*2.2. Optimization of Extraction Parameters of PSC Using RSM*

#### 2.2.1. Response Surface Analysis

After screening, RSM using BBD was used to obtain the optimal levels of the three above factors that significantly affected the yield of PSC. The experimental design and results are shown in Table 1 On the basis of the regression analysis of the data in Table 1, the effects of these three factors on the extraction yield of PSC were predicted by using a second-order polynomial function as follows: *Y* = 83.73 + 3.53*X*<sup>1</sup> + 1.47*X*<sup>2</sup> + 1.78*X*<sup>3</sup> − 0.11*X*1*X*<sup>2</sup> − 0.59*X*1*X*<sup>3</sup> − 0.80*X*2*X*<sup>3</sup> − 3.68*X*<sup>1</sup> <sup>2</sup> − 2.03*X*<sup>2</sup> <sup>2</sup> − 1.89*X*<sup>3</sup> 2 (where *Y* was the extraction yield of PSC, and *X*1, *X*2, *X*<sup>3</sup> were the pepsin concentration, solid-liquid ratio and hydrolysis time, respectively).


**Table 1.** The Box-Behnken design and the response for the extraction yield of pepsin-solubilised collagen (PSC).

In order to determine the significance of the quadratic model, the analysis of variance (ANOVA) was performed and the results are shown in Table 2. As suggested by the model *F* value and a low probability value (*p* = 0.0001), it was obvious that the model was highly significant. The lack of fit *F* value in this model was about 6.13 and it suggested that the lack of fit was not significant relative to the pure error. The determination coefficient (*R*<sup>2</sup> = 0.9920) by ANOVA of this model and the adjusted determination coefficient (Adj *R*<sup>2</sup> = 0.9777) also indicated that the model was highly significant. So, this model was selected in this study for optimizing.


**Table 2.** Analysis of variance of regression model.

Furthermore, three-dimensional response surfaces and contour plots were generated from the model equation to visualize the relationship between the extraction yield of PSC and extraction factors (Figure 2). It also show the optimal levels of each component required for the extraction of PSC (Figure 3). These three-dimensional response surfaces and contour plots provided a visual interpretation of the mutual interactions between two factors. The maximum predicted yield of PSC was 85.03% under the following conditions: concentration of enzyme was 1389 U/g, solid-liquid ratio was 1:57 and hydrolysis time was 8.67 h.

**Figure 2.** *Cont.*

**Figure 2.** Three-dimensional response surface plots (**left**) and two-dimensional contour plots (**right**) showing the effects of (**a**) enzyme concentration (*X*1) vs. liquid-solid ratio (*X*2), (**b**) enzyme concentration (*X*1) vs. hydrolysis time (*X*3) and (**c**) liquid-solid ratio (*X*2) vs. hydrolysis time (*X*3) on extraction yield of collagen from *Nibea japonica* skin.

**Figure 3.** Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of PSC from *Nibea japonica* skin. M: Protein molecular weight marker; Lane 1–3: Purified PSC from *Nibea japonica* skin.

#### 2.2.2. Validation of the Models

Three additional experiments were performed in order to verify the predicted yield under the optimal extraction conditions. The mean value of PSC yield was 84.85%, which was in excellent agreement with the predicted value, under the similar conditions.

#### *2.3. SDS-PAGE Analysis*

The protein patterns of PSC from *Nibea japonica* skin were analyzed by SDS-PAGE (Figure 3). As shown in Figure 3, PSC from *Nibea japonica* skin consisted of two α1-chains and one α2-chain. The β and γ chains as well as the cross-linked constituents were also observed in this study (Figure 3). PSC extracted from *Nibea japonica* skin may have the structure of (α1)2α2, which was classified as Type I collagen. Our results were consistent with the collagens from other marine fish skins, such as

PSC from *Aluterus monocerous* [2], PSC from *Scomberomorous niphonius* [15] and PSC from *Istiophorus platypterus* [22].

