Proteins Extracted by Water or Aqueous Ethanol During Refining of Developed Wheat Dough to Vital Wheat Gluten and Crude Starch as Determined by Capillary‐Zone Electrophoresis (CZE)

Fluids applied to large‐sale, technical separation of wheat starch and protein also extract soluble proteins. The degree and rate of extraction and the specific components extracted depend on the flour, the flour hydration and development, the starch‐displacing fluid composition, the temperature, and the mechanical processing method. This study sought to identify major extracted protein groups using high‐performance capillary zone electrophoresis (CZE) applied directly to fluids obtained during laboratory‐scale technical separations. A dough‐ball or compression separation method was applied using a Glutomatic system and a batter or dispersion method was applied using a a McDuffie mixer and Pharmasep vibratory separator. Process fluids were water at 22°C to model commercial practice and 70 vol% ethanol in water at ‐13°C to model the cold ethanol process being developed here. Data were referenced to use of 70 vol% ethanol in water at 22°C in the Glutomatic compression method. The dough processed by each method was developed by mixing to a separable state. When flooded with excess water, this dough immediately released starch and water‐soluble or albumin proteins. When flooded with excess cold aqueous ethanol, neither the albumin nor gliadin proteins appeared in significant amounts until the bulk of the starch had been displaced, regardless of the mechanical method. Even with extraction and manipulation well beyond that necessary for starch displacement, the net amount of gliadin proteins dissolved was only ≈10% of that available from wet developed dough using 70 vol% ethanol at 22°C. There was more gliadin protein in the fluids at earlier stages of processing when the batter dispersion method was applied using cold ethanol. The most common soluble proteins revealed in the electrophoresis patterns for the batter compression method using cold aqueous ethanol were initially albumins and later γ‐gliadins. Albumins not appearing as soluble in cold 70 vol% ethanol were found in the insoluble crude starch, suggesting their precipitation in the dough fluids during the change from free water to cold aqueous ethanol. These results establish that some protein is dissolved during starch displacement by cold aqueous ethanol, but that the amounts may be limited by control of the mechanical working of the dough in the presence of the displacing fluids.     

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.     

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

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

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

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

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

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

Factors Associated with Dough Stickiness as Sensed by Attenuated Total Reflectance Infrared Spectroscopy

Attenuated total reflectance (ATR) and Fourier transform infrared (FTIR) spectroscopy have been applied in the characterization of sticky dough surfaces. The characterization provides insight in the chemical distribution of gluten protein, starch, water, and fat during dough kneading. ATR is especially useful for selective sampling of dough surfaces because the depth of penetration of radiation is quite shallow. For dough, it is calculated to be in the order of 0.5–4 μm in the mid‐infrared, ideal for measurements of stickiness effects, where only the dough surface is of interest. To investigate the cohesive and adhesive properties of the individual dough constituents, dough was peeled from the ATR plate to study the material that adhered to it. The infrared spectra obtained indicate that fat and gluten protein appear to be located at the outer sticky dough surfaces, rather than water and starch. In comparison with gluten, the fatty component showed relatively strong adhesive forces to the ATR plate; a high residual fraction was measured after peeling the dough. Gluten proteins display different cohesion and adhesion properties that are strongly dependent on their hydration state. This indicates that the degree of hydration of gluten proteins contributes to the sticky properties of (overkneaded) dough. When analyzing gluten protein in D₂O instead of a dough matrix, more or less similar results were obtained. Significant differences in amide I and amide II intensities were measured for kneaded and stretched gluten protein in comparison to untreated, wet gluten. Besides changes in the vibrational properties of the amide groups, conformational changes in the tertiary protein structure also were observed. It appears that kneading and stretching of dough results in a major decrease in α‐helices content, accompanied by an increase of extended β‐sheet conformations.     

