Structural Modifications of Gluten Proteins in Strong and Weak Wheat Dough During Mixing

The network‐forming attributes of gluten have been investigated for decades, but no study has comprehensively addressed the differences in gluten network evolution between strong and weak wheat types (hard and soft wheat). This study monitored changes in SDS protein extractability, SDS‐accessible thiols, protein surface hydrophobicity, molecular weight distribution, and secondary structural features of proteins during mixing to bring out the molecular determinants of protein network formation in hard and soft wheat dough. Soft wheat flour and dough exhibited greater protein extractability and more accessible thiols than hard wheat flour and dough. The addition of the thiol‐blocking agent N‐ethylmaleimide (NEM) resulted in similar results for protein extractability and accessible thiols in hard and soft wheat samples. Soft wheat dough had greater protein surface hydrophobicity than hard wheat and exhibited a larger decrease in surface hydrophobicity in the presence of NEM. Formation of high‐molecular‐weight (HMW) protein in soft wheat dough was primarily because of formation of disulfides among low‐molecular‐weight (LMW) proteins, as indicated by the absence of changes in protein distribution when NEM was present, whereas in hard wheat dough the LMW fraction formed disulfide interaction with the HMW fraction. Fourier transform infrared spectroscopy indicated formation of β‐sheets in dough from either wheat type at peak mixing torque. Formation of β‐sheets in soft wheat dough appears to be driven by hydrophobic interactions, whereas disulfide linkages stabilize secondary structure elements in hard wheat dough.     

Gluten Structural Evolution During Pasta Processing of Refined and Whole Wheat Pasta from Hard White Winter Wheat: The Influence of Mixing,Drying, and Cooking

An attempt was made to evaluate gluten structural changes in refined and whole wheat pasta from hard white winter wheat to elucidate the impact of whole wheat components on the formation and structure of the gluten network in pasta. Attenuated total reflectance–FTIR spectroscopy was used to track gluten secondary structure through most of the major steps in pasta processing: raw material, mixing, drying, and cooking. Protein solubility, accessible thiols, and SDS‐PAGE data were also collected to provide additional information on the nature of protein interactions and network composition. Few secondary structural differences were observed between refined and whole wheat flours from hard white wheat. However, mixing induced a significant shift to β‐sheet structures in refined dough that was not equally matched by whole wheat dough. Drying under both high temperature, short time (HT) and low temperature, long time (LT) conditions resulted in a reversion to structural distributions similar to those for flour in both pastas. However, greater protein denaturation in HT samples was indicated by lower protein solubility also in the presence of denaturants and disulfide reducing agents. Cooking generated a substantial increase in β‐sheet structures for both pasta systems. This structure was greatest in refined and LT samples. Thiol accessibility data indicate the presence of a highly aggregated, compact gluten network in refined pasta, mostly driven by hydrophobic association. Conversely, the network in whole wheat pasta was more loosely associated and dependent on disulfide bonding, both of which fit well with the secondary structural data.     

Impact of Bran Addition on Water Properties and Gluten Secondary Structure in Wheat Flour Doughs Studied by Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy

The aim of this work was to elucidate the underlying physical mechanism(s) by which bran influences whole grain dough properties by monitoring the state of water and gluten secondary structure in wheat flour and bran doughs containing 35–50% moisture and 0–10% added bran. The system was studied with attenuated total reflectance (ATR) FTIR spectroscopy. Comparison of the OH stretch band of water in flour dough with that in H₂O‐D₂O mixtures having the same water content revealed the formation of two distinct water populations in flour dough corresponding to IR absorption frequencies at 3,600 and 3,200 cm^–1. The band intensity at 3,200 cm^–1, which is related to water bound to the dough matrix, decreased and shifted to lower frequencies with increasing moisture content of the dough. Addition of bran to the dough caused redistribution of water in the flour and bran dough system, as evidenced by shifts in OH stretch frequency in the 3,200 cm^–1 region to higher frequencies and a reduction in monomeric water (free water). This water redistribution affected the secondary structure of gluten in the dough, as evidenced by changes in the second‐derivative ATR‐FTIR difference spectra in the amide I region. Bran addition caused an increase in β‐sheet content and a decrease in β‐turn (β‐spiral) content. However, this bran‐induced transconformational change in gluten was more significant in the 2137 flour dough than in Overley flour dough. This study revealed that when bran is added to flour dough, water redistribution among dough components promotes partial dehydration of gluten and collapse of β‐spirals into β‐sheet structures. This transconformational change may be the physical basis for the poor quality of bread containing added bran.     

