Depolymerization of the Glutenin Macropolymer During Dough Mixing: I. Changes in Levels,Molecular Weight Distribution,and Overall Composition

Changes in the amounts, molecular weight distributions, and levels of major groups of subunits in the glutenin macropolymer (GMP) of doughs during mixing were investigated. The GMP (gel protein) is the unreduced fraction of gluten protein that remains as a layer on top of the starch after extraction of SDS-soluble proteins and centrifugation. Experiments involved doughs prepared from flours derived from one weak and one strong cultivar and lines derived from cv. Olympic that were null for specific high molecular weight glutenin subunits (HMW-GS). During mixing, the amount of GMP decreased; the major changes occurred before peak mixing time (MT, achievement of peak resistance). In addition, the average apparent molecular weight of GMP (determined by both size-exclusion HPLC and multilayer gel electrophoresis) decreased during mixing, but in this case, the major changes were seen later in the mixing process, during dough breakdown. Even after extensive mixing, polymers and oligomers were released, not free glutenin subunits. During dough breakdown, the composition of GMP also changed, such that the proportion of HMW-GS decreased but β-amylases/D low molecular weight glutenin subunits (LMW-GS) increased. Changes in the total amounts of other LMW-GS typically were smaller with a decrease in the proportion of B subunits and an increase in the proportion of C subunits. The major changes in GMP composition were observed after peak MT (peak resistance) occurring earlier and to a greater extent in the weaker dough. Our results suggest that dough breakdown during mixing may be triggered by loss of HMW-GS, leading to changes in the molecular weight distribution and composition of the disulfide-bonded GMP.     

Changes in SDS Solubility of Glutenin Polymers During Dough Mixing and Resting

An online coupling of high‐performance size‐exclusion chromatography (HPSEC) combined with multiangle laser‐light scattering (MALLS) and a reverse‐phase HPLC procedure were used to characterize and reveal the polydispersity of the glutenin polymers of doughs during mixing and resting. Experiments involved doughs prepared from several samples of a common French wheat cultivar (Soissons) differing in total amount of SDS‐unextractable glutenin polymers. During dough mixing, the amounts, size distribution of protein, and glutenin subunit composition within the SDS‐unextractable polymers changed. However, the major changes in SDS‐unextractable glutenin content and size distribution occurred before the peak mixing time (MT) was reached, whereas detectable changes in subunit composition also occurred after the peak MT. Even if sonication, which was used to solubilize the total wheat glutenin, can narrow the glutenin size distribution, HPSEC‐MALLS revealed a close relationship between the SDS solubility of the glutenin polymers and size distribution, confirming a depolymerization and repolymerization hypothesis. During the depolymerization of the SDS‐unextractable polymers, glutenin subunits were released in nonrandom order, which indicated that the polymers have a hierarchical structure. Some HMW glutenin subunits (HMW‐GS), especially 1D×5, were particularly resistant to the depolymerization mechanism. This suggested that the subunit plays a major role in forming the backbone of the SDS‐unextractable polymers, consistent with the potential to form branched structure. These studies suggest that the SDS‐unextrac‐table polymers in flours have a well‐ordered structure that can be modified by dough mixing and resting.     

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.     

Effect of Single Substitution of Glutenin or Gliadin Proteins on Flour Quality of Alpha 16, a Canada Prairie Spring Wheat Breeders' Line

The effect of genetic variation in the glutenin and gliadin protein alleles of Alpha 16, a Canada Prairie Spring (CPS) wheat line, on the dough mixing, bread, and noodle quality properties were evaluated. The presence of a gliadin component (BGGL) and the low molecular weight glutenin subunit (LMW-GS) 45 found in the selection Biggar BSR were associated with significant increases in dough strength characteristics. The results of the study showed that gliadins, LMW-GS, and high molecular weight glutenin subunits (HMW-GS) can influence bread- and noodle-making properties of wheat flour. Genotype-by-environment interactions were not significant for most of the quality parameters studied, indicating that the differences observed in quality characteristics were mainly due to the effect of genotype.     

