The impact of heating and cooling on the physico-chemical properties of wheat gluten–water suspensions

The rapid visco analysis (RVA) system was used to measure rheological behaviour in 20% (w/v) gluten-in-water suspensions upon applying temperature profiles. The temperature profiles included a linear temperature increase, a holding step, a cooling step with a linear temperature decrease to 50 °C, and a final holding step at 50 °C. Temperature and duration of the holding phase both affected RVA viscosity and protein extractability. Size-exclusion and reversed-phase HPLC showed that increasing the temperature (up to 95 °C) mainly decreased glutenin extractability. Holding at 95 °C resulted in polymerisation of both gliadin and glutenin. Above 80 °C, the RVA viscosity steadily increased with longer holding times while the gliadin and glutenin extractabilities decreased. Their reduced extractability in 60% ethanol showed that γ-gliadins were more affected after heating than α-gliadins and ω-gliadins. Enrichment of wheat gluten in either gliadin or glutenin showed that both gliadin and glutenin are necessary for the initial viscosity in the RVA profile. The formation of polymers through disulphide bonding caused a viscosity rise in the RVA profile. The amounts of free sulphydryl groups markedly decreased between 70 and 80 °C and when holding the temperature at 95 °C.     

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten II. Studies on the influence of additives

Cysteine, N-ethylmaleinimide, radical scavengers, various salts or urea were added to wheat gluten. After treatment at increasing pressure (0.1–800 MPa) and temperature (30–80 °C) the resulting material was analysed by micro-extension tests and an extraction/HPLC method to measure protein solubility. Furthermore, cysteine was added to isolated gliadin and glutenin prior to high-pressure treatment and protein solubility was determined. The resistance to extension of gluten strongly increased and the solubility of gliadin in aqueous ethanol decreased with increasing pressure and temperature. As compared to experiments without additive the observed effects were much stronger. Isolated gliadin turned largely insoluble in aqueous ethanol when cysteine was added prior to high-pressure treatment. The S-rich α- and γ-gliadins were much more strongly affected than the S-poor ω-gliadins pointing to a disulphide related mechanism. Monomeric gliadin components were completely recovered after reduction of the aggregates with dithioerythritol. In contrast, samples without free thiol groups such as isolated gliadins or with SH groups, which had been blocked by N-ethylmaleinimide, were hardly affected by high-pressure treatment. The addition of radical scavengers to gluten showed no effect in comparison to the control experiment, indicating that a radical mechanism of the high-pressure effect can be excluded. The observed effects can be explained by thiol-/disulphide interchange reactions, which require the presence of free thiol groups in the sample. The addition of salts and urea showed that unfolding of the protein due to weakening of interprotein hydrogen bonds is strongest for ions with a high radius (e.g. thiocyanate). This leads to weakening of gluten at ambient pressure but it facilitates high pressure induced reactions, e.g. of disulphide bonds.     

The impact of heating and cooling on the physico-chemical properties of wheat gluten–water suspensions

The rapid visco analysis (RVA) system was used to measure rheological behaviour in 20% (w/v) gluten-in-water suspensions upon applying temperature profiles. The temperature profiles included a linear temperature increase, a holding step, a cooling step with a linear temperature decrease to 50 °C, and a final holding step at 50 °C. Temperature and duration of the holding phase both affected RVA viscosity and protein extractability. Size-exclusion and reversed-phase HPLC showed that increasing the temperature (up to 95 °C) mainly decreased glutenin extractability. Holding at 95 °C resulted in polymerisation of both gliadin and glutenin. Above 80 °C, the RVA viscosity steadily increased with longer holding times while the gliadin and glutenin extractabilities decreased. Their reduced extractability in 60% ethanol showed that γ-gliadins were more affected after heating than α-gliadins and ω-gliadins. Enrichment of wheat gluten in either gliadin or glutenin showed that both gliadin and glutenin are necessary for the initial viscosity in the RVA profile. The formation of polymers through disulphide bonding caused a viscosity rise in the RVA profile. The amounts of free sulphydryl groups markedly decreased between 70 and 80 °C and when holding the temperature at 95 °C.     