Description of Chemical Changes Implied During Bread Dough Mixing by FT‐ATR Mid‐Infrared Spectroscopy

The aim of the present study was to investigate the ability of mid‐infrared (MIR) spectroscopy to identify physicochemical changes in the French bread dough mixing process. An ATR FT‐MIR spectrometer at 4000–800 cm^–1 was used. The MIR spectra collections recorded during mixing were analyzed after standard normal variate using principal component analysis (PCA) and after second‐derivative treatment. The results were interpreted in terms of chemical changes involved in dough development and more particularly in terms of secondary structural protein changes (amide III). The loading spectrum associated with principal component 1 (PC1) allows three MIR wave number regions of variations (3500–3000, 1700–1200, and 1200–800 cm^–1) to be identified. The loading spectrum associated with PC1 describes an increase in the relative protein band intensities and a decrease in relative water and starch band intensities. The variation during bread dough mixing time of the different amide III bands identified after the second‐derivative show that α‐helical, β‐turn, and β‐sheet structures increase while random coil structure decreases, suggesting that the gluten structure is becoming a more ordered structure. The MIR mixing time identified as being the maximum scores value on the PC1 scores plots was associated with the time at which the dough apparent torque begin to collapse, suggesting that the MIR spectroscopy could monitor bread dough development.     

Effect of Damaged Starch and NaCl Level on the Dough Handling Properties of a Canadian Western Red Spring Wheat

The effects of damaged starch and NaCl (1 and 2% w/w [flour weight]) on the dough handling properties of a wheat flour (Triticum asetivum L. ‘Roblin’) were investigated with rheological and textural methods. Damaged starch levels of the base flour and three remilled flours (using reduction rolls with decreasing gap sizes) were 5.42, 6.23, 7.30, and 8.43%. Rheological measurements on the dough showed that the complex modulus increased and the loss tangent (tan δ) decreased with increasing damaged starch levels in the flour, indicating that greater amounts of damaged starch produced stiffer dough. The base flour produced doughs with the highest creep compliance value (J _max), whereas the flour with the most damaged starch deformed the least. Higher levels of salt produced stiffer dough that deformed less, as evident by the higher complex modulus and lower creep compliance, compared with 1% NaCl. Damaged starch overall decreased dough stickiness (N), work of adhesion (N·s), and cohesiveness (mm). Increasing the salt content decreased the stickiness of the doughs. Increasing the damaged starch greatly increased dough extensibility at 1% NaCl. The greater amounts of damaged starch in the remilled flour mitigated some of the negative effects of reducing the salt content on the dough machinability.     

Effect of Cross‐Linked Resistant Starch on Wheat Tortilla Quality

Resistant starch (RS) ingredients are an attractive option to increase dietary fiber in baked products. This study determined the effect of two forms of cross‐linked and pregelatinized cross‐linked RS, Fibersym‐RW (Fsym) or FiberRite‐RW (FRite), respectively, from wheat on dough and tortilla quality and acceptability. Refined wheat tortillas with 0% (control) to 15% RS (flour basis) were made using a standard baking process. Tortillas with 100% whole white wheat were also made. Physical and rheological properties of dough and tortillas, and sensory profile of tortillas were evaluated. Dough with whole wheat and 15% FRite were significantly harder and less extensible than the control dough; this was related to high water absorption of these doughs. Tortillas with whole wheat and 10–15% FRite were less puffed and denser than the control; however these levels of FRite significantly increased tortilla weight (by up to 6.2%). Dough and tortillas with Fsym were comparable to the control. Dietary fiber (g/100 g, db) increased from 2.8 ± 0.3 in control to 14.3 ± 0.5 and 13.6 ± 0.5 in 15% Fsym and 15% FRite tortillas, respectively. Tortillas with whole wheat were less acceptable than the control in appearance, flavor, and texture, while tortillas with 15% Fsym had higher overall acceptability than the control. Incorporation of 15% cross‐linked wheat RS to increase tortilla dietary fiber is feasible without negatively affecting dough handling and tortilla quality.     