Effect of Transglutaminase,Citrate Buffer,and Temperature on a Soft Wheat Flour Dough System

Transglutaminase (TGase) can improve the functional characteristics of proteins by introducing covalent bonds inter‐ or intrachains. Temperature and pH interfere with the protein structure and the catalytic activity of enzymes. Because these three factors can act synergistically, TGase, citrate buffer, and temperature were evaluated for their effects on the rheological and chemical changes in low‐protein wheat flour dough. Dough strength, measured by microextension test, significantly increased with increasing levels of TGase (8 U/g of protein), with changes in pH of the citrate buffer (pH 6.5), and by the effect of interaction between these factors. The same trend was observed in the size‐exclusion HPLC measurements, indicating that these two parameters have the effect of increasing gluten protein aggregation. Temperature had a significant effect on dough extension, measured by microextension test. The changes in secondary structure of gluten protein were investigated by FTIR second‐derivative spectra (amide I region, 1,600–1,700 cm⁻¹) and showed an increase in β‐sheet structures initiated by TGase, citrate buffer pH, and their interaction.     

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.     

Polymer Conformation Structure of Wheat Proteins and Gluten Subfractions Revealed by ATR‐FTIR

The polymer conformation structure of gluten extracted from a Polish wheat cultivar, Korweta, and gluten subfractions obtained from 2 U.K. breadmaking and biscuit flour cultivars, Hereward and Riband, was investigated using attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR). The results showed the conformation of proteins varied between flour, hydrated flour, and hydrated gluten. The β‐sheet structure increased progressively from flour to hydrated flour and to hydrated gluten. In hydrated gluten protein fractions comprising gliadin, soluble glutenin, and gel protein, β‐sheet structure increased progressively from soluble gliadin and glutenin to gluten and gel protein; β‐sheet content was also greater in the gel protein from the breadmaking flour Hereward than the biscuit flour Riband.     

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

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

Microbial Transglutaminase as a Tool to Restore the Functionality of Gluten from Insect‐Damaged Wheat

In some wheat‐growing countries, considerable quantities of commercial wheat are rendered unusable in standard baking because of preharvest damage of the grain by protease‐injecting bugs. In the present study, we studied the ability of transglutaminase (TG) treatment of damaged wheat flour to return the functionality of the gluten network. To confirm the TG cross‐linking, the degree of protein hydrolysis, the amount of free thiol groups, and the electrophoresis properties of glutenin subunits were determined. The effectiveness of the TG treatment on insect‐damaged wheat was analyzed by measuring the dough mixing behavior and the gluten quality. A decrease in the degree of hydrolysis (or free amino groups), a reduction in thiol group concentration, and a decrease of extractable high molecular weight glutenin subunits (HMW‐GS) (measured by high‐performance capillary electrophoresis) confirmed the protein cross‐linking catalyzed by TG, the simultaneous formation of disulfide bonds by the proximity of the cross‐linked polypeptide chains, and the formation of aggregates of high molecular weight. The TG treatment of the damaged wheat flour led to a recovery of the consistograph parameters and gluten index value, and the covalent nature of the bonds ensured the stability of the protein changes.     

Comparison of Quality Characteristics and Breadmaking Functionality of Hard Red Winter and Hard Red Spring Wheat

Various whole‐kernel, milling, flour, dough, and breadmaking quality parameters were compared between hard red winter (HRW) and hard red spring (HRS) wheat. From the 50 quality parameters evaluated, values of only nine quality characteristics were found to be similar for both classes. These were test weight, grain moisture content, kernel size, polyphenol oxidase content, average gluten index, insoluble polymeric protein (%), free nonpolar lipids, loaf volume potential, and mixograph tolerance. Some of the quality characteristics that had significantly higher levels in HRS than in HRW wheat samples included grain protein content, grain hardness, most milling and flour quality measurements, most dough physicochemical properties, and most baking characteristics. When HRW and HRS wheat samples were grouped to be within the same wheat protein content range (11.4–15.8%), the average value of many grain and breadmaking quality characteristics were similar for both wheat classes but significant differences still existed. Values that were higher for HRW wheat flour were color b*, free polar lipids content, falling number, and farinograph tolerance. Values that were higher for HRS wheat flour were geometric mean diameter, quantity of insoluble polymeric proteins and gliadins, mixograph mix time, alveograph configuration ratio, dough weight, crumb grain score, and SDS sedimentation volume. This research showed that the grain and flour quality of HRS wheat generally exceeds that of HRW wheat whether or not samples are grouped to include a similar protein content range.     

