## 3 Results and Discussion
### Physical Appearances of Collagen Hydrolysate before and after Sterilization
These changes in molecular structure can profoundly affect collagen hydrolysate's physical and biological properties, including its solubility, gelling properties, and interactions with cells and tissues [19; 20; 21; 26]. Therefore, the selection of a sterilization method for protein-based biomaterials has to consider these structural changes to preserve the original characteristics of the material. The \({}^{13}\)C-NMR spectral analysis of collagen hydrolysate after various sterilization methods is shown in Figure 5. It presents a remarkable change in chemical structure; focusing specifically on the regions around 180 ppm and 31-33 ppm for samples exposed  
Figure 4: \({}^{1}\)H-NMR spectra of the chemical structure of collagen hydrolysate before and after sterilization. The red frame indicates the position of the peak that had shifted and/or changed. to beta and gamma radiation, it did not show any significant peaks on the spectrum, indicating that the sterilization process did not alter the methylene groups in the aliphatic chains of the collagen and therefore gave rise to the same results as the control group that was not sterilized. This is in contrast to ethylene oxide sterilization. The sample displayed a peak at 180 ppm, indicating oxidative changes, and a peak in the 31-33 ppm range. This illustrates the impact of ethylene oxide sterilization on the structure of collagen hydrolysate, as EIO reacts with NH\({}_{2}\), potentially causing changes in the chemical structure or generating significant heat, which leads to protein aggregation [10; 18; 19; 20; 21; 22]. MALDI-TOF-MS Characterization of the Molecular Weight of Collagen Hydrolysate before and after Sterilization  
MALDI-TOF-MS was employed to investigate the potential changes in the structure and composition of the collagen hydrolysate that were induced by different sterilization processes. Figure 6 shows the mass spectra of collagen hydrolysate when using MALDI-TOF mass spectrometry techniques to examine the molecular weight of the collagen hydrolysate before and after sterilization. The sums of the \(m/z\) values of collagen hydrolysate before and after sterilization using various techniques are listed in Table 1.  
Figure 5: The spectral results of \({}^{13}\)C-NMR technique for examining the chemical structure of collagen hydrolysates before and after sterilization. The red frame indicates the position of the peak that had shifted and/or changed. The molecular weight profiles of collagen hydrolysate from tuna tendon were analyzed using MALDI TOF mass spectrometry and the results are shown in Figure 6, which reveals the effects of various sterilization methods on molecular structure. The mass-to-charge ratio (\(m/z\)) was used as an indicator of approximate molecular weight [27]. While initial analysis showed a maximum peak at 1401 Da across all sample groups, in-depth analysis using Equation (1) to calculate the percentage difference in total molecular weight for each sample group compared to the control or non-sterilized group in Table 1 demonstrated subtle changes. Beta radiation resulted in a 0.987% decrease in the total molecular weight, indicating partial degradation of amino acids in some protein structures [10; 28; 29]. Conversely, gamma radiation and ethylene oxide treatment slightly increased the total molecular weight by 0.626% and 0.118%, respectively, possibly due to crosslinking, molecular aggregation, or residual EtO particles [10; 19; 20; 21; 22; 28; 29; 30]. These minor alterations suggest that the backbone structure of collagen hydrolysate was not significantly affected by the sterilization processes in this study. However, to confirm safety for medical applications or consumption, additional cytotoxicity testing is necessary. Furthermore, if radiation