Influence of sulphur fertilisation on quantities and proportions of gluten protein types in wheat flour

Although different supplies of sulphur (S) during wheat growth are known to influence the quantitative composition of gluten proteins in flour, an effect on the amount and on the proportions of single protein types has yet not been determined. Therefore, wholemeal flours of the spring wheat ‘Star’ grown on two different soils and at four different levels of S fertilisation (0, 40, 80, 160 mg S per container) were analysed in detail using an extraction/HPLC procedure. The results demonstrated that the amount of total gluten proteins as well as of the crude protein content of flour was little influenced, whereas amounts and proportions of single protein types were strongly affected by the different S fertilisation. The changes were clearly dependent on the Cys and Met content of each protein type. The amount of S-free ω-gliadins increased drastically, and that of S-poor high-molecular-weight (HMW) glutenin subunits increased moderately in the case of S deficiency. In contrast, the amounts of S-rich γ-gliadins and low-molecular-weight (LMW) glutenin subunits decreased significantly, whereas the amount of α-gliadins was reduced only slightly. S deficiency resulted in a remarkable shift of protein proportions. The gliadin/glutenin ratio increased distinctly; ω-gliadins became major components, and γ-gliadins minor components, whereas the ratio of HMW to LMW glutenin subunits was well-balanced.     

Effect of temperature, time and wheat gluten moisture content on wheat gluten network formation during thermomolding

A plastic-like material can be obtained by thermomolding wheat gluten protein which consists of glutenin and gliadin. We studied the effect of molding temperature (130-170 °C), molding time (5-25 min) and initial wheat gluten moisture content (5.6-18.0%) on the gluten network. Almost no glutenins were extractable after thermomolding irrespective of the molding conditions. At the lowest molding temperature, the extractable gliadin content decreased with increasing molding times and moisture contents. This effect was more pronounced for the α- and γ-gliadins than for the ω-gliadins. Protein extractabilities under reducing conditions revealed that, at this molding temperature, the cross-linking was predominantly based on disulfide bonds. At higher molding temperatures, also non-disulfide bonds contributed to the gluten network. Decreasing cystine contents and increasing free sulfhydryl and dehydroalanine (DHA) contents with increasing molding temperatures and times revealed the occurrence of β-elimination reactions during thermomolding. Under the experimental conditions, the DHA derived cross-link lanthionine (LAN) was detected in all gluten samples thermomolded at 150 and 170 °C. LAN was also formed at 130 °C for gluten samples containing 18.0% moisture. Degradation was observed at 150 °C for samples thermomolded from gluten with 18.0% moisture content or thermomolded at 170 °C for all moisture contents.     

Glutathione and related thiol compounds II. The importance of protein bound glutathione and related protein-bound compounds in gluten proteins

Protein-bound glutathione (PSSG) and protein-bound related thiol compounds, i.e. cysteine (PSSCys), glutamyl-cysteine (PSSGlu-Cys) and cysteinyl-glycine (PSSCys-Gly), were analysed in proteins of Osborne fractions, i.e. gliadin, glutenin and gliadin-, glutenin-subfractions separated by gel filtration chromatography, gel protein and the total gluten proteins separated from wheat varieties with varying breadmaking performances. The results showed that PSSG and some protein-bound related thiol compounds were found in monomeric gliadins, indicating that glutathione and some related thiol compounds are able to form disulphide bonds (SS) with sulphydryl group (SH) of those proteins and the formation of those disulphide bonds may prevent those monomeric proteins from binding to other proteins. It was also observed that a larger amount of PSSG in glutenin proteins was negatively correlated with the molecular weight (M_w) distribution of glutenin polymers, suggesting that PSSG and protein-bound related thiol compounds may play an important role in controlling polymerisation of glutenin. Furthermore, it was found that the level of PSSG in gel protein from flours with poor breadmaking performances was constantly higher and significantly different (p<0.05) from that of flours with good breadmaking performance. The same trend was observed with gluten samples from breadmaking and biscuitmaking flours.     

Effects of ultrasound-assisted freezing on the water migration of dough and the structural characteristics of gluten components

The effects of ultrasound-assisted freezing on the freezing time and water migration of dough, and the structural characteristics of gluten components were investigated. The effects of ultrasound-assisted freezing in the whole immersion freezing process (UWF) on the freezing time were better than those of ultrasound-assisted freezing in the maximum ice crystal generation zone. The shortest freezing time was obtained at 80 W/L, and was 577 s shorter than that with traditional immersion freezing. The UWF treatment at 80 W/L significantly (p < 0.05) affected the absorption enthalpy, freezable water content and water migration of frozen dough. In UWF compared with traditional immersion freezing, the SH content of gluten, glutenin and gliadin was significantly (p < 0.05) higher, by 12.06%, 27.55% and 21.65%, respectively. The surface hydrophobicity of gluten, glutenin and gliadin in UWF treated samples significantly (p < 0.05) decreased, by 19.67%, 13.21% and 9.17%, respectively. The secondary structure of gluten components was also significantly changed by UWF. The network of gluten, the chain structure of glutenin and the gliadin particles were all changed by UWF treatment. These findings indicated that UWF is an effective method to improve the moisture distribution in dough and reduce the damage to protein molecular structure caused by freezing.     

