Thermoanalytical characterization of high molecular weight glutenin subunits: Water effect on their glass transition
Introduction
Gluten, the major storage protein fraction of cereal grains, is composed of two protein groups: gliadins and glutenins, which are respectively soluble and insoluble in 70–90% aqueous ethanol [1], [2].
Gliadins are present as monomers and are responsible for the extensibility and cohesive properties of gluten, while the glutenins form high molecular weight polymers and contribute to the elasticity of gluten [3].
The combination of these two protein groups is, therefore, responsible for the viscoelastic properties that allow wheat doughs to be processed into bread and other foods. Both the gliadins and glutenins are mixtures of proteins that can be divided into groups. One such group of glutenin proteins, called the high molecular weight (high Mr) subunits of glutenin, appears to be particularly important in relation to determining the viscoelastic properties of gluten and the differences in this property between cultivars (cv) of good and poor breadmaking performance [4], [5]. They have therefore been studied in detail to determine their structures and biophysical properties [6], [7], [8], [9].
The gluten proteins, like all polymers, are characterized by a temperature dependent equilibrium between two phases, a semiliquid, prevalent at high temperature, and a glassy solid, prevalent at low temperature. This type of physical change has been called ‘glass transition’ [10]. The glass transition temperature (Tg) is the principal parameter for understanding the mechanical properties of gluten proteins [11]. Details of this parameter should, therefore, provide useful information relevant for the comprehension of the rheological properties of wheat-flour-water based foods.
A powerful tool for studying the thermal behaviour of these proteins is the Thermal Analysis carried out by Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) techniques [11], [12], [13], [14].
The first technique measures the change in weight caused by the loss of water and eventual product degradation, allowing the total amount of water present in the protein structure to be calculated. The second technique allows the observation of all phenomena that involve heat exchange. Recent studies have shown that the glass transition temperature can be influenced by the presence of a plasticizing agent such as water, increasing the mobility of polymeric chains and lowering the Tg value [3], [12].
The aim of this work was to determine the thermal behaviour of different high Mr glutenin subunits and to correlate their glass transition temperatures with different water contents.
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Materials
High Mr glutenin subunits, 1Dx5 and 1Bx6 were obtained from bread wheat cv. Mercia, 1Dx2 and 1Dy12 from cv. Longbow, 1Bx7 from cv. Galatea, 1Dy10 from an isogenic Gabo/Olympic line, L 88-22 [15] and 1Bx20 from durum wheat cv. Bidi 17. The Mr 58 000 peptide, derived from the repetitive domain of subunit 1Dx5, was expressed in Escherichia Coli and isolated as described below.
Purification of the high Mr glutenin subunits from wheat
Proteins were extracted and purified according to the method of Shewry et al. [16] with minor modifications:
- 1.
fractions
Thermogravimetric analysis
TGA of the as-received samples showed different weight losses for each protein. The water contents (w/w) were 6.0, 6.8, 8.8, 7.0, 7.3, 6.0, 7.8 and 6.1% for 1Dx5, 1Bx7, 1Dx2, 1Bx20, 1Dy12, 1Bx6, 1Dy10 and Mr 58 000 peptide, respectively. These losses occurred in a single step as the proteins were heated to 120°C. As an example, the TG curve (curve a) and the DTG curve (curve b) of the glutenin subunit 1Dx5 are shown in Fig. 1.
Differential scanning calorimetry
Fig. 2, Fig. 3 show the calorimetric curves of the high Mr glutenin
Discussion
The abrupt change in slope of the calorimetric curves of the HMW glutenin subunits (Fig. 2, Fig. 3) can be attributed to the well-known glass transition typical for amorphous materials like biopolymers. The glass transition temperature (Tg), defined as the temperature at which a glassy polymer is converted into a softer rubbery one upon heating, can be readily calculated by extending the tangents of the curve immediately preceding and following the transition and then measuring the temperature
Conclusions
From the results, we can infer that:
- 1.
the high Mr glutenin subunits exhibit a different thermotropic behaviour. 1Dx5, 1Bx7, 1Dx2, 1Bx20, 1Dy12, 1Dy10 subunits and Mr 58 000 peptide show, during the first scan, an endothermic peak due to a relaxation process that superimposes on the glass transition. This relaxation phenomenon does not appear in studies on the 1Bx6 subunit;
- 2.
since the second calorimetric scan, all the proteins present a similar behaviour, exhibiting a reproducible glass transition
Acknowledgements
This work was supported by the EU FAIR program CT 96–1170, the Italian C.N.R. (Progetto Finalizzato Biotecnologie) and M.I.R.A.F. (Progetto Finalizzato Nazionale sulle Biotecnologie Vegetali).
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