The Combination of Rhizopus chinensis Lipase and Transglutaminase Affects the Rheology and Glutenin Macropolymer Properties of Frozen Dough

The combination of Rhizopus chinensis lipase (RCL) and transglutaminase (TG) was previously reported to improve the quality of frozen dough bread. In this study, the effects of RCL, TG, and their combination on the modification of glutenin macropolymer (GMP) and rheological properties of dough during frozen storage were investigated. Frozen storage changed both GMP and rheology properties of dough. TG treatment significantly decreased the ratio of high‐molecular‐weight glutenin subunits to low‐molecular‐weight glutenin subunits and GMP content in fresh dough, and GMP particle size increased. The effect of RCL on GMP properties was not significant, but its combination with TG dramatically increased the proportion of the larger particles and weighted average volume (D_4.3) in GMP. The treatment with the enzyme combination could have inhibited the depolymerization of GMP, which slowed down the decrease rate of some parameters such as GMP content, proportion of larger particles, D_4.3, and release of free amino and thiol groups during frozen storage. The modification of GMP properties by enzyme treatment weakened the effect of the freezing process on rheological properties of dough, especially TG treatment and its combination with RCL. Correlation between GMP particle size and dough properties (dough tensile force and elastic modulus) after freezing and enzyme treatment were confirmed.     

Glutenin Macropolymer in Salted and Alkaline Noodle Doughs

An attempt was made to understand the physicochemical attributes that are the basis of physical differences between alkaline and salted noodle doughs. Flour and dough properties of one soft and three hard‐grained wheat cultivars were observed. Doughs were made with either sodium chloride or sodium carbonate. Each formulation variant was tested at both high and low water additions. Samples for glutenin macropolymer (GMP) isolation were taken at selected noodle dough processing stages. When a 1.67% w/v Na₂CO₃ solution was used for mixograph testing, dough characteristics were radically altered and differences between cultivars were masked. In lubricated squeezing flow (LSF) testing, hard wheat noodle doughs had significantly (P < 0.01) longer relaxation times and higher % residual force values than soft wheat doughs in both the salted and alkaline variants. LSF maximum force and biaxial viscosity were significantly higher in alkaline doughs than salted. GMP extracted from alkaline doughs was gummy and sticky, and was more opaque than GMP from salted doughs. GMP weight decreased sequentially when extracted from samples taken in the active phase (mix, compound, sheet) of noodle dough processing and decreased more in alkaline doughs. GMP weight increased more after 24 hr of dough rest in salted doughs. GMP gel strength was noticeably higher in GMP extracted from alkaline doughs. After dough resting, alkaline GMP gel strength significantly increased, whereas it decreased in GMP from salted doughs, suggesting a role for GMP in the increased stiffness of alkaline noodle doughs.     

Influence of Salts and Aggregation of Gluten Proteins on Reduction and Extraction of High Molecular Weight Glutenin Subunits of Wheat

High-molecular weight glutenin subunits (HMW-GS) of wheat were extracted by various combinations of reducing agents, salt solutions, and solvents. Preferential extraction of 1D-encoded HMW-GS occurred when flours were extracted with Tris-HCl SDS buffer at pH 6.8 containing 6% mercaptoethanesulfonic acid sodium salt (MESNA) and analyzed by SDS-PAGE. Similar effects were also found when dithiothreitol or β-mercaptoethanol were used in conjunction with nonchaotropic salts. If flours were first extracted with 50% 1-propanol, the extraction procedure yielded all HMW-GS, even in the presence of MESNA or high levels of salts. Addition of alcohols or chaotropes to the Tris buffer solutions containing MESNA or of solutions containing salt also extracted all HMW-GS. The HMW-GS reported most important in baking quality were found preferentially extracted by nonchaotropic salts and reducing agents. This is related to gluten aggregation and the gliadin-glutenin interaction and structure.     

