## 3 Results and Discussion
### Decalcification of Extracted Residues  
After extracting the gelatin from the mixed tissues of the Kalamtraurgeon, the residues were composed of a mixture of semi-soft (small cartilage pieces) and hard tissues (bones and scales). Decalcification methods for the re-extraction of gelatin were investigated. The \(\alpha\)- and \(\beta\)-bands of gelatin were significantly more intense after decalcifying the extracted residues with 0.05 M HCl for 3 h for both types of gelatin (Figure 3a,b and Figure S3a,b). The yields for both types of gelatin were also higher in the 3 h decalcified groups (1.64% in type A and 1.59% in type B, Table 3). In SDS-PAGE, no low molecular-weight bands were observed in either type of gelatin in the 3 h groups (Figure S3a,b); thus, gelatin did not seem to break down under this treatment. Thus, 3 h is the best decalcification period.  
After re-extraction, the re-extracted residues were decalcified for 3 h and gelatin was extracted again. Although the yield was similar to the re-extracted samples, SDS-PAGE showed faint gelatin bands in both types of gelatin (data not shown). Thus, gelatin from the re-extracted residues was not suitable for future studies and applications.  
The optimized methods of tissue separation, pretreatment, extraction, decalcification, and re-extraction of type A and type B gelatin from the mixed tissues of Kalamtra sturgeon are summarized in Figure 4.  
Figure 3: Effects of the decalcification period on the intensities of \(\alpha\)-, \(\beta\)-, \(\gamma\)-, and low molecular-weight (<100 k) bands of gelatin in the re-extracts in SDS-PAGE (7.5% gel). Gelatins were re-extracted from the Type-A extracted residues (**a**) and Type-B extracted residues (**b**) of the mixed tissue from Kalamtra head after the decalcification in 0.05 M HCl. Control groups were re-extracted without decalcification. Each lane contained 3 \(\mu\)L extract. Columns and bars represent means and standard errors of three replicates, respectively. Different letters in each band indicate a significant difference (\(p<0.05\)).  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \multirow{2}{*}{**Gelatin**} & \multirow{2}{*}{**Decalcification**} & \multirow{2}{*}{**Decalcification**} & \multicolumn{3}{c}{**Extraction Conditions**} & \multirow{2}{*}{**Yields (\%)**} \\ \cline{3-3} \cline{5-6}  & & **Time (h)** & & & **pH** & **Temperature (\({}^{\circ}\)C)** & **Time (h)** \\ \hline \multirow{4}{*}{Type A} & \multicolumn{3}{c}{Control} & \multirow{4}{*}{35} & \multirow{4}{*}{3} & \(1.10\pm 0.02\) \\  & & 1 & & & \(1.53\pm 0.15\) \\ \cline{1-1}  & & 3 & & & \(1.64\pm 0.03\)\({}^{*}\) \\ \cline{1-1}  & & 6 & & & \(1.31\pm 0.17\) \\ \hline \multirow{4}{*}{Type B} & \multicolumn{3}{c}{Control} & \multirow{4}{*}{8} & \multirow{4}{*}{50} & \(0.95\pm 0.03\) \\  & & 1 & & & \(1.28\pm 0.13\) \\ \cline{1-1}  & & 3 & & & \(1.59\pm 0.03\)\({}^{*}\) \\ \cline{1-1}  & & 6 & & & \(1.26\pm 0.08\) \\ \hline \end{tabular} All values are the mean \(\pm\) standard error of three replicates. Control samples were extracted without decalcification.  
\({}^{*}\) Significantly different from control group.  
\end{table}
Table 3: Yields (%, dry weight gelatin/wet weight sample) of gelatins after decalcification of extracted residues from the mixed tissue of the Kalamtra sturgeon head using 0.05 M HCl at different times.