General Discussion
Conclusions
Future work

A model structure has been proposed for gliadin and deamidated gliadin based on the results from chapters 3 and 4. Molecules of gliadin which are not spherical in shape aggregated through hydrogen bonding to form a unit with a diameter of 32nm. Formation of the unit caused exposure of hydrophobic groups making it insoluble in water. This unit then aggregated with other units through hydrophobic interactions to form large aggregates It is being proposed that this might be a mechanism that nature uses to store proteins so that they do not dissolve and diffuse away from their storage site. Deamidation of the gliadin molecule changed the glutamine residues to glutamic acid which caused electrostatic repulsion between the molecules. The hydrogen bonding holding the molecules together in gliadin were replaced by electrostatic repulsion which caused deamidated gliadin to aggregate through hydrophobic interaction exposing carboxyl groups making the deamidated gliadins soluble in water.

The addition of beta-mercaptoethanol to deamidated gliadin caused a reduction of the disulphide bonds with the exposure of cys-299 and cys-307 which subsequently formed disulphide bonds with other deamidated gliadin molecules. In the presence of both beta-mercaptoethanol and urea, the whole molecule unfolded and fibrils of 343nm thickness were formed.

Below 16% (w/v) concentration the viscosity of SWP decreased with an increase in temperature (Figure 3.25). In addition, exposed hydrophobicity showed greater values than surface hydrophobicity (Figure 3.27). The large aggregates of SWP formed on hydration at room temperature (Plates 3.22 and 3.24) were attributed to polymerisation and interactions between the glutenin fraction (Figure 2.9). The reason for the reduction in viscosity on heating was caused by the breakdown of aggregates, which was observed by microscopy (Plates 3.23 and 3.25). Further evidence by differential scanning calorimetry (DSC) indicated that there was an absence of conformational transition during the heating of SWP proteins (Figure 3.28). This stability can be attributed to intra-disulphide bonds in deamidated gliadins and both intra- and inter-disulphide bonds in deamidated glutenins. A reduction of the disulphide bonds by beta-mercaptoethanol promoted the gelation of SWP at room temperature (Figure 3.35) which otherwise would not gel (Figure 3.34).

The addition of 2% NaCl to SWP at 20°C caused a decrease in viscosity (Figure 6.14). This decrease was proposed to be due to the formation of a diffuse double layer and also to an increase in the dielectric constant of the medium.

Small quantities of SWP (1%) caused a major increase in the storage modulus of BSA (Figure 5.17). It is proposed that SWP electrostatically interacted with domain III of BSA which can be saturated. Above the critical value at which saturation of domain III occurs, the elastic modulus decreased because of repulsion between SWP and negatively charged domains I and II.

In the absence of salt, 50% SWP/WPI mixtures were found to have greater apparent viscosities than the individual proteins (Figures 6.16, 6.17 and 6.19). It was proposed that since SWP is a sodium salt of a protein, WPI scavenged part of the sodium ions to induce its own aggregation and the shielding effect of sodium ions also helped to reduce the repulsion between WPI and SWP leading to aggregation. The gels formed in the SWP/WPI system at 95°C are most likely to be phase separated gels made up of viscous SWP sol trapped in WPI gel network.

The addition of small amounts of basic proteins like clupeine or lysozyme (0.1%) led to the gelation of 19% (w/v) SWP aqueous dispersion at 80°C. SWP on its own, normally gels above 105°C. The lysozyme-induced gelation was proposed to be electrostatic in nature involving proteins which did not unfold prior to gelation (Figure 8.3). SWP was found to protect lysozyme from heat induced denaturation and, therefore, could be useful in the preservation of products to which lysozyme has been added as an anti-bacterial agent.

SWP was found to bind iron, copper, zinc and cadmium. The highest affinity was found to be for cadmium.