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

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

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

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

Influence of Bran Particle Size on Bread‐Baking Quality of Whole Grain Wheat Flour and Starch Retrogradation

The influence of bran particle size on bread‐baking quality of whole grain wheat flour (WWF) and starch retrogradation was studied. Higher water absorption of dough prepared from WWF with added gluten to attain 18% protein was observed for WWFs of fine bran than those of coarse bran, whereas no significant difference in dough mixing time was detected for WWFs of varying bran particle size. The effects of bran particle size on loaf volume of WWF bread and crumb firmness during storage were more evident in hard white wheat than in hard red wheat. A greater degree of starch retrogradation in bread crumb stored for seven days at 4°C was observed in WWFs of fine bran than those of coarse bran. The gels prepared from starch–fine bran blends were harder than those prepared from starch–unground bran blends when stored for one and seven days at 4°C. Furthermore, a greater degree of starch retrogradation was observed in gelatinized starch containing fine bran than that containing unground bran after storage for seven days at 4°C. It is probable that finely ground bran takes away more water from gelatinized starch than coarsely ground bran, increasing the extent of starch retrogradation in bread and gels during storage.     

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

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

Effects of Oxido-Reductants on Rheological Properties of Wheat Flour Dough and Comparison with Some Characteristics of Extruded Noodles

The effects of oxido-reductants on the rheological properties of wheat flour dough were evaluated by using a capillary rheometer and an oscillatory rheometer at three temperatures. The oxidants potassium iodate (KIO₃) and l -ascorbic acid (l -AA) significantly increased the apparent viscosity and G′ and decreased loss tangent at low temperatures of 30 and 60°C due to enhanced formation of disulfide bonds. The reductant glutathione (GSH) had the opposite effect. Heating caused the gelatinization of starch, which diminished the effects of the oxido-reductants and produced doughs with similar rheological properties at 80°C. The correlation between dough rheology and characteristics of extruded noodles was also studied.     

Surface Lipids Play a Role in the Interaction of Puroindolines with Wheat Starch and Kernel Hardness

Kernel hardness is an important quality characteristic of common wheat. In this study, we investigated the role of starch surface lipids on the interaction of puroindoline proteins and starch granules through in vitro starch–protein binding experiments and flour reconstitution. SDS‐PAGE showed that there were no puroindoline proteins on the starch granule surface when surface lipids were removed or when defatted starch was incubated with puroindoline proteins. However, the puroindoline protein bands were present when defatted starch was incubated with lipids followed by purified puroindoline proteins, which indicated that starch surface lipids play a role in the binding of puroindolines to starch granules. The hardness of flour tablets and dough sheets made from reconstituted flour, which combined defatted starch incubated with lipids and puroindolines with gluten, was lower than for the control reconstituted flour, which was made from defatted starch and gluten. The results of scanning electron microscopy also showed that starch granules were embedded in the gluten in the gluten + defatted starch + lipids + puroindolines treatment. These results confirmed that starch surface lipids are involved in the interaction of puroindolines with wheat starch and kernel hardness.     

Starch Participation in Durum Dough Linear Viscoelastic Properties

The contribution of starch to dough rheological properties has been largely overshadowed by the role of gluten, receiving much less attention in comparison. The influence of starch granule surface properties on durum wheat dough linear viscoelasticity was investigated, and surface interactions between starch granules and gluten were assessed using a model system. Proportions of starch were substituted in dough on a volume basis with an inert filler (glass powder) with a similar particle size range. The doughs were subjected to dynamic and creep measurements. Dough linear viscoelastic properties were weakened on substitution of starch with glass powder at ≤50% substitution, inferring a reduction in adhesion at the matrix‐filler (starch and glass powder) interface with declining proportions of starch granules. Surface modification of starch granules or glass powder altered dough rheological properties, confirming the importance of starch granule surface characteristics and the nature of protein‐starch bonding on durum dough linear viscoelastic behavior.     

Effects of Baking Temperature on Crumb-Staling Kinetics

This study evaluated the effects of bread baking temperature on the staling kinetics of crumb. Bread dough was leavened and baked in sealed molds. Cooking trials were performed at various temperatures ranging from 90 to 110°C. The crumb samples were then stored at 20°C at constant moisture, and staling was evaluated by measuring crumb elastic modulus (using an Instron dynamometer) and starch retrogradation degree (using differential scanning calorimetry). Results show that the cooking temperature greatly influences bread staling. The lower the cooking temperature, the lower the staling rate, both in terms of crumb hardening and of starch retrogradation. Starch and protein solubility was evaluated on crumb cooked at 90 and 110°C. An increase in cooking temperature resulted in an increase in protein insolubilization and starch granule disruption.     