Tyrosine cross-links: molecular basis of gluten structure and function

The formation of the large protein structure known as "gluten" during dough-mixing and bread-making processes is extremely complex. It has been established that a specific subset of the proteins comprising gluten, the glutenin subunits, directly affects dough formation and breadmaking quality. Glutenin subunits have no definitive structural differences that can be directly correlated to their ability to form gluten and affect dough formation or breadmaking quality. Many protein structural studies, as well as mixing and baking studies, have postulated that disulfide bonds are present in the gluten structure and contribute to the process of dough formation through the process of disulfide-sulfhydryl exchange. Evidence presented here indicates that tyrosine bonds form in wheat doughs during the processes of mixing and baking, contributing to the structure of the gluten network. The relative contributions of tyrosine bonds and disulfide--sulfhydryl interchange are discussed.     

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.     

Effect of Transglutaminase on Protein Electrophoretic Pattern of Rice,Soybean, and Rice‐Soybean Blends

The interactions taking place in composite dough containing rice flour and soybean proteins (5% w/w) in the presence of transglutaminase, an enzyme with cross‐linking activity, were studied using different electrophoretic analyses. The interaction between rice proteins and soybean proteins was intensified by the formation of new intermolecular covalent bonds catalyzed by transglutaminase and the indirect formation of disulfide bonds among proteins. The main protein fractions involved in those interactions were both β‐conglycinin and glycinin of soybean and the glutelins of the rice flour, although albumins and globulins were also cross‐linked. The addition of soybean proteins to rice flour improves the amino acid balance and they also might play an important role on the rice dough properties because soybean proteins interact with rice proteins, yielding protein aggregates of high molecular weight.     

Protein Allelic Composition,Dough Rheology,and Baking Characteristics of Flour Mill Streams from Wheat Cultivars with Known and Varied Baking Qualities

Flour mill streams obtained by milling grain of 10 bread wheat cultivars grown in the Skopje region of Macedonia were analyzed for rheological and breadmaking quality characteristics and for composition of gliadins and HMW‐GS. The objective of this study was to examine the relationships between the composition of gluten proteins and breadmaking quality, as well as to determine the importance of gluten proteins for technological quality of flour mill streams. The grain was milled in an experimental mill according to a standardized milling procedure, with three break and three reduction passages. The addition of two vibratory finishers in the milling scheme enabled better separation of bran. A small‐scale baking method for evaluation of the breadmaking properties was developed, and electrophoretic methods including acid‐PAGE and SDS‐PAGE were used to determine the composition of the gluten proteins. There were significant differences in the degree of dough softening of individual and total flour fractions of the flour mill streams for cultivars with different alleles from six loci, for farinograph water absorption from seven loci, and for bread loaf volume and crumb quality score from six loci. The Glu‐1 quality scores for the wheat cultivars investigated were 3–9 and proved to be a useful indicator of breadmaking quality. The novel feature of the investigation related to the breadmaking potential of the flour mill streams compared with straight‐run flours.     

Comparison of Small and Large Deformation Rheological Properties of Wheat Dough and Gluten

