*4.1. Chemicals*

Sodium hydroxide (NaOH), acetic acid, sodium chloride (NaCl), hydrochloric acid (HCl), ethanol, and potassium bromide (KBr) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Pepsin from porcine stomach mucosa (EC 3.4.23.1; 1:3000 U) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Trypsin (EC 3.4.21.4, 1:250), dithiothreitol (DTT), and Coomassie brilliant blue R-250 were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Dual Color protein standard marker with MW of 37–250 kDa (Catalog No. 1610374), 4× Laemmli Sample Buffer (Catalog No. 1610747), 10× Tris/Glycine/SDS (Catalog No. 1610732) were purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). All reagents were used at an analytical grade unless otherwise specified.

### *4.2. Raw Materials and Pre-Treatment*

The skin of the blacktip reef shark (*Carcharhinus melanopterus*) was purchased from M/s. Yueqing Ocean Biological Health Care Product Co., Ltd. Zhejiang, China. The skins were washed thoroughly with tap-water and cut into small pieces, then stored in plastic bags in the freezer at −80 ◦C until further use. The skin pieces of blacktip reef shark were mixed with 0.1 M NaOH at a sample-to-solution ratio of 1:10 (*w*/*v*) at 4 ◦C with stirring for 24 h to remove water-soluble substances and non-collagenous protein, respectively, and the alkali solution was refreshed every 8 h. The samples were then repeatedly washed with distilled water until a neutral pH of washing water was obtained.

### *4.3. Preparation of Acid-Soluble and Pepsin-Soluble Collagen*

All procedures were carried out at 4 °C with stirring. Acid-soluble (ASC) and pepsinsoluble collagens (PSC) were extracted from blacktip reef shark skin according to our earlier method with some modifications [38]. For extraction of acid-soluble collagen, the cleaned skins were soaked with 0.5 M acetic acid in a ratio of 1:20 (*w*/*v*) for 48 h. The mixture was then centrifuged at 20,000× *g* for 30 min at 4 ◦C using a Himac CR 21G High-speed floor centrifuge (Hitachi, Tokyo, Japan).

At the same time, the centrifugation pellet from the first extraction was redissolved in 0.5 M acetic acid for second extraction for 48 h, and then the supernatant was combined with the earlier extraction. After centrifuge, the collagen was precipitated by mixing the supernatant with 1.0 M NaCl. The salting-out precipitates were redissolved in 10 volumes of 0.5 M acetic acid and dialyzed using dialysis membranes (MWCO: 10 kDa) against distilled water until a neutral pH was obtained. The dialyzed sample was lyophilized using a freeze-dryer (Labconco Freezone 2.5L, Kansas City, MO, USA), and concentrates were stored at −80 ◦C. The procedure for the extraction of pepsin soluble collagen was the same as the extraction of acid-soluble collagen by using 1% pepsin in 0.5 M of acetic acid. A detailed flow chart of the blacktip reef shark skin collagen preparation procedure is presented in Figure 7.

**Figure 7.** Schematic representation of steps involved in collagen extraction.

### *4.4. Molecular Pattern*

The molecular pattern of purified collagen was determined by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli's method [39]. The ASC and PSC samples were prepared with SDS to obtain 0.5 mg/mL and 1 mg/mL, respectively. The collagen samples were mixed with (3:1) 4 × Laemmli Sample Buffer (SDS, Tris-HCl, bromophenol blue, glycerol, and DTT) and oscillated slightly with a scroll oscillator (TianGen Biotech Co, Ltd., Beijing, China). The samples were boiled for 5 min and the boiled mixture was briefly centrifuged at 1890 g. The test samples and standard protein standard marker (MW ranging from 37 kDa to 250 kDa) (Bio-Rad Laboratories Inc., Hercules, CA, USA) were loaded onto 4.5% stacking polyacrylamide gel with 7.5% separating gel (Cat# PG112, EpiZyme Biotechnology, Shanghai, China). The electrophoresis unit was set at a constant voltage of 200V in order to efficiently separate the collagen samples using a mini-PROTEAN Tetra Cell (Bio-Rad Laboratories Inc., Richmond, CA, USA). After the electrophoresis, the gel was stained with 0.25% Coomassie brilliant blue R-250 solution for 30 min and discolored with the mixture of 20% (*v*/*v*) ethanol and 10% (*v*/*v*) acetic acid twice, each for 1 h until clear protein bands were observed. The protein bands were then captured using the gel documentation system (Clinx GenoSens 2100(T), Shanghai, China).

### *4.5. Amino Acid Composition*

The amino acid content of collagen samples was analyzed by using an amino acid analyzer (Hitachi LA-8080, Tokyo, Japan) [14]. The freeze-dried collagens were completely hydrolyzed in 6M HCl at 110 ◦C for 24 h. The excess amount of solvent was evaporated under the vacuum incubator until the dried sample then dissolved in distilled water and the drying process was repeated three to four times. In the end, the dried sample was dissolved with a minimum amount of sodium citrate buffer solution (pH 2.2) and filtered through a 0.45 nm hydrophilic membrane. The amino acid analyzer was calibrated with a standard reagent, and a positive control of all amino acids was run as a reference before analyzing the test sample. The retention time of each amino acid peak was equalized with the respective positive control amino acid peak and the content of amino acid is expressed as the number of residues/1000 residues.

