*4.1. Materials*

Type I collagen from rat tail and protein markers (26,634) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sodium dodecyl sulphate (SDS), Coomassie Brilliant Blue R-250, and N,N,N,N-tetramethylethylenediamine (TEMED) were obtained from Bio-Rad Laboratories (Hercules, CA, USA). HaCaT cell line (Cat No. CBP60331) and MC3T3-E1 cell line (Cat No. CBP60946) were provided by Cobioer (Nanjing, Chian). All chemicals were of analytical grade.

### *4.2. Preparation of Collagen*

Collagen extraction from lizardfish scales was in accordance with the method of Chen et al. (2019) [29] with slight modifications. Lizardfish scales were purchased from a food processing factory in Zhangzhou, Fujian Province, China. The scales were cleaned several times with water to remove bones, spines, shellfish, shrimp feet, and offal, and then dried naturally indoors and stored at −20 ◦C until use. To remove noncollagenous proteins and pigments from the scales, the scales were soaked in 0.1 M NaOH at a ratio of 1:8 (*w*/*v*) at 4 ◦C. The mixture was continuously stirred for 12 h (EUROSTAR 20 digital, IKA, Germany), with 0.1 M NaOH solution being changed every 6 h. The scales residues were washed with cold distilled water until the pH was neutral. Thereafter, the scales residues were treated with a ratio of 1:10 (*w*/*v*) of 0.5 M Na2EDTA (pH 7.5) for 24 h under stirring, changing the solution at an interval of 6 h. The decalcified materials were washed with cold distilled water to achieve the neutral pH and dried, followed by crushing under liquid nitrogen. The samples were then stored at −20 ◦C until further processing of collagen extraction.

Pretreated scales' samples were extracted with 0.5 M acetic acid at ratio of 1:10 (*w*/*v*) for 24 h under stirring to obtain ASC, while PSC was obtained by extracting with 0.5 M acetic acid (1:10, *w*/*v*) containing 1% (pepsin 1:3000) pepsin for 24 h. The two suspensions were centrifuged at 14,334× *g* for 30 min at 4 ◦C using an Avanti J-26 XP centrifuge (Beckman Coulter, Inc., Brea, CA, USA), and the collagen in the supernatant was precipitated by adding NaCl to the final concentration of 2.5 M. After stirring for 2 h, the precipitates were collected by centrifugation at 14,334× *g* for 30 min at 4 ◦C. The precipitates were dissolved in 0.5 M acetic acid at a ratio of 1:20 (*w*/*v*) and dialyzed (molecular weight cutoff: 10 kDa, MD 77 MM, Viskase, Lombard, IL, USA) against 40 volumes of 0.1 M acetic acid for 24 h, and then dialyzed against 40 volumes of cold distilled water for 48 h; the dialysis water was changed every 6 h. All of the procedures were carried at 4 ◦C. The dialyzed solution was freeze-dried (Telstar, lyoobeta-25, Spain) and stored at −40 ◦C.

The yield of collagen was calculated using the following equation:

$$\text{Yield } (\%) = \frac{\text{m}\_1}{\text{m}\_2} \times 100 \tag{1}$$

where m1 is the weight of lyophilized collagen, and m2 is the dry scales weight after pretreatment.

### *4.3. SDS-PAGE Characterization*

The SDS-PAGE of the sample was conducted in accordance with the method of Laemmli (1970) [51] with slight modifications. The samples (2 mg/mL) were dissolved in cold distilled water and mixed at a 4:1 *v*/*v* ratio with sample loading buffer (277.8 mM Tris-HCl, pH 6.8, 44.4% (*v*/*v*) glycerol, 4.4% SDS, and 0.02% bromophenol blue), followed by boiling for 10 min. Then, 10 μL of the samples' solution was loaded onto a gel consisting of 7.5% separating gel and 3% stacking gel at a constant voltage of 110 V for electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis for 90 m, the gel was soaked using a solution consisting of 50% (*v*/*v*) methanol and 10% (*v*/*v*) acetic acid followed by staining with 0.125% Coomassie Brilliant Blue R-250 that contained 50% (*v*/*v*) methanol and 10% (*v*/*v*) acetic acid. The gel was finally destained with a mixture of 50% (*v*/*v*) ethanol and 10% (*v*/*v*) acetic acid for 30 m. The Marker of 46,634 was used to estimate the molecular weight of the collagen, and the type I collagen from rat tail was used as standard.

### *4.4. Spectral Characterization*

### 4.4.1. UV Spectrum

The lyophilized collagen was dissolved in 0.5 M acetic acid to produce a 1 mg/mL sample solution, followed by centrifugation at 9729× *g* for 5 min at 4 ◦C (Neofuge 15R, Shanghai Lishen Scientific Equipment Co., Ltd., Shanghai, China). The supernatant was analyzed by UV-visible spectrophotometer (UV-2550 Spectrophotometer, Shimadzu, Japan) at a wavelength range of 600–190 nm with a scan speed of 400 nm min−<sup>1</sup> with a data interval of 1 nm per point. The baseline was set with 0.5 M acetic acid.