Isolation and Functionality Testing of Low Molecular Weight Glutenin Subunits

Various protein fractionation techniques have been applied to the isolation and purification of milligram quantities of low molecular weight glutenin subunits (LMW-GS). No single technique was applicable to the purification of the majority of the subunits. Partial purification of certain LMW-GS was obtained using ion-exchange chromatography and reversedphase HPLC. Preparations containing α- and γ-type subunit sequences did not strengthen dough when incorporated into a base flour, whereas preparations containing a subunit with an N-terminal methionine residue (METSHIPGL-) did. Using preparative isoelectric focusing over a narrow pH range, it was possible to purify (to ≈90% purity) a B subunit that also had the N-terminal sequence of METSHIPGL-. This polypeptide, when incorporated into a base flour, had a dough strengthening effect in mixing trials, but less so than an equivalent amount of a high molecular weight glutenin subunit.     

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.     

Purification and Characterization of the Glutenin Subunits of Triticum tauschii,Progenitor of the D Genome in Hexaploid Bread Wheat

N-terminal amino acid sequences and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) molecular weights have been determined for high-performance liquid chromatography (HPLC)-purified high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits (GS) of Triticum tauschii ssp. strangulata, contributor of the D genome to hexaploid bread wheat. The use of three different extraction procedures resulted in similar glutenin preparations. On the basis of N-terminal sequences, the same types of glutenin subunits that have been reported in bread and durum wheats (HMW-GS of both the x and y types and LMW-GS of the LMW-s, LMW-m, α-, and γ-types) were found in T. tauschii. However, the HMW-GS in T. tauschii were in greater proportion relative to LMW-GS when compared to reported values for a bread and durum wheat. Our results support the likelihood that differences in the proportions of the various subunits contributed by the A, B, and D genomes, rather than qualitative differences in the types of subunits, are responsible for the major differences in quality characteristics between bread wheat and durum wheat.     

Molecular Weight Distribution of Wheat Proteins

The molecular weight distribution (MWD) of wheat proteins is becoming recognized as the main determinant of physical dough properties. Studies of high polymers have shown that properties such as tensile strength are related to a fraction of polymer with molecular weight above a critical value and the MWD of this fraction. Elongation to break is treated as a kinetic process with energies of activation for breaking noncovalent bonds and for chain slippage through entanglements. These considerations are related to tensile properties of wheat flour doughs such as those measured by the extensigraph. The MWD of wheat proteins is determined by the relative amounts of monomeric and polymeric proteins and the MWD of the polymeric proteins. The latter, in turn, depends on the ratio of high molecular weight glutenin subunits (HMW-GS) to low molecular weight glutenin subunits (LMW-GS), the specific HMW-GS that result from allelic variation, and the presence of modified gliadins that act as chain terminators. The role of these compositional variables in determining dough extensional properties is discussed in terms of present knowledge. Determination of MWD of wheat proteins is hindered by the difficulty of their solubilization and the lack of methods for reliably measuring very high molecular weights. Among the promising techniques for achieving these measurements are multiangle laser light scattering (MALLS) and field flow fractionation (FFF).     

施氮量对不同品质类型小麦子粒蛋白质组分含量及加工品质的影响

选用强筋小麦济麦20、中筋小麦泰山23和弱筋小麦宁麦9号,利用反相高效液湘色谱（RP-HPLC）方法测定了施氮量对不同品质类型小麦子粒蛋白质组分含量和高分子量谷蛋白亚基（HMW-GS）、低分子量谷蛋白亚基（LMW-GS）含量的影响,并分析其与子粒加工品质的关系。结果表明,随施氮量增加,强筋小麦济麦20和中筋小麦泰山23的子粒蛋白质含量及各组分含量均呈先增加后降低的趋势,施氮量为N 240 kg/hm2时,蛋白质各组分含量较高,加工品质较好; 过量施氮抑制了HMW-GS合成,这是过量施氮导致强筋和中筋小麦子粒蛋白质品质变劣的原因之一。随施氮量增加,弱筋小麦宁麦9号子粒的蛋白质各组分含量显著增加,加工品质变劣。增施氮肥,3个品种的谷蛋白和醇溶蛋白含量的增加幅度显著高于清蛋白+球蛋白含量,这是施氮改善强筋和中筋小麦子粒加工品质的主要原因。济麦20和泰山23两品种的总蛋白质含量及醇溶蛋白含量无显著差异,但强筋小麦济麦20的谷蛋白含量、贮藏蛋白、HMW-GS、LMW-GS、谷蛋白大聚合体（GMP）含量及谷蛋白与醇溶蛋白含量的比值（Glu/Gli）和HMW-GS与LMW-GS含量的比值（HMW/LMW）高于中筋小麦泰山23,这是强筋小麦济麦20加工品质形成及其面团形成时间和稳定时间显著高于泰山23的重要原因。     

