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Wheat grain is recognized as the most suitable raw material for bread and pasta making due to the unique viscoelasticity of its storage proteins, and in particular its glutenin polymers. During grain development, low molecular weight (LMW) and high molecular weight (HMW) glutenin subunits (GS) gradually assembled through inter-chain disulfide bonds. Despite the impact of the final glutenin polymer size distribution on wheat technological quality, little is known concerning the oxidative folding of GS. In particular, which of their cysteine (Cys) residues participate to GS inter-chains remains putative. In this study GS from immature Triticum durum wheat grain were separated (10 SDS-PAGE) and digested into peptides, which were analyzed by MALDI-TOF/TOF. Differential alkylation of free and disulfide bonded Cys allowed us to identify the oxidative folding state of five types of LMW-GS and of the 1Bx20 HMW-GS. GS as monomers or as part of small oligomers carried a high number of free cysteine residues. In addition, some Cys residues, hitherto assumed as involved in intra-chain disulfide bonds, appeared simultaneously in free and oxidized forms. The last result could be assigned to their partial-blocking by glutathione. We concluded that the complete oxidative folding of LMW-GS is a late event, subsequent to GS inter-chain pairing. (C) 2014 Elsevier Ltd. All rights reserved.

The glass transition temperature (Tg) and change in heat capacity (ΔCp at Tg) of processed wheat gluten samples, including wet (casting) and dry processes (thermal treatment, mixing, thermomoulding and mixing followed by thermomoulding) were determined by modulated differential scanning calorimetry (MDSC) as a function of water content. An increase of Tg and a drastic decrease of ΔCp were observed when gluten was dry processed. Casting process gave a film with calorimetric properties close to those of native gluten. The molecular size distribution of proteins in native and processed glutens was measured by size exclusion chromatography. The thermomoulding and the use of chemical cross-linker during casting resulted in a drastic drop of SDS-soluble proteins. In contrast, mixing of gluten, even using high specific mechanical energies, gave only a slight polymerisation of the proteins. Therefore, except for treatments where a high temperature was applied to gluten, the modification of calorimetric parameters accounting for reticulation phenomena was not related to the SDS-insoluble protein content. Tg, ΔCp and protein SDS extractability could account for different kinds of protein networks.

International audience; Gluten-glycerol dough was extruded under a variety of processing conditions using a corotating self-wiping twin-screw extruder. Influence of feed rate, screw speed, and barrel temperature on processing parameters (die pressure, product temperature, residence time, specific energy) were examined. Use of flow modeling was successful for describing the evolution of the main flow parameters during processing. Rheological properties of extruded samples exhibited network-like behavior and were characterized and modeled by Cole-Cole distributions. Changes in molecular sizes of proteins during extrusion were measured by chromatography and appeared to be correlated to molecular size between network strands, as derived from the rheological properties of the materials obtained. Depending on operating conditions, extrudates presented very different surface aspects, ranging from very smooth-surfaced extrudates with high swell to completely broken extrudates. The results indicated that extrudate breakup was caused by increasing network density, and some gliadins may have acted as cross- linking agents. Increasing network density resulted in decreasing mobility of polymeric chains, and 'protein melt' may no longer have been able to support the strain experienced during extrusion through the die. Increasing network density was reflected in increased plateau modulus and molecular size of protein aggregates. Increasing network structure appeared to be induced by the severity of the thermomechanical treatment, as indicated by specific mechanical energy input and maximum temperature reached.

A simple method allowing the determination of the number of cysteine residues of the high molecular weight glutenin subunits (HMW-GS) is presented. The method was adapted from that of Creighton (T. E. Creighton, Nature 1980, 2840, 487-489) with modified reagents for alkylation of the cysteine residues and the electrophoretic system. The acid-urea-polyacrylamide gel electrophoresis (PAGE) method, developed by Morel (M. H. Morel, Cereal Chem. 1994, 713, 238-242), was adopted and mixtures of iodoacetic acid and 4-vinylpyridine were used to alkylate the glutenin subunits. The accuracy of the method was checked with some HMW-GS whose number of cysteine residues was already known from molecular biology approaches. In subunits 5*, 2.2, 2.2* and 4.1 (of Dx types) from cultivars Ben and Fiorello, MG 7249, MG 315, and Kador, respectively, only 4 cysteine residues were demonstrated. Subunit 20 was found to exist as a regular Bx and By subunit pair, with the Bx subunit containing only 2 cysteine residues, which is unusual.