Influence of Milk,Corn Starch,and Baking Conditions on the Starch Digestibility,Gelatinization, and Fracture Stress of Biscuits

Dough for nontraditional semisweet biscuits—prepared with wheat flour or replacing part of the wheat flour with corn starch, with or without skim milk—was baked at two oven temperatures, 120 or 170°C, until reaching moisture content and water activity lower than 6% and 0.5, respectively. Assays of fracture stress, differential scanning calorimetry, X‐ray diffraction, and starch digestibility were performed. Results showed that biscuits containing milk had the highest fracture stress, and biscuits baked at low temperature were harder than biscuits baked at high temperature. The degree of starch gelatinization during baking was higher when dough was baked at 170°C, compared with dough baked at 120°C. The decrease in gelatinization coincides with the decrease in the height and surface of peaks at 15 and 23° in the X‐ray diffraction patterns. Milk and corn starch did not affect the starch digestibility of biscuits, but biscuits baked at 170°C presented lower fracture stress and higher starch digestibility than biscuits baked at 120°C.     

Role of Water in Pretzel Dough Development and Final Product Quality

The relationship between flour quality or processing conditions and pretzel quality has not been extensively investigated. The objective of this study was to elucidate the role of water in pretzel dough development and the consequent impact on pretzel integrity. Control pretzel and pretzels made with lower or higher levels of added water in the dough were produced under standard processing conditions at Reading Bakery Systems' pilot plant in Robesonia, PA. Dough samples were evaluated for their appearance, moisture content, and extensibility and were viewed under a microscope to evaluate the gluten network. Pretzels before and after the kiln were evaluated for moisture content, pasting properties, and hardness and were viewed under a microscope to evaluate the extent of starch gelatinization. The structural and functional attributes of dough and pretzels were significantly different for the three treatments. The degrees of gluten development during mixing and starch gelatinization during baking were influenced by the levels of water added and consequently influenced pretzel quality. Pretzels made using low‐water treatment were brittle due to a lack of gluten development in the dough and inadequate starch gelatinization during baking, while pretzels made using high water treatment were unacceptable due to extensive gelatinization and retrogradation of starch. Pretzel quality therefore appeared to be a function of appropriate gluten development and starch gelatinization in the product.     

Effects of Mixing Conditions on Pasta Dough Development and Biochemical Changes

Mixing of commercial durum wheat semolina with water was performed under different conditions in a Brabender micromixer equipped with pastamaking shafts. Semolina filling of the mixing chamber was 30.4–42.9% (v/v), shaft speed was 10–110 rpm, temperature was 10–40°C, and hydration level was 47–52.5% (db). The blend of water and semolina evolved from individualized hydrated particles (HP) to a dough product (DP) as a function of these conditions. Torque values (T) and the specific mechanical energies (SME) were recorded during mixing as a function of time. Terms from these curves were defined to characterize the mixing process: t_o (starting time of dough development), t_d (time to reach the maximum dough consistency), T_m (mean torque value after dough development), and SME_f (total energy applied to the dough during mixing). Transformation of HP into DP and the mixing temperature were the main parameters affecting t_o, t_d, T_m, and SME_f. Protein aggregate distribution was measured by size-exclusion HPLC, protein solubility in 0.01N acetic acid, free -SH content, soluble arabinoxylans, reducing sugars, ferulic acid, carotenoid content, and oxidase activities to characterize the biochemical changes that occurred during pasta dough formation. DP was characterized by lower amounts of insoluble glutenin aggregates, lower protein solubility in dilute acetic acid, lower free -SH content, ferulic acid, carotenoid content, and lower oxidoreductase activities as compared to HP. Once the dough was developed, the effects of mixing speed, temperature, or hydration level on the biochemical composition of the blend were null or low compared to the modifications that were observed when the blends changed from HP to DP. The t_o and SME_f were the most significant parameters in characterizing the pasta dough mixing process in relation to biochemical changes.