*3.1. Materials and Chemical Reagent*

*Nibea japonica* skins were provided by Zhejiang Marine Fisheries Research Institution (Zhoushan, China). The age of these fish was about one year and the average weight is about 0.4–0.5 kg. The fish skins were thawed at 4 ◦C and the residues under skins were removed. The cleaned fish skins were cut into small pieces and then stored at −20 ◦C until used for extracting of collagen. Pepsin was purchased from YTHX Biotechnology Co., Ltd. (Beijing, China). L-hydroxyproline and chloramine T trihydrate were purchased from Aladdin (Shanghai, China). All other reagents used were analytical grade.

#### *3.2. Extraction of PSC from Nibea japonica Skin*

The PSC from *Nibea japonica* skin was extracted according to the previous methods with slight modification [11,32]. Ten grams of fish skins were weighed precisely and in order to remove other proteins, the fish skins were then treated with 10 volumes (*v*/*w*) of 0.1 M NaOH for 24 h at 4 ◦C with continuous stirring. The alkali-treated fish skins were neutralized by washing repeatedly with pre-cooling distilled water and extracted by 1200 U/g pepsin in a ratio of 1:55 (*w*/*v*) for 10 h at 4 ◦C in 0.5 M acetic acid. The extractions were then centrifuged at 12,000 rpm for 10 min at 4 ◦C and the supernatants were collected. The supernatants were dialyzed against cold distilled water until the neutral pH was reached by using a dialysis bag with molecular weight cut-off of 25 kDa at 4 ◦C with a gentle stirring. The final solution was then lyophilized using d freeze dryer (ALPHA 1-2 LD plus, Christ, Germany). The Hyp content in the PSC or fish skins was calculated according to the previous study [33] and the extraction yield of PSC was calculated using the equation as follows:

$$\text{PSC extraction yield} \left( \% \right) = \frac{\text{Hydroxyline content in PSC}}{\text{Hydroxyline content in fish skin}} \times 100\% \tag{1}$$

#### *3.3. Experimental Design and Statistical Analysis*

Single factor experiments were carried out for establishing the preliminary range of the extraction variables, such as pepsin concentration, solid-liquid ratio and hydrolysis time. Then, RSM and BBD were applied to optimize the three extraction parameters for improving the yield of PSC from *Nibea japonica* skin. The range and levels of the variables investigated in the present study were given in Table 4.


**Table 4.** Independent factors and their levels used in the response surface design.

RSM with BBD was performed to obtain the optimum conditions for PSC extraction. For statistical calculations, the factors were coded according the equation as follows:

$$\chi\_i = \frac{X\_i - X\_o}{\Delta X} \tag{2}$$

where, χ*<sup>i</sup>* is the coded value of the independent factor, *Xi* is the actual value of the independent factor, *X*<sup>o</sup> is the actual value of *Xi* at the center point and Δ*X* is the step change value. As shown in Table 2, the Box-Behnken design in the experiment design consists of 15 experimental points and the data from experiment design were explained by multiple regressions to fit the second-order polynomial equation as follows:

$$\mathcal{Y} = \mathfrak{G}\_{\diamond} + \sum \mathfrak{B}\_{i} X\_{i} + \sum \mathfrak{B}\_{ij} X\_{i} X\_{j} + \sum \mathfrak{B}\_{ii} X\_{i} X\_{i} \tag{3}$$

where, *Y* is the dependent variable (PSC yield, %); β<sup>o</sup> is the intercept term, β*<sup>i</sup>* is the linear regression coefficient, β*ii* is the aquared coefficient and β*ij* is the interaction coefficient. *Xi* and *Xj* are levels of the independent variables. Each experiment design was determined in triplicate and the data were analyzed using the software Design-Expert 8.0.5 (State-Ease Inc., Minneapolis, MN, USA).

#### *3.4. SDS-PAGE Analysis*

The PSC samples from *Nibea japonica* skin were then analyzed by using SDS-PAGE according to the method described by Tang et al. [34]. The PSC samples were firstly dissolved in 0.5 M acetic acid and mixed with the loading buffer. Electrophoresis was performed on 7.5% gels and high protein molecular weight marker (Takara, Dalian, China) was used to estimate the molecular weight of PSC.
