## 3 Results and Discussion  
### Physical Appearances of Collagen Hydrolysate before and after Sterilization  
The physical appearances of the collagen hydrolysates were visibly altered after undergoing the various sterilization methods, as shown in Figure 2. Ethylene oxide sterilization caused the collagen hydrolysate to change from a light brown powder to a burnt brown and sticky mass. When dissolved in pure distilled water, the resulting solution appeared dark brown. This transformation can be attributed to the high temperatures experienced during the ethylene oxide sterilization method. In contrast, both beta and gamma radiation sterilization methods did not result in any observable physical changes to the collagen hydrolysate, which maintained its pre-sterilization appearance. The visual appearances of the samples before and after the sterilization of collagen hydrolysate can be quantitatively analyzed by image histograms from the ImageJ program, as presented in Figure 3. The histogram of the non-sterilized collagen hydrolysate exhibited a normal distribution of pixel intensities, indicating a homogeneous appearance. However, the histogram of the ethylene-oxide-sterilized sample showed a significant shift toward darker pixel intensities, confirming the visual observation of a darkened/burnt zone of the specimen. The histograms of both the beta- and gamma-radiation-sterilized samples almost resembled that of the non-sterilized sample, suggesting minimal impact on the physical appearance of the collagen hydrolysate. These findings demonstrate that the choice of sterilization method substantially influences the physical appearance of collagen hydrolysate. Ethylene oxide sterilization, likely due to the high temperatures involved, causes significant changes in color and texture, potentially affecting the material's properties and applications. In contrast, the beta and gamma radiation sterilization methods preserve the original physical appearance of the collagen hydrolysates, making them more suitable for maintaining the material's integrity. NMR Characterization of the Chemical Structure of Collagen Hydrolysate before and after Sterilization  
The \({}^{1}\)H-NMR spectral analysis from Figure 4 indicates visible changes in the chemical structure of collagen hydrolysate after sterilization. It was found that sterilization with beta and gamma radiation at a dose of 17.5 kGy did not change the chemical structure of collagen hydrolysate. This means that the energy of beta and gamma particles does not cause molecular fragmentation or change. In contrast, ethylene oxide sterilization showed pronounced changes and new peaks in the 2-3 ppm range, indicating changes in the methylene and methyl groups [17]. This could be caused by breakdown products  
Figure 3: Image histogram of collagen hydrolysate before and after different sterilization methods obtained from ImageJ analysis. Figure 2: Physical characteristics of collagen hydrolysate after different methods of sterilization image. The red frame was used as the outer edge where the tip of the arrow cross cursor met for measurement accuracy. or the crosslinking of protein chains, with a slightly more pronounced peak shift. This may indicate chemicals forming between ethylene oxide and amino acids in the collagen hydrolysate [10; 18; 19; 20; 21; 22]. The \({}^{1}\)H-NMR technique clarified the effect of sterilization on the chemical structure of collagen hydrolysate. Sterilization with ethylene oxide appears to have a marked effect on the secondary structure of the protein [23]. This can be seen from the change in the chemical shift in the aliphatic region. This includes protons attached to carbon atoms in the protein's backbone and side chains. The use of high heat during sterilization can lead to molecular damage, causing more significant structural changes compared to beta and gamma radiation [20; 24; 25]. A few small peaks can be observed, indicating the potential to preserve the original collagen structure.