Assembly of glutenin polymers was examined for two contrasted durum wheat cultivars in connection with changes in the redox status of the endosperm cells that accompanied grain development. The evolutions of the redox state of ascorbate and glutathione, as well as the activities of antioxidant enzymes were measured. Changes in the size distribution profile and redox state of storage proteins were evaluated, with particular emphasis on protein-bound glutathione (PSSG). At the beginning of grain filling phase, the size distribution profile of proteins included an extra peak shoulder at about 40,000 g mol(-1). The shoulder was assimilated to free glutenin subunits as it disappeared concomitantly with the upturn in glutenin polymers accumulation. Irrespective of cultivars, small SDS-soluble polymers accumulated first, followed by larger and insoluble ones, attesting for a progressive polymerization. During the grain filling phase, catalase (EC 1.11.1.6) activity dropped, reaching a very low level at physiological maturity. During the same period, superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) activities increased steadily while the equilibrium constant between GSSG and PSSG shifted from 10(-2) to unity. These results demonstrated that grain filling was accompanied by a continuous decrease in cellular redox potential. In this context, formation of protein-bound glutathione would represent a protective mechanism against irreversible thiol oxidation. Storage protein S-gluta-thionylation instead of limiting glutenin polymer assembly as it has been proposed might be a required intermediate step for glutenin subunits pairing. (C) 2012 Elsevier Masson SAS. All rights reserved.

During pasta processing, structural changes of protein occur, due to changes in water content, mechanical energy input, and high temperature treatments. The present paper investigates the impact of successive and intense thermal treatments (high temperature drying, cooking, and overcooking) on aggregation of gluten protein in pasta. Protein aggregation was evaluated by the measurement of sensitivity of disulfide bonds toward reduction with dithioerythritol (DTE), at different reactions times. In addition to the loss in protein extractability in sodium dodecyl sulfate buffer, heat treatments induced a drastic change in disulfide bonds sensitivity toward DTE reduction and in size-exclusion high-performance liquid chromatography profiles of fully reduced protein. The protein solubility loss was assumed to derive from the increasing connectivity of protein upon heat treatments. The increasing degree of protein upon aggregation would be due to the formation of additional interchain disulfide bonds.

International audience; Size exclusion high-performance liquid chromatography analysis was carried out on wheat gluten-glycerol blends subjected to different heat treatments. The elution profiles were analyzed in order to follow the solubility loss of protein fractions with specific molecular size. Owing to the known biochemical changes involved during the heat denaturation of gluten, a mechanistic mathematical model was developed, which divided the protein denaturation into two distinct reaction steps: (i) reversible change in protein conformation and (ii) protein precipitation through disulfide bonding between initially SDS-soluble and SDS-insoluble reaction partners. Activation energies of gluten unfolding, refolding, and precipitation were calculated with the Arrhenius law to 53.9 kJ x mol(-1), 29.5 kJ x mol(-1), and 172 kJ x mol(-1), respectively. The rate of protein solubility loss decreased as the cross-linking reaction proceeded, which may be attributed to the formation of a three-dimensional network progressively hindering the reaction. The enhanced susceptibility to aggregation of large molecules was assigned to a risen reaction probability due to their higher number of cysteine residues and to the increased percentage of unfolded and thereby activated proteins as complete protein refolding seemed to be an anticooperative process.

Gluten films were prepared by casting an acidic and ethanolic solution of gluten previously treated with sodium sulfite. The effects of sulfitolysis on proteins were investigated by SE-HPLC and thiol/disulfide content measurements. During sulfitolysis, insoluble glutenin macropolymer was converted into its constitutive subunits. About 10% of gluten disulfide bonds were cleaved, of which three-fourths originated from interchain disulfide bonds. Oxidation of thiol groups released during sulfitolysis was followed for various temperatures (T) and relative humidities. Oxidation was shown to be a second-order rate process occurring below the glass transition temperature (T(g)) and related to T - T(g). Thiol oxidation ensured the formation of interchain bonds between specific classes of gluten proteins according to an ordered process. Intrachain bonds were also formed and through thiol/disulfide-exchange reactions were finally converted to interchain bonds. Thus, fully oxidized gluten films had more insoluble glutenin macropolymers than native gluten.

International audience; The rheological behaviour of a gluten plasticized with glycerol has been studied in oscillatory shear. The mixing operation in a Haake batch mixer leads to a maximum torque for a level of specific energy (500-600 kJ/kg) and temperature (50-60 degrees C) quite independent of mixing conditions (rotor speed, mixing time, filling ratio). The gluten/glycerol dough behaves as a classical gluten/water dough, with a storage modulus higher than the loss modulus over the frequency range under study. A temperature increase induces a decrease of moduli, but the material is not thermorheologically simple. Glycerol has a plasticizing effect, which can be classically described by an exponential dependence. Mixing conditions influence the viscoelastic properties of the material, mainly through the specific mechanical energy input (to 2000 kJ/kg) and temperature increase (to 80 degrees C). Above 50 degrees C, specific mechanical energy highly increases the complex modulus. The aggregation of proteins, as evidenced by size-exclusion chromatography measurements, occurs later as the dough temperature reaches 70 degrees C. The nature of network interactions and the respective influence of hydrophobic and disulphide contribution is discussed. A general expression is proposed for describing the viscous behaviour of a gluten/glycerol mix, which could seem simplistic for such a complex rheological behaviour, but would remain sufficient for modelling the flow behaviour in a twin screw extruder.