## 3 Results and Discussion
### Determining the Optimum Extraction Conditions
#### 3.3.2 Extraction Temperature and Time  
In SDS-PAGE, a slight tendency towards dominant gelatin bands, especially in \(\beta\)-bands, were observed at 35 \({}^{\circ}\)C compared to 50 and 65 \({}^{\circ}\)C for type A gelatin (Figure 2c and Figure S2c). Effects of extraction time were small, except for the extraction at 65 \({}^{\circ}\)C, at which 3 h extraction clearly decreased the intensities of gelatin bands (Figure 2c and Figure S2c). The low molecular-weight bands were obvious when gelatin was extracted at 65 \({}^{\circ}\)C (Figure 2c and Figure S2c), suggesting that the quality of type A gelatin was low when extracted at this temperature.  
In type B gelatin extraction, a tendency of stronger gelatin bands was observed at 50 \({}^{\circ}\)C (Figure 2d and Figure S2d). Moreover, the band intensities showed a tendency to increase as the extraction time extended at this temperature (Figure 2d and Figure S2d). The low molecular-weight bands were observed at all the examined conditions in type B gelatin extraction (Figure 2d and Figure S2d). The variation of extraction temperature between type A and type B gelatins might be due to pretreatment effects; the acid-pretreatment increases H+ that promote water access to the tissues and evokes better swelling of the tissues [20], resulting in easier gelatin extraction at lower temperatures than alkali-pretreatment.  
When the extraction temperature increased from 35 to 65 \({}^{\circ}\)C, the impact on gelatin yield was minimal (Table 2). However, slightly higher yields were obtained at lower temperatures for type A gelatin, and at higher temperatures for type B gelatin. This difference might be attributed to the effects of acid and alkali pretreatments. Compared to the alkali-pretreatment, the acid-pretreatment increases the loss and breakdown of collagen molecules, even at low concentrations and low temperature [14]. This may affect the extractability of gelatin at lower temperatures. The broken collagen fragments with non-collagenous fractions were probably absorbed into the acid solution during pretreatment; consequently, type A gelatin contained fewer low molecular-weight bands in SDS-PAGE (Figure S2c,d) and showed lower yields compared to type B gelatin.  
Yields tended to increase when extraction time increased for both types of gelatin. The optimum extraction temperature and extraction time were 35 \({}^{\circ}\)C for 3 h for type A gelatin and 50 \({}^{\circ}\)C for 1 h for type B gelatin, in which relatively higher yields and stronger gelatin bands were recorded. Higher yields but weaker bands of gelatin, suggesting lower gelatin quality such as lower intensity of \(\alpha\)-, \(\beta\)- and \(\gamma\)-bands as well as higher content of low molecular-weight bands, were obtained when extraction temperature and time were increased in squid [21] and the skin of featherback [22].  
Based on these experiments, the best extraction conditions were: (1) for type A gelatin, DW (_w/v_ 1:5) at 35 \({}^{\circ}\)C, pH 7 when stirring at 200 rpm for 3 h, and (2) for type B gelatin, DW (_w/v_ 1:5) at 50 \({}^{\circ}\)C, pH 8 when stirring at 200 rpm for 1 h.