#### *2.4. Amino Acid Composition of PSC*

The amino acid composition of PSC from *Nibea japonica* skin was determined and the results are shown in Table 3 and compared with collagen from calf skin, type I collagen from porcine skin and human [23–25]. The most abundant amino acids found in PSC from *Nibea japonica* skin were glycine (Gly), alanine (Ala), proline (Pro) and hydroxyproline (Hyp). In this study, Gly was found to be the major amino acid in PSC (348 residues/1000 residues), the result is accordance with the (Gly-Xaa-Yaa) n repeat structure in all collagen molecules. It is known that the Xaa and Yaa positions can be occupied by any other amino acid, but the most common residue for Xaa is Pro and for Yaa is Hyp [26], forming the most common triplet repeats that found in most collagens (Gly-Pro-Hyp) n [22]. The Pro and Hyp contents of the PSC was 116 residues/1000 residues and 75 residues/1000 residues, respectively, which is similar to that of PSC from skin of *Aluterus monocerous* [2]. The rate of proline hydroxylation was about 39.3% for PSC from *Nibea japonica* skin. There were no tryptophan and cysteine residues in the PSC from *Nibea japonica* skin.



### *2.5. UV-Visible Spectroscopy*

It is known that collagen has a single absorption peak at 230 nm because of its triple helical structure, so UV-visible spectroscopy of collagen can be used to evaluate its purity [14,27]. As shown in Figure 4, PSC extracted from *Nibea japonica* skin showed a single absorption peak at 230 nm. Our result was similar to the collagen that has been isolated from other fish species. It is also necessary to point out that no any other obvious peaks were found at 280–300 nm while other proteins usually have absorption peaks at 280 nm. This is because the tyrosine content in collagen was very low. Finally, the UV-visible spectroscopy of PSC indicated that the extracted proteins using pepsin extraction was collagen and it also shown that pepsin extraction was the efficient methods to obtain purity collagens.

**Figure 4.** UV-visible spectroscopy of PSC from *Nibea japonica* skin.

#### *2.6. Fourier Transforms Infrared Spetroscopy (FTIR) Analysis*

The FTIR spectra of PSC from *Nibea japonica* skin is shown in Figure 5. These peaks correspond to five main amide bonds (amide A, B, I, II and III). The amide A bands of PSC was measured at 3305.90 cm−1. The value is associated with N-H stretching frequency and indicate the presence of hydrogen bonds. The free N-H frequency vibration occurs at 3400–3440 cm−<sup>1</sup> and shifts lower to 3300 cm−<sup>1</sup> [28]. The amide B band of PSC was measured at 2928.38 cm−1, which was consistent with asymmetrical stretch of CH2 [29]. The amide I band of PSC was detected at 1641.35 cm−1, fitting well with the range of 1600–1700 cm−<sup>1</sup> for general amide I band position. The amide II band of PSC was measured at 1550.26 cm−1, fitting well with the range of amide II band position (1550–1600 cm<sup>−</sup>1). Finally, the amide III band of PSC was measured at 1240.47 cm−1, which indicated the helical arrangement existed in the PSC from *Nibea japonica* skin [29,30].

**Figure 5.** FTIR analysis of PSC from *Nibea japonica* skin.

### *2.7. Effects of pH and Sodium Chloride on PSC Solubility*

The effects of pH and sodium chloride on the solubility of PSC from *Nibea japonica* skin were also investigated in the present study. As shown in Figure 6a, the PSC was dissolved in the acidic pH range of 1.0–4.0. The decrease in solubility was observed in the pH range of 5.0–7.0 and the dissolved protein was found to be deposited in this pH range. However, the slight increase in solubility was shown in the pH range of 8.0–10.0. Our results were consistent with the collagens from the skin of *Spanish* *mackerel* [15], bone and skin of *Hypophthalmichthys molitrix* [14] and skin of *Ictalurus punctatus* [31]. As shown in Figure 6b, all PSCs had a slight decrease in solubility with the concentrations of sodium chloride lower than 2% and drastic decrease was observed with the concentrations of sodium chloride higher than 2%. Similar reports were reported from the skin of *Ictalurus punctatus* [31] and skin of *Aluterus monocerous* [2].

**Figure 6.** Effects of pH (**a**) and sodium chloride (**b**) on PSC solubility.
