GENERAL DISCUSSION AND CONCLUSIONS
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
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
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
SWP was found to bind iron, copper, zinc and cadmium. The highest affinity
was found to be for cadmium.
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
- 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
- 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
- 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
- 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.
Use of neutron scattering to study the interaction of SWP and lysozyme under
varying temperatures and the gelation of SWP above 100°C.
- Investigation of the number of molecules and the forces involved in the
formation of the gliadin unit and why hydrogen bonding was preferred to
- 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
- 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
- Purification of deamidated gliadin.
- Production of polyclonal antibodies against deamidated gliadin and
- 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.