Differing Effects of Mechanical Dough Development and Sheeting Development Methods on Aggregated Glutenin Proteins

Dough development using sheeting and mechanical dough development (MDD) were compared with respect to the effect the mixing method had on the molecular size distribution and degree of protein thiol exposure of the aggregated glutenin proteins. Although sheeting imparts a lower rate of work input on doughs than does MDD mixing, changes in protein aggregation patterns during mixing were similar for both methods of dough development, indicating that protein disaggregation was important in the process of dough development. In both systems, a reduced rate of change in the protein aggregation patterns was associated with optimum dough development. The MDD mixing was characterized by increasing exposure of the thiol groups on the SDS‐insoluble glutenin during mixing while the sheeting process resulted in fewer exposed thiol groups on both SDS‐soluble and SDS‐insoluble glutenin proteins. This suggested that disulfide bond rupture may not be a required process in dough development and that high effective stresses per se may not be required to develop doughs. This is consistent with a model for dough development that does not require extensive covalent bond rupture but instead involves mainly rupture and reformation of noncovalent interactions such as hydrophobic bonds and hydrogen bonds between protein chains.     

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.     

Development of a Panel of Specific Monoclonal Antibodies to High Molecular Weight Glutenin Subunits and Their Application in Genetic Screening

High molecular weight glutenin subunits (HMW-GS) encoded by different chromosomal loci and alleles (1, 2, 5, 7, 10, and 12) were purified using reversed-phase HPLC from reduced, aqueous propanol extracts of flour from aneuploid or null wheat lines. Unlike previous libraries of monoclonal antibodies developed in our laboratory to SDS-extracted or alkylated HMW-GS, several of the monoclonal antibodies (mAb) developed in this study had a range of specificity patterns for HMW-GS in enzyme-linked immunosorbent assay (ELISA) and on immunoblots. A subset of the mAb bound either x- or y-type HMW-GS but not other gluten proteins, while a few antibodies bound one (mAb 110622, 110421, 140820), or two (mAb 101319, 110804, 140705, 1410460) HMW-GS expressed in each cultivar tested. In most cases, antibodies bound equally to the subunits encoded by different HMW-GS alleles. The more specific antibodies should be useful in research on the quantitative variation of HMW-GS expression and in studies of the role of particular HMW-GS in dough structure. The mAb 101319, which was prepared to subunit 1, bound to HMW-GS 1Bx subunits in ELISA and on immunoblots. This antibody also provided a higher absorbance value in ELISA with extracts of wheat lines expressing the Glu-Ble allele (HMW-GS 20) compared with the Glu-Bli allele (HMW-GS 17+18). Another mAb (110622) detected subunit 2 more strongly than subunit 5 in ELISA and produced a higher signal in immunoblots with subunit 2 even though these subunits are >98.7% homologous in amino acid sequence. An ELISA assay using this antibody was optimized for discrimination of wheat lines with the allelic pairs of subunits 1Dx5-1Dy10 from those with 1Dx2-1Dy12, with the former lines providing stronger dough properties and superior breadmaking quality. The performance of this assay was unaffected by other variations at HMW-GS loci and was demonstrated in sets of biotypes, doubled haploid, and cross-bred breeder's lines.     

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.     