Conclusions

In any broad, applied area like Food Science and Technology, an integrated approach comprising several techniques is required to investigate complex systems. With this in mind, the problem of solving the mechanisms of interaction of the selected systems was attacked from different angles using different techniques. The findings using this approach are listed as follows:

• Molecules of gliadin (with a radius of gyration of 32Å) which are not spherical in shape aggregated to a compact spherical form of diameter 32nm, which can be called a unit. This unit then aggregated with other units to form massive aggregates.
• Although gliadin was soluble in 50-70% ethyl alcohol, the solvent was partitioned into an inner layer of alcohol around the gliadin molecule with an outer layer of water.
• Deamidated gliadin was found to be very stable between 50-94°C. Although there were changes in the secondary structural elements upon heating, the changes were reversible on cooling.
• The disulphide bonds in both gliadin and deamidated gliadin were found to be in the interior of the protein.
• Although SWP did not gel below 100°C, reduction of the disulphide bonds with beta-mercaptoethanol promoted the gelation of SWP at room temperature.
• SWP aggregated at room temperature when hydrated through hydrophobic interactions which were broken on heating at 60°C.
• Below 3% (w/v), SWP was found to be Newtonian whereas it was shear thinning above 3% (w/v). The intrinsic viscosity of SWP was found to be 0.9 dlg-1.
• A sequence in gliadin responsible for coeliac disease was not found in deamidated gliadin; therefore, it is possible that SWP may not be associated with the disease.
• The secondary structure prediction showed that deamidation caused an increase in alpha-helix but a decrease in beta-sheet content of gliadin.
• A possible metal binding function was predicted by the supersecondary structure prediction method.
• BSA gelation is mainly due to the presence of a reactive cys34.
• Although SWP did not form a gel at 90°C, it enhanced the gelation of BSA when added in small quantities (BSA:SWP ratio 11:1).
• The mechanism of SWP - BSA interaction was considered to be electrostatic, between SWP carboxyl groups and basic groups on BSA and hydrophobic interaction between exposed hydrophobic residues.
• Partial unfolding of BSA prior to heating gave stronger gels.
• The gelation of beta-lactoglobulin influenced the gelation of Bipro whey protein isolate. Beta-Lactoglobulin dimers dissociated on heating to give monomers which unfolded after a reduction of disulphide bonds causing the molecules to polymerise through disulphide interchange reactions thus forming a network.
• NaCl when added to SWP at room temperature caused a decrease in viscosity due to the formation of a diffuse double layer and an increase in the dielectric constant of the medium, whereas, the viscosity of WPI increased in the presence of NaCl because salt induced structural changes in beta-lactoglobulin with an increase in apparent hydrophobicity.
• Heating of SWP in the presence of NaCl increased the viscosity due to a reduction of the Debye-Hückel parameter.
• WPI gels formed in the absence of salt were translucent, whilst those formed with salt were opaque due to a higher refractive index of the dense salt-formed aggregates.
• SWP interacted with WPI through both an indirect and a direct mechanism. In the indirect mechanism, WPI scavenged sodium salts associated with SWP to induce its own gelation. In the direct mechanism, disulphide bonds are thought to form between WPI and SWP on heating at 95°C.
• SWP binds metals (iron, copper, zinc and cadmium) with a very high affinity for cadmium and, therefore, has a potential as a detoxifying agent.
• The gelation of SWP can be induced with basic proteins such as lysozyme and clupeine at temperatures lower than (80°C) those required for SWP to gel on its own (105°C).
• SWP protected lysozyme from heat denaturation.

• Investigation of the number of molecules and the forces involved in the formation of the gliadin unit and why hydrogen bonding was preferred to hydrophobic interaction.
• What is the difference between the enthalpy of hydrogen bonding between glutamine residues and between glutamine residues and water molecules and conditions which give one preference over the other?
• Analysis of the structure and function of protein motifs found in section 4.6.2.
• A study to determine the number and location of binding sites in deamidated gliadin and deamidated glutenin for specific metals and the effect of metal binding on the structure of the protein molecules.
• Effect of various metals on SWP gelation.
• Contrast variation studies of gliadin and deamidated gliadin using deuterated alcohol.
• Purification of deamidated gliadin.
• Production of polyclonal antibodies against deamidated gliadin and deamidated glutenin.
• The use of fluorescent, radioactive or gold labeled antibodies to locate lysozyme, deamidated gliadin and deamidated glutenin in lysozyme induced gelation of SWP.
• Toxicity studies of SWP.
• Phase contrast and electron microscopy studies of SWP/BSA mixtures.