2.1.1. Effect of Enzyme Concentration on the Extraction Yield of PSC

In the previous studies, collagens were often extracted by using acid extraction and enzymatic extraction [15,16]. However, the acid solubilisation process gives a low yield of collagen. Since pepsin or papain is able to cleave peptides in the telopeptide region of collagen and the helical arrangement can exist in the PSC or papain digested collagen. However, there are some other proteins (above 97.4 kDa) in the papain digested collagen when compared to the PSC [21]. So, pepsin was chosen for extracting collagen in this study. Then, different pepsin concentrations (800, 1200, 1600, 2000 and 2400 U/g) were used to investigate the effect of pepsin concentration on the extracting yield of PSC. The other two extraction parameters were set as follows: solid-liquid ratio was 1:45 and hydrolysis time was 8 h in 0.5 M acetic acid buffer. As shown in Figure 1a, the PSC extraction yield significantly increased from 66.35% to 79.93% when pepsin concentration varied from 800 to 1200 U/g and then slightly increased when pepsin concentration exceeded 1200 U/g. Considering the higher-cost industrial extraction process, the amount of 1200 U/g pepsin was used for further optimization.

**Figure 1.** Effects of enzyme concentration (**a**), liquid-solid ratio (**b**) and hydrolysis time (**c**) on extraction yield of collagen from *Nibea japonica* skin.