Relationship between endogenous protein disulfide isomerase family proteins and glutenin macropolymer

The effects of endogenous protein disulfide isomerase (PDI) family proteins on the properties of gluten proteins in dough during breadmaking were determined using bacitracin, an inhibitor of PDI. Bread loaf volume in the presence of bacitracin was increased to 118% of that in the absence of bacitracin. The addition of bacitracin caused a decrease in the extension tolerance of the dough. The amount of sodium dodecyl sulfate (SDS)-insoluble glutenin macropolymer (GMP) in dough decreased to approximately 70% of that in flour during the 20 min of mixing for doughmaking. The addition of bacitracin to dough caused a dramatic GMP decrease, corresponding to ～20-30% of that in flour during the 20 min of mixing. The decrease in GMP was compensated by an increase in SDS-soluble glutenin polymer. Taken together, these results suggest that the endogenous PDI family proteins in flour suppress the depolymerization of GMP during dough mixing.     

Quantitative Determination of High Molecular Weight Glutenin Subunits of Hard Red Spring Wheat by SDS-PAGE. I. Quantitative Effects of Total Amounts on Breadmaking Quality Characteristics

Thirteen hard red spring wheat genotypes in which seven genotypes had the same high molecular weight (HMW) glutenin subunits (2*, 7+9, 5+10) were compared for their physical-chemical and breadmaking properties. These samples were categorized into three groups based on their dough mixing and baking performances as follows: the strong dough (SD) group (six genotypes), characterized by the strongest dough mixing (average stability, 35 min); the good loaf (GL) group (four genotypes), characterized by the largest loaf volume; and the poor loaf (PL) group (three genotypes), characterized by the smallest loaf volume. Total flour proteins were fractionated into 0.5M salt-soluble proteins, 2% SDS-soluble proteins, and residue proteins (insoluble in SDS buffer). SDS-soluble proteins, residue proteins, and total flour proteins were analyzed by SDS-PAGE and densitometry procedures to determine the proportions of HMW glutenin subunits, medium molecular weight proteins, and low molecular weight proteins in relation to the total amount of proteins. No differences in the amount of salt-soluble proteins were found among the different groups of samples. Solubilities of gluten proteins (total proteins minus salt-soluble proteins) in SDS buffer were related to the differences in dough strength and baking quality among the three groups. The SD group had the lowest solubility and the PL group had the highest. SDS-PAGE analysis showed that SDS-soluble proteins of the SD group contained a smaller amount of HMW glutenin subunits than those of the GL and PL groups. The highest proportions of HMW glutenin subunits in total flour proteins were found in the SD group, while the PL group had the lowest percentage of HMW glutenin subunits in their total flour proteins. These results showed that the total quantities of HMW glutenin subunits played an important role in determining the dough mixing strength and breadmaking performance of hard red spring wheats.     

Proteolysis in model sourdough fermentations

Model wheat doughs started with six different lactic acid bacteria (LAB), with or without a commercial baker's yeast culture, were used to study proteolysis in sourdough fermentations. Cell counts, pH, and free amino acid concentration were measured. Sequential extraction of dough samples was performed to separate wheat proteins. The salt-soluble protein fraction (albumins and globulins) was analyzed by RP-HPLC and SDS-PAGE, whereas propanol-soluble (gliadins) and insoluble (glutenins) protein fractions were analyzed by SDS-PAGE only. Multivariate statistical methods were used for the analysis of results. The presence of yeasts and LAB affected RP-HPLC and SDS-PAGE patterns of the salt-soluble fraction in a complex way. The only changes in the gluten proteins that could be related to the presence of LAB were the appearance of new protein fragments (20 and 27 kDa) from gliadins and the degradation of high molecular weight glutenin subunits.     

卫星搭载小麦SP3代高分子麦谷蛋白亚基的SDS-PAGE分析

利用十二烷基磺酸钠-聚丙烯胺凝胶电泳（SDS-PAGE）技术对返回式卫星搭载小麦两个品种SP3代籽粒的高分子麦谷蛋白亚基（HMW-GS）进行了分析,并按照相关评分方法计算了高分子量麦谷蛋白Glu-1位点的品质得分。结果表明,经卫星搭载可产生较高频率的HMW-GS基因变异,陕253和西农1043 两个品种SP3代HMW-GS组成的变异频率分别为27.08%和27.45%。陕253和西农1043 SP3代的小麦品质得分分别为7分和6分,陕253 SP3代变异株为优质小麦。