## 2 Materials and Methods
### Extraction Yield, Hydroxproline Content, pH, Colour and Thermal Transition Measurement
The hydroxyproline component is likewise found exclusively in collagen and is in negligible quantities compared to other proteins. As a result, it can provide a quantitative estimate of the amount of collagen present. Furthermore, the more the hydroxyproline content value, the greater the extractable collagen content [2]. Table 1 shows the results of the pH test for acid-soluble collagen, which indicates that there were no appreciable differences between the various types of acid extraction. The pH value of AAC was 4.85, which was higher than that of LAC (4.81) and CAC (4.51). Typically, a pH measurement of between 6 and 7 is considered ideal for collagen [41]. In contrast, the measurements of the pH of AAC, LAC, and CAC in this study did not exceed pH 6, which indicates that these compounds are acidic. This means that during the dialysis process, salts and acids could be removed completely, so there are still salts and acids left [42]. Additionally, the type of acid utilised and the amount of acid during the extraction process may contribute to differences in the pH of collagen [30]. This could also be the result of a pH neutralisation process that is not working to its full capacity. Another factor that could contribute to the low pH is that the pH of the collagen in the fish varies according to species [15]. In terms of colour, the quality of collagen can be measured. There are three values on the colour scale: L* (brightness), a* (green-reddish), and b* (brightness) (blue-yellowish). Table 1 shows the L* a* b* values for AAC, LAC, and CAC collagen samples. LAC had a higher L* colour scale value than AAC and CAC, with significant differences at (\(p<0.05\)) of 71.89, 69.77, and 57.14, respectively. The colour scale a* revealed that LAC had the greatest value of 0.92, followed by CAC (0.73) and AAC (0.15), with a significant difference of (\(p<0.05\)) between them. Furthermore, b* colour showed that the AAC sample recorded a reading of 5.52 that was slightly higher than CAC (5.20) and LAC (4.69), with no significant difference at (\(p<0.05\)). In the thermal conversion of collagen, when collagen is heated, its triple helix breaks up into a random coil through a series of changes in viscosity, sedimentation, diffusion, light scattering, and other properties [43]. Protein denaturation is associated with the breakdown of tertiary structure and the loss of biological activity. Collagen denatures by detaching from its alpha chain and melting from its triple helix crystal [44]. According to several studies, marine collagen has less heat stability than mammalian collagen. The thermal stability of collagen is also closely connected to the environment and body temperature [45]. According to Table 1, the maximal transition \begin{table}
\begin{tabular}{c c c c} \hline
**Parameters** & **AAC** & **LAC** & **CAC** \\ \hline Yield (\% dry weight basis) & 3.13 \(\pm\) 0.02 c & 0.56 \(\pm\) 0.011 & 1.03 \(\pm\) 0.03 b \\ Swelling (\%) & 226.37\(\pm\) 0.03 c & 198.37\(\pm\) 0.03 a & 210.41\(\pm\) 0.16 b \\ Hydroxyproline content (mg/g dry sample) & 81.69 \(\pm\) 2.01 b & 79.07 \(\pm\) 0.3 a & 79.95 \(\pm\) 0.09 a \\ pH & 4.85 \(\pm\) 0.49 a & 4.81 \(\pm\) 0.19 a & 4.51 \(\pm\) 0.07 a \\ Colour scale, L* & 69.77 \(\pm\) 4.04 b & 71.89 \(\pm\) 1.52 c & 57.14 \(\pm\) 4.13 a \\ a* & 0.15 \(\pm\) 0.16 a & 0.92 \(\pm\) 0.22 b & 0.73 \(\pm\) 0.09 b \\ b* & 5.52 \(\pm\) 1.23 a & 5.20 \(\pm\) 0.02 a & 4.69 \(\pm\) 0.65 a \\ T\({}_{\rm max}\) (\({}^{\circ}\)C) & 39.00\(\pm\) 0.25 a & 38.60\(\pm\) 0.19 a & 38.15 \(\pm\) 0.12 a \\ Enthalpy (AH) (m/g) & 0.0398 & 0.0346 & 0.0218 \\ \hline Values are reported as mean \(\pm\) 50 (n = 3). Different lowercase letters indicate significant difference (\(p<0.05\)). The colour scale: L* (brightness), a* (green-reddish), and b* (brightness) (blue-yellowish). AAC: acetic acid collagen; LAC: lactic acid collagen; CAC: citric acid collagen. \\ \hline \end{tabular}
\end{table}