## 3 Results and Discussion
### Circular Dichroism  
Circular dichroism was used to study the effect of the HIU treatments on the secondary structure of the gelatins because this technique allows for the calculation of the proportion of each component (\(\alpha\) helix, \(\beta\) sheet, and random coil) in gelatins. Figure 2A shows the proportions of components in all gelatins at 25 \({}^{\circ}\)C; all gelatins showed a higher proportion of random coils, which was followed  
Figure 1: SDS-PAGE profile of tilapia (_Oreochromis niloticus_) skin gelatins that were treated with high intensity ultrasound pulses.  
by the \(\alpha\)-helix, and finally by the \(\beta\)-sheets in a smaller proportion. This is consistent with changes resulting from the denaturation of collagen (triple helix) to gelatin. The changes in the secondary structure of gelatins were caused by the conformations acquired by the atoms of the backbone of the polypeptide chain induced by cavitation. Previous studies have related the reduction of the \(\alpha\)-helix with protein unfolding and the increase of the \(\beta\)-sheet with protein aggregation [12]. Additionally, it can be seen that the HIU treatments generated significant differences in the secondary structure at increasing temperatures from 10 to 90 \({}^{\circ}\)C, as shown in Figure 2B-F, where although the random coil was the major secondary structure for all gelatins, UPG-71 and UPG-84 had a substantial change in the \(\beta\)-sheet/\(\alpha\)-helix ratio at around 60 \({}^{\circ}\)C.  
For the control gelatin, the most substantial changes occurred at approximately 40 \({}^{\circ}\)C, where there was a 37% increase in the \(\alpha\)-helix (Figure 2B), while the random coils decreased; this may have been due to the unfolding of the remaining collagen, since the collagen from marine species presented little cross-linking, confirmed by its low thermal denaturation temperature at \(\sim\)40 \({}^{\circ}\)C, corresponding to a poorly cross-linked collagen [23]. Additionally, as the temperature rose, it could be seen that the \(\beta\)-sheet structure began to have a progressive increase until reaching 64% of this structure; as this  
Figure 2: Secondary structure composition of ultrasound pulsed tilapia (_Orochromis niloticus_) skin gelatin. (**A**): Secondary structure composition of gelatins at 25 \({}^{\circ}\)C. (**B–F**): Secondary structure composition in a temperature range of 10 to 90 \({}^{\circ}\)C of the control gelatin (CG), ultrasound-pulsed gelatin (UPG)-42, UPG-52, UPG-71, and UPG-84, respectively.  
happened, and while the random coil remained constant, the \(\alpha\)-helix could have been transforming into a \(\beta\)-sheet.  
As for UPG-42 (Figure 2C), as the temperature rose, the random coil remained the dominant component; however, the \(\beta\)-sheets began to predominate over the \(\alpha\)-helix, reaching their lowest point around the denaturation temperature. For UGP-52 (Figure 2D), there was a 21% increase in the \(\alpha\)-helix at around 30 instead of 40 \({}^{\circ}\)C [24]. In the case of UPG-71 (Figure 2E), the denaturation temperature was low, around 37 \({}^{\circ}\)C, which implied drastic changes in the \(\beta\)-sheet and the \(\alpha\)-helix, resulting in a loss of 87.5% of the \(\alpha\)-helix and an increase in the \(\beta\)-sheet. Finally, in the case of UPG-84 (Figure 2F), the denaturation took place at around 40 \({}^{\circ}\)C. However, changes in the secondary structures began to occur from 30 \({}^{\circ}\)C in the \(\alpha\)-helix, even reaching temperatures of 60 \({}^{\circ}\)C, at which almost all of the \(\alpha\) structures were lost.  
These \(\alpha\)-helix decreases and \(\beta\)-sheet increases are consistent with those that have been reported for scallops [9], but they contrast with those that have been reported for lactic proteins [25]. These results indicate that the high-intensity ultrasonic pulses that were used in this work affect even the secondary structure and denaturation temperature of gelatins due to the alteration in interactions between polypeptide chains.