Figure 3. (a) Th-T fluorescence intensity anddH values for the samples sheared at constant
shear rate of 300s-1 at 55°C (black circle), 65°C (red
square) and 75°C (blue diamond); and (b) Th-T fluorescence
intensity comparison between thermal and thermomechanical treatments
(sheared at 300 s-1) at the above three temperatures.
Dotted lines indicate the fitted data using a single exponential
expression.
The secondary structural changes in the protein solutions for the shear
experiments carried out at 300 s-1 and at 55°C, 60°C
and 65°C have been recorded for the samples collected at different time
intervals. Figure 4 shows the CD spectra of the sheared samples. It was
observed that shearing at 55°C caused no change in secondary structure
as compare to the native BSA. Although, a slight increase in the helical
conformation was seen, presumably due to the formation of new intra and
inter molecular bonds under the influence of dissipation energy below
the melting point of the protein. As the temperature increased, the loss
of the initial helical conformation was clearly visualized as the
negative band at 222 nm gradually shifts towards the positive direction.
This increase in ellipticity became more prominent as the temperature
increase as well as the duration of shear increased. These result
correlated with the Th-T fluorescence and hydrodynamic data as shown in
Figure 3.
The change in MRE values at 222 nm is plotted as a function of time for
all the three temperatures (Figure 4 (d)). The increase in the MRE
signals at 60 and 65 °C correlated with the formation of β-sheet rich
aggregates. The rate was increase in MRE data at 65 °C was greater than
that at 60 °C. This corresponded to the enhanced rate of aggregation at
65 °C (Figure 3 (b)). The loss of helicity was found to be about 40%
and 90% at 60°C and 65°C, respectively. This indicated that the
thermomechanical treatment at even lower 60 °C resulted a similar loss
of helicity as thermal treated at a higher temperature of 75 °C
(Bogahawaththa and Vasiljevic 2020).