion of albumins/globulins did not significantly differ between transgenic lines and wildtype. The proportions of alcohol‐insoluble glutelins and SDS‐insoluble proteins drastically increased in transgenic rye due to a shift of HMW and γ‐75k secalins into the polymeric fractions. Significant differences in the proportion of highly polymeric proteins between nonsprouted and sprouted flours could not be detected. The quantitative data demonstrated that the expression of HMW‐GS led to a higher degree of polymerization of storage proteins in rye flour. The HMW‐GS combination 1Dx5+1Dy10 showed stronger effects than 1Dy10 alone. The analyzed flours contained two HMW secalins (R1, R2), whose amino acid compositions were closely related to those of 1Dy10 and 1Dx5, respectively. The amounts of R1 in Lwt flours determined by RP‐HPLC were 221 mg (ns) and 186 mg (s) per 100 g and those of R2 were 344 mg (ns) and 298 mg (s), respectively. These amounts increased to 240 mg (ns)/201 mg (s) (R1) and 479 mg (ns)/432 mg (s) (R2) in L10 flours. In L5+10 flours, the amount of R1 decreased to 150 mg (ns)/132 mg (s) while R2 increased to 432 mg (ns)/338 mg (s). The amount of HMW‐GS 1Dy10 was almost the same as that of R2 in L10 flours but was strongly increased in L5+10 flour (633 mg [ns]/538 mg [s]). HMW‐GS 1Dx5 was, by far, the major subunit in L5+10 flours (987 mg 7[ns]/896 mg [s]). The summarized amounts of all HMW subunits increased from ≈0.5 g (Lwt) to ≈1.1 g (L10) and ≈2.0 g (L5+10). Thus only L10 flours were similar to wheat flours in HMW subunit content. The baking performance of L10 flour determined by a microbaking test was improved compared with Lwt flour, whereas L5x10 flour showed very poor properties obviously due to the strongly increased proportion of highly cross‐linked glutelins. The breadmaking quality of flours from 1Dy10 seeds and wildtype seeds was reduced by the same degree when flours from sprouted seeds were analyzed.     

Quantitative Variation of HMW Glutenin Subunits from Hard Red Spring Wheats Grown in Different Environments

The objective of this study was to investigate the quantitative variation of HMW glutenin subunits in relation to glutenin polymers and hence breadmaking quality across different environments. Six genotypes of hard red spring (HRS) wheat were grown at seven locations in North Dakota in 1998 in a randomized complete‐block experimental design with three replicates at each location. Unreduced SDS‐soluble glutenins of flour were fractionated by multistacking SDS‐PAGE into different sized glutenin polymers, followed by SDS‐PAGE and imaging densitometry to determine the quantitative variation of HMW glutenin subunits. SDS‐insoluble glutenin polymers also were examined for their quantitative composition of HMW glutenin subunits. The results showed that the percentage of HMW glutenin subunits was significantly affected by growing locations. The quantity of HMW glutenin subunits in SDS‐insoluble glutenins was significantly and positively correlated with loaf volume. SDS‐insoluble glutenin polymers had a higher percentage of HMW glutenin subunits than did SDS‐soluble glutenins. SDS‐insoluble glutenin polymers in flour were positively and significantly correlated in proportions of both total and individual HMW glutenin subunits in total SDS glutenins. SDS‐insoluble glutenin polymers also were positively and significantly correlated with the combined proportion of HMW glutenin subunits 2* + 5. The results of this study indicated that either subunit 2* or 5 might be more important in forming a greater quantity of larger SDS‐insoluble glutenin polymers than other subunits. SDS‐insoluble glutenin polymers from different cultivars or locations could have different quantities of HMW glutenin subunits in their composition. SDS‐insoluble glutenin polymers with more HMW glutenin subunits might be larger sized than those with less HMW glutenin subunits. Environment significantly influenced the quantitative variation of HMW glutenin subunits, which in turn affected the size distribution of glutenin polymers, and hence breadmaking quality.     

Effect of Aelia spp. and Eurygaster spp. Damage on Wheat Proteins

The effect of Aelia spp. and Eurygaster spp. wheat bugs on the protein fractions of different wheat cultivars has been studied by size‐exclusion high‐performance liquid chromatography (SE‐HPLC) and free‐zone capillary electrophoresis (FZCE). Those methods were used to quantify and characterize the extent of protein modification. A decrease in the amount of alcohol‐insoluble polymeric proteins along with an increase in the alcohol‐soluble polymeric proteins and gliadins were observed in damaged wheat. The high molecular weight (HMW) and low molecular weight (LMW) glutenin fractions were barely detected in the incubated damaged wheat from some cultivars, which indicated hydrolysis of those proteins by the bug proteinases. In damaged wheats, both incubated and unincubated, gliadin electrophoregrams revealed the presence of some new peaks with mobilities similar to the ω gliadins. The overall results suggest that the bug proteinases are potent enzymes that appear to be nonspecific because they hydrolyze all gluten proteins.     

Identification of Wheat Protein Components Involved in Polymer Formation on Incubation with Transglutaminase

Transglutaminase (TG) catalyzes acyl‐transfer reactions, introducing covalent cross‐links between l ‐lysine and l ‐glutamine residues. As a result, peptides are connected and the structure of a stabilized protein network is formed, thereby improving protein strength. In this study, wheat flour was incubated with TG for different time intervals (0, 30, 60, 120, and 240 min) and the extent of polymer formation and proteins involved were investigated by SE‐HPLC, SDS‐PAGE, and RP‐HPLC. Results indicated that the amount of polymers formed increased with incubation time. TG induced the cross‐linking of HMW glutenin subunits more so than of other proteins in wheat.     

Comparative study of the effect of incorporated individual wheat storage proteins on mixing properties of rice and wheat doughs

The aim of this work was to compare the effects of incorporated wheat storage proteins on the functional properties of rice and wheat flours. The advantage of rice as a base flour compared to wheat is that it does not contain any wheat flour components and, therefore, has no interactive effect between wheat glutenin proteins. The incorporation of individual HMW glutenin subunit proteins (Bx6, Bx7, and By8) in different ratios had significant positive effects on the mixing requirements of both rice and wheat doughs. Reconstitution experiments using two x+y type HMW-GS pairs together with a bacterially expressed LMW-GS have been also carried out in this study. The largest effects of polymer formation and mixing properties of rice flour dough were observed when Bx and By subunits were used in a 1:1 ratio and HMW and LMW glutenin subunits in a 1:3 ratio. However, using the same subunit ratios in wheat as the base flour, these synergistic effects were not observed.