The rheological properties of dough and gluten are important for end‐use quality of flour but there is a lack of knowledge of the relationships between fundamental and empirical tests and how they relate to flour composition and gluten quality. Dough and gluten from six breadmaking wheat qualities were subjected to a range of rheological tests. Fundamental (small‐deformation) rheological characterizations (dynamic oscillatory shear and creep recovery) were performed on gluten to avoid the nonlinear influence of the starch component, whereas large deformation tests were conducted on both dough and gluten. A number of variables from the various curves were considered and subjected to a principal component analysis (PCA) to get an overview of relationships between the various variables. The first component represented variability in protein quality, associated with elasticity and tenacity in large deformation (large positive loadings for resistance to extension and initial slope of dough and gluten extension curves recorded by the SMS/Kieffer dough and gluten extensibility rig, and the tenacity and strain hardening index of dough measured by the Dobraszczyk/Roberts dough inflation system), the elastic character of the hydrated gluten proteins (large positive loading for elastic modulus [G′], large negative loadings for tan δ and steady state compliance [J_e⁰]), the presence of high molecular weight glutenin subunits (HMW‐GS) 5+10 vs. 2+12, and a size distribution of glutenin polymers shifted toward the high‐end range. The second principal component was associated with flour protein content. Certain rheological data were influenced by protein content in addition to protein quality (area under dough extension curves and dough inflation curves [W]). The approach made it possible to bridge the gap between fundamental rheological properties, empirical measurements of physical properties, protein composition, and size distribution. The interpretation of this study gave indications of the molecular basis for differences in breadmaking performance.     

Structural Modification of Gluten Proteins in Strong and Weak Wheat Dough as Affected by Mixing Temperature

The effects of temperature (≥25°C) on dough rheological properties and gluten functionality have been investigated for decades, but no study has addressed the effect of low temperature (<30°C) on gluten network attributes in flours with strong and weak dough characteristics. This study monitored changes in protein extractability in the presence and absence of reducing agents, the contents of readily accessible and SDS‐accessible thiols, and the secondary structural features of proteins in doughs from commercial hard wheat flour (HWF) and soft wheat flour (SWF) mixed at 4, 15, and 30°C. SWF mixed at 4 and 15°C showed similar mixing properties as HWF mixed at 30°C (which is the standard temperature). The effect of mixing temperature is different at the molecular level between the two flours studied. Protein features of HWF did not change as mixing temperature decreased, with the only exception being an increase in SDS‐accessible thiols. Decreasing mixing temperature for SWF caused an increase in SDS protein solubility and SDS‐accessible thiols as well as an increase in β‐turn structures at the expense of β‐sheet structures. Thus, noncovalent interactions appear to drive protein network at low temperatures (4 and 15°C), whereas covalent interactions dominate at standard mixing temperature (30°C) in doughs from both flours.     

Qualitative Effect of Added Gluten on Dough Properties and Quality of Chinese Steamed Bread

Glutens isolated from 15 soft red winter (SRW) wheat flours were added into a SRW wheat flour to obtain protein levels of 9.6 and 11.3% for determination of the qualitative effect of added gluten on the dough properties and quality of northern‐style Chinese steamed bread (CSB). Sodium dodecyl sulfate sedimentation (SDSS) volume of the gluten source flour exhibited positive relationships with mixograph absorption, midline peak time (MPT), and midline peak value (MPV) of the gluten‐added flours and with surface smoothness, crumb structure, and total score of CSB prepared from the gluten‐added flours regardless of protein content. Positive correlations were also observed between SDSS volume of the gluten source flour and specific volume and stress relaxation score of CSB prepared from the gluten‐added flours of 11.3% protein. The increase in protein content from 9.6 to 11.3% by gluten addition raised mixograph absorption, MPT, and MPV but had no apparent effect on resistance breakdown, dough maximum force for extension, and extensibility, and it increased CSB specific volume and crumb structure score without affecting surface smoothness, stress relaxation, and total score. Mixograph parameters exhibited significant relationships with CSB total score, indicating that they could be effective predictors of the CSB‐making quality of flours.     