Characteristics of enzymatic hydrolysis of thermal-treated wheat gluten

Effect of thermal treatment at 50–90 °C on wheat gluten hydrolysis by papain was evaluated in this study. Thermal treatment decreased the amount of sodium dodecyl sulfate (SDS) extractable protein. The treatments at 80 and 90 °C had a strong impact on protein extractability. Thermal treatment for 30 min resulted in a significant reduction in SDS extractable glutenin level in wheat gluten. A significant drop in free sulphydryl level was found in wheat gluten treated at 70 °C for 30 min. It indicated that cross-linking of glutenin through S–S occurred during thermal treatment. The treatments at 70–90 °C led to significant decreases in soluble and nitrogen level, while significant increases in peptide nitrogen amount in the hydrolysates from treated gluten were found. A time-dependent effect was observed for the changes in soluble forms of nitrogen and PN. Thermal treatment resulted in molecular mass distribution change according to gel permeation chromatography analysis. Thermal treatment significantly increased the amount of fractions with molecular mass beyond 10 K (67.2%) in the hydrolysates and greatly decreased the amounts of fractions with MM of 10–5 K and below 5 K in hydrolysates.     

Using the Gluten Peak Tester as a tool to measure physical properties of gluten

The functional properties of wheat are largely dictated by composition and interactions of the gluten proteins. All flours contain gliadin and glutenin, but produce baked products of varying quality, which provides evidence that gluten proteins from different wheats possess different properties. A common method to study differences in gluten properties, which is utilized in this study, is fractionation/reconstitution experiments to understand how various gliadin to glutenin ratios and how fractions from different wheat sources affect gluten aggregation properties. Gliadin and glutenin from a vital wheat gluten were fractionated with 70% ethanol and reconstituted at various gliadin to glutenin ratios. Gliadin and glutenin from a Canadian eastern soft, eastern hard and western hard wheat (14% moisture) were fractionated and substituted between flours at the native gliadin to glutenin ratio. Gluten combinations were evaluated with a Gluten Peak Tester at constant temperature and mixing. Varying gliadin to glutenin ratio showed that 50:50 is optimal for fast gluten aggregation while amount of glutenin dictates strength. Substitution experiments showed that replacing good quality gluten fractions with those from a lower quality wheat decreases gluten quality, and vice versa. Data also showed that cultivar specific differences in gliadin and glutenin are more important in dictating gluten strength (torque), while gliadin to glutenin ratio dictates aggregation time (PMT) independent of the source of fractions. The study demonstrated the ability of the improved method to evaluate gluten aggregation by controlling for all variables except the one being tested. The data also revealed information about gluten aggregation properties never before seen.     

Using the Gluten Peak Tester as a tool to measure physical properties of gluten

The functional properties of wheat are largely dictated by composition and interactions of the gluten proteins. All flours contain gliadin and glutenin, but produce baked products of varying quality, which provides evidence that gluten proteins from different wheats possess different properties. A common method to study differences in gluten properties, which is utilized in this study, is fractionation/reconstitution experiments to understand how various gliadin to glutenin ratios and how fractions from different wheat sources affect gluten aggregation properties. Gliadin and glutenin from a vital wheat gluten were fractionated with 70% ethanol and reconstituted at various gliadin to glutenin ratios. Gliadin and glutenin from a Canadian eastern soft, eastern hard and western hard wheat (14% moisture) were fractionated and substituted between flours at the native gliadin to glutenin ratio. Gluten combinations were evaluated with a Gluten Peak Tester at constant temperature and mixing. Varying gliadin to glutenin ratio showed that 50:50 is optimal for fast gluten aggregation while amount of glutenin dictates strength. Substitution experiments showed that replacing good quality gluten fractions with those from a lower quality wheat decreases gluten quality, and vice versa. Data also showed that cultivar specific differences in gliadin and glutenin are more important in dictating gluten strength (torque), while gliadin to glutenin ratio dictates aggregation time (PMT) independent of the source of fractions. The study demonstrated the ability of the improved method to evaluate gluten aggregation by controlling for all variables except the one being tested. The data also revealed information about gluten aggregation properties never before seen.     

