## 3 Results and Discussion
### Determining the Optimum Extraction Conditions  
Extraction processes such as extraction pH, temperature and time strongly affect the yield and quality of gelatin [(9)]. The effects of extraction conditions were assessed based on the intensities of the \(\alpha\)-, \(\beta\)-, and \(\gamma\)-bands of gelatin in SDS-PAGE (Figure 2 and Figure S2) and the yields (Table 2) for type A and type B gelatins. Gelatin is a denatured, uncoiled form of collagen but sometimes contains hydrolyzed collagen (peptides) and other substances simultaneously extracted from the material tissues. Therefore, the intensities of \(\alpha\)-, \(\beta\)-, and \(\gamma\)-bands in SDS-PAGE reflect amount of un-hydrolyzed, complete form of gelatin, which shows gelatin purity of the extracts.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Gelatin**} & \multicolumn{2}{c}{**Pretreatments**} & \multicolumn{3}{c}{**Extraction Conditions**} & \multirow{2}{*}{**Yields (\%)**} \\ \cline{2-2} \cline{4-6}  & **Reagents** & **Time (h)** & **pH** & **Temperature (\({}^{\circ}\)C)** & **Time (h)** & \\ \hline \multirow{3}{*}{Type A} & \multirow{3}{*}{0.05 M HCl} & 1 & & & 6.81 \(\pm\) 0.56 \\  & & 3 & 7 & 65 & 1 & 5.60 \(\pm\) 0.40 \\  & & 6 & & & 3.81 \(\pm\) 0.32 \({}^{*}\) \\ \hline \multirow{3}{*}{Type B} & \multirow{3}{*}{0.1 M} & 1 & & & & 10.56 \(\pm\) 0.39 \\  & & 3 & 7 & 65 & 1 & 9.79 \(\pm\) 0.68 \\  & & 6 & & & & 8.92 \(\pm\) 0.52 \\ \hline \multirow{3}{*}{Combined} & \multirow{3}{*}{0.1 M} & 1 & & & & \\  & NaOH & & 7 & 65 & 1 & 5.38 \(\pm\) 0.60 \\ \cline{1-1}  & & 0.05 M HCl & 1 & & & \\ \hline Control & - & - & 7 & 65 & 1 & 3.97 \(\pm\) 0.14 \\ \hline \hline \end{tabular} All values are the mean \(\pm\) standard error of three replicates. Control samples were extracted without any pretreatments. * Significantly different (\(p<0.05\)) compared with 1 and 3 h.  
\end{table}
Table 1: Yields (%, dry weight gelatin/wet weight sample) of gelatins extracted from the head skin of Kalamtra sturgeon after different pretreatment conditions.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Factors**} & \multirow{2}{*}{**Gelatins**} & \multicolumn{2}{c}{**Extraction Conditions**} & \multirow{2}{*}{**Yields (\%)**} \\ \cline{3-3} \cline{5-5}  & & **pH** & **Temperature (\({}^{\circ}\)C)** & **Time (h)** & \\ \hline \multirow{8}{*}{Extraction pH} & 2 & & & 5.22 \(\pm\) 0.17 \({}^{\text{b}}\) \\  & 6 & & & 5.04 \(\pm\) 0.22 \({}^{\text{b}}\) \\ \cline{3-5}  & Type A & 7 & 65 & 1 & 6.99 \(\pm\) 0.54 \({}^{\text{a,b}}\) \\ \cline{3-5}  & 8 & & & 5.24 \(\pm\) 0.79 \({}^{\text{a,b}}\) \\ \cline{3-5}  & 12 & & & 7.74 \(\pm\) 0.66 \({}^{\text{a}}\) \\ \cline{3-5}  & 2 & & & 7.25 \(\pm\) 0.25 \({}^{\text{b}}\) \\ \cline{3-5}  & 6 & & & 8.29 \(\pm\) 0.41 \({}^{\text{a,b}}\) \\ \cline{3-5}  & Type B & 7 & & 8.17 \(\pm\) 0.25 \({}^{\text{a,b}}\) \\ \cline{3-5}  & Type B & 8 & 65 & 1 & 8.41 \(\pm\) 0.28 \({}^{\text{a,b}}\) \\ \cline{3-5}  & 9 & & & 8.23 \(\pm\) 0.53 \({}^{\text{a,b}}\) \\ \cline{3-5}  & 10 & & & 9.05 \(\pm\) 1.15 \({}^{\text{a,b}}\) \\ \cline{3-5}  & 12 & & & 10.60 \(\pm\) 0.60 \({}^{\text{a}}\) \\ \hline \hline \end{tabular}
\end{table}
Table 2: Yields (%, dry weight gelatin/wet weight sample) of gelatins extracted from the head skin of Kalamtra surgeon using different extraction pH, temperature (\({}^{\circ}\)C), and time (h).  
Figure 2: Effects of the extraction pH (2–12) (**a,b**) and extraction temperature (35 \({}^{\circ}\)C–65 \({}^{\circ}\)C) and time (0.5 h–3 h) (**c,d**) on the intensities of \(\alpha\)-, \(\beta\)-, \(\gamma\)-, and low molecular-weight (<100 k) bands of gelatin in the extracts in SDS-PAGE (7.5% gel). Gelatins were extracted from the head skin at different conditions after 0.05 M HCl for 1 h (Type A gelatin) or 0.1 M NaOH for 1 h (Type B gelatin). Each lane contained 1 \(\mu\)L extract. Columns and bars represent means and standard errors of three replicates, respectively. Different letters in each band indicate a significant difference (\(p<0.05\)).