Quantitative and Qualitative Role of Added Gluten on White Salted Noodles

A commercial gluten and glutens isolated from four soft and four hard wheat flours were incorporated into a hard and a soft white flour by replacement to directly determine the quantitative and qualitative role of gluten proteins in making noodles. Gluten incorporation (6%) decreased water absorption of noodle dough by 3%, shortened the length of the dough sheet by 15 and 18%, and increased the thickness of the dough sheet by 18 and 20% in soft and hard wheat flour, respectively. Noodles imbibed less water and imbibed water more slowly during cooking with gluten incorporation, which resulted in a 3‐min increase in cooking time for both soft and hard wheat noodles. Despite the extended cooking time of 3 min, noodles incorporated with 6% gluten exhibited decreases in cooking loss by 15% in soft wheat. In hard wheat flour, cooking loss of noodles was lowest with 2% incorporation of gluten. Tensile strength of fresh and cooked noodles, as well as hardness of cooked noodles, increased linearly with increase in gluten incorporation, regardless of cooking time and storage time after cooking. While hardness of cooked noodles either increased or showed no changes during storage for 4 hr, tensile strength of noodles decreased. There were large variations in hardness and tensile strength of cooked noodles incorporated with glutens isolated from eight different flours. Noodles incorporated with soft wheat glutens exhibited greater hardness and tensile strength than noodles with hard wheat glutens. Tensile strength of cooked noodles incorporated with eight different glutens negatively correlated with SDS sedimentation volume of wheat flours from which the glutens were isolated.     

Differential recovery of lupin proteins from the gluten matrix in lupin-wheat bread as revealed by mass spectrometry and two-dimensional electrophoresis

Bread made from a mixture of wheat and lupin flour possesses a number of health benefits. The addition of lupin flour to wheat flour during breadmaking has major effects on bread properties. The present study investigated the lupin and wheat flour protein interactions during the breadmaking process including dough formation and baking by using proteomics research technologies including MS/MS to identify the proteins. Results revealed that qualitatively most proteins from both lupin and wheat flour remained unchanged after baking as per electrophoretic behavior, whereas some were incorporated into the bread gluten matrix and became unextractable. Most of the lupin α-conglutins could be readily extracted from the lupin-wheat bread even at low salt and nonreducing/nondenaturing extraction conditions. In contrast, most of the β-conglutins lost extractability, suggesting that they were trapped in the bread gluten matrix. The higher thermal stability of α-conglutins compared to β-conglutins is speculated to account for this difference.     

Stress Relaxation Behavior of Wheat Dough,Gluten, and Gluten Protein Fractions

Relaxation behavior was measured for dough, gluten and gluten protein fractions obtained from the U.K. biscuitmaking flour, Riband, and the U.K. breadmaking flour, Hereward. The relaxation spectrum, in which relaxation times (τ) are related to polymer molecular size, for dough showed a broad molecular size distribution, with two relaxation processes: a major peak at short times and a second peak at times longer than 10 sec, which is thought to correspond to network structure, and which may be attributed to entanglements and physical cross‐links of polymers. Relaxation spectra of glutens were similar to those for the corresponding doughs from both flours. Hereward gluten clearly showed a much more pronounced second peak in relaxation spectrum and higher relaxation modulus than Riband gluten at the same water content. In the gluten protein fractions, gliadin and acetic acid soluble glutenin only showed the first relaxation process, but gel protein clearly showed both the first and second relaxation processes. The results show that the relaxation properties of dough depend on its gluten protein and that gel protein is responsible for the network structure for dough and gluten.     

Changes in Disulfide and Sulfhydryl Contents and Electrophoretic Patterns of Extruded Wheat Flour Proteins

Twin‐screw extrusion of wheat flour and the effects on the flour proteins were studied using flour samples containing 9, 20, and 30% protein. Vital gluten containing 70% protein was used to achieve the flour protein levels. The three flour samples were extruded with a twin‐screw extruder at a combination of processing parameters (exit die temperatures of 120, 140, and 160°C, and screw speeds of 240, 320, and 400 rpm). Increasing extruder exit die temperatures resulted in increased sulfhydryl content of the 9 and 20% protein content flour samples, but appeared to have little or no effect on the 30% protein content flour sample. Similarly, disulfide content decreased, albeit disproportionately, following the same trend. Both sulfhydryl and disulfide contents of extruded samples were lower than those of the nonextruded samples and could imply denaturation of protein, aggregation through intermolecular disulfide bonds, or oxidation during extrusion processing. Total cysteine content of extruded samples decreased by ≈16% relative to nonextruded samples, but otherwise remained almost unchanged among all extruded samples. The loss of total cysteine in extruded samples could represent the generation of hydrogen sulfide, volatile organic compounds, or flavor compounds during extrusion. SDS‐PAGE analysis of total proteins showed a shift from the higher to lower molecular weight regions for certain protein bands. Both depolymerization and protein aggregation occurred at higher shear forces during extrusion.