The dynamic rheological properties of glutens and gluten sub-fractions from wheats of good and poor bread making quality

The dynamic rheological properties of glutens and gluten fractions (gliadin and glutenin) of two U.K.-grown wheat cultivars, Hereward and Riband, having good and poor bread quality, respectively, were studied. Gluten and glutenin doughs from cv. Hereward had higher G' and lower tan δ values than those from cv. Riband at all frequencies studied. A more pronounced difference in G' and tan δ was observed between the glutenin doughs of the two wheats than between their respective gluten doughs. The rheological properties, i.e. G' and tan δ values, of gliadin doughs were similar for both wheats. Varying the gliadin/glutenin ratio by adding the isolated gliadin or glutenin sub-fractions to the parent glutens showed that the G' values decreased and the tan δ values increased as the gliadin/glutenin ratio was increased for both cultivars, indicating a considerable decrease in elasticity as the gliadin/glutenin ratio increased. The decrease in G' may be attributed to a plasticising effect of gliadin and ‘interference’ of gliadin with glutenin-glutenin interactions. The reduction in G' was much more pronounced when the gliadin/glutenin ratio was increased between 0.15 and 1.0 than between 1.0 and above. Gluten from cv. Hereward had higher G' and lower tan δ values than cv. Riband gluten at all gliadin/glutenin ratios, indicating that cv. Hereward gluten had greater elastic character than cv. Riband gluten. Although significant effects of other non-protein hydrocolloid components cannot be discounted, these observations are consistent with the view that the viscoelasticity of the glutenin sub-fraction of gluten and differences in the ratio of gliadin to glutenin are the main factors governing inter-cultivar differences in the viscoelasticity of wheat gluten.     

The Role of Gluten Proteins in the Baking of Arabic Bread

Fractionation and reconstitution/fortification techniques were utilised to study the role of gluten in Arabic bread. Glutens from two wheat cultivars of contrasting breadmaking quality were fractionated by dilute HCl into gliadin and glutenin. Gluten, gliadin and glutenin doughs from the good quality flour had higher G ′ and lower tan δ values than those from the poor quality flour at all the frequencies examined. Interchanging the gliadin and glutenin fractions between the reconstituted flours showed that the glutenin fraction is largely responsible for differences in the breadmaking performance. Fortification of an average quality flour with the gliadin and glutenin fractions from the poor and good quality flours, at the levels of 1% and 2% (protein to flour mass), induced marked differences in the mechanical properties of bread. The resilience of the loaves was not adversely affected by the addition of gliadins and increased, with a concomitant significant (p<0·05) improvement in quality, at the 2% level of fortification with gliadins from the good quality flour. Addition of glutenin resulted in loaves with leather-like properties that became particularly apparent at the higher level of fortification; the observed deterioration in quality paralleled the increase in the elastic character of the doughs. It is suggested that highly-elastic doughs are not compatible with the rapid expansion of gases at the high-temperature short-time conditions employed in the baking of Arabic bread and that there exists a threshold in dough elasticity beyond which a rapid decline in quality takes place.     

Enhanced separation and characterization of gluten polymers by asymmetrical flow field-flow fractionation coupled with multiple detectors

Asymmetrical flow field-flow fractionation (AsFlFFF) coupled with refractive index (RI) and multi-angle light scattering (MALS) detectors was used for macromolecular characterization of four different industrial wheat protein preparations (native, enzymatically hydrolyzed, physically separated, and denatured). The fractionation conditions were optimized separately for each protein sample and molar masses were determined from RI and MALS signals. Decaying cross-flow gradient seemed to produce best results for most of the gluten samples in terms of resolution and sample recovery. Sonication of the samples enabled the solubilization of the high-molar mass components with molar mass ranging from 8 × 10⁶ to 3.5 × 10⁸ g/mol. In case of lower-molar mass glutenins (α-gliadins, ω-gliadins, and high molecular weight glutenin subunits), AsFlFFF results were also compared with the results obtained with capillary gel electrophoresis.     

