## 2 Materials and Methods
### Extraction Yield, Hydroxproline Content, pH, Colour and Thermal Transition Measurement  
Table 2 shows that the collagen yields of needlefish skin samples treated with different organic acids AAC, LAC, and CAC, all of which were significantly different at (\(p<0.05\)). AAC produced the most collagen (3.13%), followed by CAC (1.03%) and LAC (0.56%). Due to the use of different acids during the extraction, the interaction of aldehydes with lysine and hydroxylin at the telopeptide helix region may account for differences in collagen yield among AAC, LAC, and CAC [15]. This may be due to the fact that various types of acids were employed to extract collagen from fish skin, and the pH of the combination was not kept at the same level as it was at the beginning of the process. This resulted in a shift in the amount of collagen that was extracted from the fish skin. A major reason that organic acids are used so frequently in collagen production studies is that the acid-collagen molecule interaction can split collagen crosslinks and is able to boost extraction efficiency. For instance, homogenization, mixing, acid dissolution, and extraction time can affect collagen yield [7]. Furthermore, based on previous studies, AAC should extract more collagen than CAC because the acid employed has a high capacity for collagen extraction [33]. This may be connected to the excess salt left behind after cellulose-tube dialysis, which increases collagen mass. This is clear when CAC has a rough surface and removable crystals. In addition, [PERSON] and [PERSON] [34] reported that synthesised collagen from Baltic cod skin (_Gadus morhua_) using various acids produced different percentages of collagen, with the greatest collagen content formed from acetic acid and lactic acid, with a maximum yield of 90%, followed by citric acid with a yield of 60%. In contrast, the production of collagen with hydrochloric acid yielded the lowest percentage (18%). The yield of collagen extracted from marine fish skin has been reported in ranges from 49.8% to 51.4% [35]. However, [PERSON] et al. [36] revealed a final yield of eel skin collagen was around 4.2%. In collagen extraction, the swelling percentage is as significant as the collagen yield content. This is because collagen can swell when the pH is decreased to 4 or elevated to 10 due to its inability to connect between portions of its molecular structure [37]. According to Table 1, there was a significant difference at (\(p<0.05\)), indicating that the largest swelling percentage for CAA (226.37%), followed by CAC (210.41%), and LAC (198.37%). This significant swelling percentage indicates that the weight of the needlefish skin expanded fourfold from its original weight of 50 g. Furthermore, swelling is an important factor to consider in collagen extraction because it can impact the ability of collagen's internal molecular structure to connect to other proteins and enhance protein fragmentation by breaking non-covalent interactions [38]. Furthermore, according to Table 1, the value of \begin{table}
\begin{tabular}{c c} \hline \hline
**Composition (\%)** & **Skins Sample** \\ \hline Moisture & 61.65 \(\pm\) 1.15 d \\ Protein & 27.39 \(\pm\) 0.08 c \\ Lipid & 8.59 \(\pm\) 0.07 b \\ Ash & 2.16 \(\pm\) 0.03 a \\ \hline \hline \end{tabular} Values are reported as mean \(\pm\) SD (n = 3). Different lowercase letters indicate significant difference (\(p<0.05\)).  
\end{table} 
Table 1: Proximate composition of needlefish skin. hydroxproline content for all collagens demonstrated a significant difference (\(p<0.05\)). AAC had a slightly higher hydroxyproline content of 81.69 mg/g, than LAC (79.07 mg/g) and CAC (79.95 mg/g). [PERSON] et al. [39] determined the hydroxyproline concentration in the skin and bones of a giant eye fish (_Priaanthus tayenus_) to be 58.5 mg/g and 42.4 mg/g, respectively. Additionally, hydroxyproline is an amino acid marker that supports collagen's triple helical structure and a substance used to quantify tissue collagen [40].