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).