Impact of redox agents on the physico-chemistry of wheat gluten proteins during hydrothermal treatment

The impact of the oxidants potassium bromate and potassium iodate and the reducing agent dithiothreitol (DTT) on the rheological behaviour of 20% (w/v) gluten-in-water suspensions during thermal treatment was monitored with the rapid visco analyser (RVA). The suspensions were subjected to a linear temperature increase from 40 to 95 °C in 14 min, a holding step of 40 min at 95 °C, a cooling step (7 min) with a linear temperature decrease to 50 °C, and a final holding step at 50 °C (13 min). Potassium iodate (1.18 and 1.77 μmol/g protein) and potassium bromate (1.52 and 15.2 μmol/g protein) decreased RVA viscosities in the holding step and increased sodium dodecyl sulphate (SDS) protein extractabilities suggesting a greater heat resistance and decreased gliadin–glutenin cross-linking. In contrast, in the presence of DTT (1.65 and 3.30 μmol/g protein) RVA viscosity increased at lower temperatures and lowered SDS extractabilities. It is postulated that low concentrations of reducing agent facilitate gliadin–glutenin cross-linking during heating while oxidants hinder gluten polymerization due to decreased levels of free sulphydryl groups and less flexibility of the glutenin chains.     

Salt-Induced Disaggregation/Solubilization of Gliadin and Glutenin Proteins in Water

The effect of salt concentration used in preparing gluten, on the subsequent dissolution of gluten in water, was examined. Flour from a Canadian hard red spring wheat cultivar, Katepwa, was used to prepare glutens using three different solvents, i.e. distilled deionized water (DDW), 0·2% NaCl solution and 2% NaCl solution. The isolated wet glutens were extracted sequentially with DDW, providing four water soluble fractions and an insoluble residue. The amount of protein in each fraction was determined and respective compositions were assessed electrophoretically under reducing and non-reducing conditions. Surprisingly, DDW extracts of gluten prepared with 2% NaCl contained almost all the gliadins, except some ω-gliadin components, and most of the polymeric glutenin. For the gluten prepared with 0·2% NaCl, most of the gliadin, but only a small portion of glutenin, was extracted. For gluten prepared with DDW, only part of the gliadins and almost no glutenin was extractable with water. The DDW solubilities of gluten proteins prepared in DDW, 0·2% NaCl and 2% NaCl were 27, 52, and 85%, respectively, after four sequential extracts with DDW. The large increases in the solubility of gliadin and glutenin proteins in DDW when the gluten is prepared in salt solution (after removal of most of the salt) can be explained on the basis of a salt-induced conformational change of the proteins, which renders water a more effective solvent.     

Functional Properties of Wheat Gliadins. II. Effects on Dynamic Rheological Properties of Wheat Gluten

The effects of addition of purified total gliadin and its subgroups (α-, β-, γ- and ω-gliadins) on the dynamic rheology of gluten were investigated. The frequency sweeps of gluten with added α-, β-, γ- and ω2-gliadins showed unexpected increases in the magnitude of G′ and G′′, suggesting stiffening of the native gluten. Conversely, a reduction in the magnitude of G′ and G′′ occurred upon addition of the total gliadin fraction and the ω1-gliadin, implying softening of the gluten. Addition of individual gliadin fractions increased the values of slope log G′ vs log frequency, suggesting increased concentrations of uncrossed-linked material compared with the native gluten. There were significant differences in the slope values for individual gliadin fractions. The increasing order of slopes for different gliadins was: β- >γ- >α- =ω1>ω2, indicating that glutens containing ω- and α- gliadins are relatively less crossed-linked than those containing β- and γ-gliadins. The dynamic moduli, G′ and G′′, of cv. Hereward gluten showed significant positive relationships with Mixograph parameter peak dough resistance (PDR), and loaf volume for gliadin subgroups added to cv. Hereward flour.     

The final established physicochemical properties of steamed bread made from frozen dough: Study of the combined effects of gluten polymerization,water content and starch crystallinity on bread firmness

The gluten polymerization behavior, water content, starch crystallinity and firmness of Chinese steamed bread made from frozen dough were investigated and their correlations were also established in this study. The decreased degree of gluten polymerization in steamed bread was observed by the enhanced SDS-extractable proteins (SDSEPs) upon frozen storage. Less incorporation of glutenin in the glutenin–gliadin crosslinking of steamed bread mainly contributed to the decreased degree of gluten polymerization. The decreased moisture of steamed bread had a significant negative correlation with the sublimated water in frozen dough (r = −0.8850, P < 0.01). Frozen storage also induced an increase in starch crystallinity and bread firmness. A multiple linear regression model with SDS-extractable proteins, water content and melting enthalpy of starch crystals of steamed bread accounted for 86% of the variance in the natural logarithm of firmness and further revealed that starch crystallinity mainly contributed to bread firmness.     

