Insight into gluten structure in a mild chaotropic solvent by asymmetrical flow field-flow fractionation (AsFlFFF) and evidence of non-covalent assemblies between glutenin and ω-gliadin

Abstract

Wheat gluten, one of the most complex viscoelastic protein networks in nature, is unique to get the specific texture of bread. Due to its complex protein composition, its insolubility in most solvents and the very high molar mass of half of the proteins (glutenin, the other half being gliadin), the architecture of the network is still not well understood. In this work, we have investigated model gluten protein extracts with contrasted compositions in glutenin and gliadin solubilized in a mild chaotropic solvent: ethanol/water (50/50 v/v). The samples display a liquid-liquid phase separation with an upper critical solution temperature that depends on the protein composition. The phase diagrams are consistent with the presence of supramolecular assemblies of proteins. To confirm the presence of these assemblies and fully characterize the objects dispersed in ethanol/water, we have used an asymmetrical flow field-flow fractionation (AsFlFFF) setup coupled with differential refractive index, multi-angle light scattering and dynamic light scattering detections to probe very dilute protein suspensions. We have identified three classes of objects, with distinctive molar mass, characteristic size and conformation: protein monomers, polymeric structures, and very loose protein assemblies with molar mass larger than 2.10 6 g/mol. A molecular characterization of the species by size exclusion chromatography in a denaturing solvent shows that polymers and assemblies are mainly composed of glutenin and -gliadin. The high content of 2 -gliadin, devoid of cysteines, indicates the importance of non-covalent interactions involved in protein assemblies and might play a major role in gluten rheology.

Impact of the protein composition on the structure and viscoelasticity of polymer-like gluten gels

“…The fit with OZ (line in the inset Fig. 2) gives ξ = 3.1 nm, a numerical value consistent with the size of gliadin [17,34]. Note that data are equally well fitted using a Debye function [35], which is the form factor for Gaussian chains, yielding a comparable size (4.6 nm).…”

“…Interestingly, above 30 %, the proportion of polymer is roughly constant and one observes the emergence of large protein assemblies as a third species, whose amount increases with GLU . [17]. The relative error, as evaluated from 3 replicated measurement with the extract with GLU = 47 % is less than 5 %.…”

“…Gluten protein extracts are prepared from an industrial gluten (courtesy of TEREOS-SYRAL, Aalst, Belgium), following a protocol described elsewhere [17,22]. In brief, gluten is first dispersed at room temperature in a water/ethanol 50/50 v/v solvent.…”

“…Figure 1 reports the composition of the species present in very dilute suspensions (C = 4 mg/mL) of the different protein extracts as determined thanks to asymmetrical flow field-flow fractionation [17]. Three classes of objects, monomeric gliadins, glutenin polymers and protein assemblies, are identified depending on their average size, < R >, and molar mass < M w >.…”

“…Mass fraction of monomers, polymers and assemblies (see text) in dilute suspensions of the different protein extracts. Adapted from[17]. The relative error, as evaluated from 3 replicated measurement with the extract with GLU = 47 % is less than 5 %.…”