Effect of jet-cooked wheat gluten/lecithin blends on maize and rice starch retrogradation

Vital wheat gluten and lecithin (GL) (50:50, w/w) were dry blended in a coffee grinder and a 9.5% (w/v) aqueous slurry was jet-cooked (steam pressures of 65 psi/g inlet and 40 psi/g outlet) to disaggregate wheat gluten and facilitate better dispersion of the two components. The jet-cooked material was freeze-dried and stored at 0 °C for future use. The GL blend was added to pure food grade common maize and rice starch at concentrations of 0 (control), 6, 11, 16, and 21%. Starch gelatinization and retrogradation temperature transitions were determined using Differential Scanning Calorimetry (DSC). From the DSC profiles, the change in the ΔH value was used as an indication of starch retrogradation, where a higher ΔH value indicated higher retrogradation. The ΔH values of the blends at 4 °C had higher values than the −20 °C and the ambient (25 °C) storage temperatures. Overall, the 21% GL/starch blends reduced retrogradation by 50%. The lower amylose content of rice starch relative to maize starch was reflected in Rapid Visco Amylograph (RVA) measurements of peak viscosity, and similarly, Texture Analyzer (TA) measurements indicated that maize starch gel is firmer than rice starch gel. Retrogradation was also evaluated by observing G′, the shear storage modulus, as a function of time after running a standard pasting curve. Using this method, it appears that GL has a significant effect on maize starch retrogradation, since low concentrations (<0.4%, w/w) reduced G′ up to 40%. The opposite behavior was seen in rice starch, where G′ increased directly with added GL. It appears that the amylose level in the rice starch is too low to be affected by the GL, and the increase seen in G′ is most likely due to added solids.     

Phospholipids and wheat gluten blends: interaction and kinetics

A model system comprising of lysophosphatidylcholine (LPC) and isolated gluten were used help understand the positive effect of PL on bread-loaf volume. The kinetics of the effect of gluten on the thermal properties of LPC were determined using DSC. Blends of PL and 3, 6, and 10% gluten were heated from 0 to 70 °C at rates between 3 and 19 °C/min and cooled to 0 °C. The onset and peak temperatures and ΔH were recorded. The peak temperature was used to calculate the activation energy (E_a) and Z value. The transition for pure LPC vesicle formation was detectable by DSC in the presence of gluten. Gluten increased the activation energy of LPC during vesicle formation and disruption. The increase in gluten content from 3 to 6% and then to 10% had a slight effect on the activation energy value of LPC during vesicle disruption, whereas during formation a steady increase was noticed with higher gluten additions. Overall, the ΔH of the blends showed a decrease at higher heating rate. The change in the PL activation energy in the presence of gluten is indicative of a form of interaction.     

Mixing history affects gluten protein recovery, purity, and glutenin re-assembly capacity from optimally developed flour–water batters

Batters, from three wheat cultivars, were mixed up to their maximal consistency (t_peak) at different mixing speeds (N) and flour/water ratios [Auger, F., Morel, M.H., Lefebvre, J., Dewilde, M., Redl, A., 2008. A parametric and microstructural study of the formation of gluten network in mixed flour–water batter. Journal of Cereal Science 48, 349–358]. Gluten and starch were extracted from those batters using a process which included two successive steps: dilution and sieving. In order to reveal the specific influence of the mixing step, a standardized gentle washing and sieving procedure was selected. Mixing the batters at t_peak guaranteed a high and stable gluten protein recovery (ca. 82%) irrespective of mixing conditions. SE-HPLC analysis of protein, from flours and batters sampled at t_peak, demonstrated that mixing led to the almost total breakdown of the unextractable glutenin polymers (ca. 80%), whereas their re-assembly occurred during gluten extraction. The extent of glutenin re-assembly in gluten was influenced by the batter mixing history and was mainly related to the number of mixing rotations (N.t_peak). Gluten protein contents were also found related to N.t_peak. We proposed that the leaching of starch from the batter during gluten extraction was controlled by the elasticity of the protein network, i.e. the gluten content in unextractable glutenin. An innovating scheme relating the glutenin re-assembly capacity to the irreversible thiol protein oxidation is proposed.