### *4.6. UV Absorption*

The maximum absorption of collagen in the UV range was determined using a UV-vis spectrophotometer in order to confirm the purity and contamination of other proteins [33]. The ultraviolet absorption spectra of blacktip reef shark skin collagens were recorded by a spectrophotometer (MAPADA UV-3000PC, Shanghai, China). Freeze-dried collagen was dissolved in 0.5 M acetic acid and the absorption from 190 to 400 nm was measured at a scan speed of 2 nm/s with an interval of 1 nm. An aliquot of 0.5 M acetic acid was used as blank.

### *4.7. Fourier Transform Infrared Spectroscopy (FTIR)*

The structural and amide differences between ASC and PSC were determined by using the Spotlight 400 FT-IR Imaging System (PerkinElmer, Waltham, MA, USA) equipped with a deuterated triglycine sulfate detector. The 2 mg freeze-dried sample was mixed with dried KBr (100 mg) in order to make a 13 × 1 mm thin transparent disk by subjecting a pressure of approximately 5 × 10<sup>6</sup> Pa. The transparent disk was then placed in a sample holder in FTIR and the spectra in a range of 4000 to 600 cm<sup>−</sup>1, with automatic signal gain collected in 32 scans at a resolution of 2 cm<sup>−</sup>1. The absorption intensity of the peaks was calculated using the baseline method. The resultant spectra were analyzed using Origin Pro 2021 software. The secondary structures of the collagen were analyzed through the areas of 1600–1700 cm<sup>−</sup><sup>1</sup> in the amide I region using PeakFit Version 4.12 software (SeaSolve software Inc., Framingham, USA) and the Gaussian peak fitting algorithm. Finally, the secondary structure percentage was calculated by dividing the peak area of the secondary structure by the whole peak area of all the secondary structures.

### *4.8. Relative Solubility*

The relative solubility of collagen was determined by varying NaCl and pH by following our previous protocol [40]. The freeze-dried collagen sample (10 mg/mL) was prepared with 0.5 M acetic acid with gentle stirring at 4 ◦C for 12 h and used for solubility experiments.

### 4.8.1. Effect of pH

The collagen solution (5 mL) was transferred into a series of centrifuge tubes, adjusted to pH values ranging from 1 to 11 by the addition of the appropriate amount of 6 M NaOH or 6 M HCl. The resulting sample solution totaled 10 mL of distilled water. The solution was stirred gently for 30 min at 4 ◦C and centrifuged at 5000× *g* for 30 min. An aliquot (1 mL) of the supernatant was collected from each tube and the protein content was measured. The relative solubility of collagen was calculated compared with the pH rendering the highest solubility.

### 4.8.2. Effect of NaCl

The collagen solution (5 mL) was mixed with 5 mL of cold NaCl in acetic acid of various concentrations (0–12%, *w*/*v*) to obtain final concentrations of 1–6% ( *w*/*v*). The mixture was stirred gently at 4 ◦C for 30 min and centrifuged at 10,000× *g* for 30 min at 4 ◦C. The relative solubility was calculated compared with that of the salt concentration exhibiting the highest solubility.

### *4.9. Peptide Mapping*

The peptide mapping pattern of collagen against the proteolytic enzyme, trypsin, was determined by following the method of Li et al., with some modification [31]. The freeze-dried collagen sample was dissolved in 0.5 M acetic acid, pH 2.5, at a concentration of 1.5 mg/mL. In parallel, another set of collagen samples was dissolved in a 0.2 M sodium phosphate buffer, pH 7.8, at a concentration of 1.5 mg/mL. After the addition of trypsin with an enzyme/substrate ratio of 1/2 ( *w*/*w*) to collagen solutions, the reaction mixtures were incubated at 37 ◦C for 3 h (pH 2.5) and 3 min (pH 7.8), respectively. The SDS-PAGE sample buffer was added to the digestion samples, and the mixtures were boiled for 5 min to terminate the reaction. Using 4.5% stacking gel, 7.5% and 12.5% separating gels, SDS-PAGE was performed to separate peptides generated by the protease digestion and compared.

### *4.10. Microstructural Analysis*

The changes in the structural properties between ASC and PSC were determined by scanning electron microscopy (SEM). The freeze-dried collagen samples were pasted on an aluminum plate with conductive adhesive tape in the desired orientation and placed in an ion coater for the gold coating to increase the electrical conductivity. The metalized collagen samples on the SEM sample holder (20-s glow discharged carbon support adhesive films) were mounted and then introduced into the specimen chamber for analysis. The surface morphology of collagen was captured with different magnifications of 100 μm, 50 μm and 10μm by using SEM (Hitachi SU5000, Tokyo, Japan) at an accelerating voltage of 15 kV.

### *4.11. Atomic Force Microscope Analysis*

In order to evaluate the surface morphology, collagen fiber alignment, topography and superficial properties of the collagen, an atomic force microscope (AFM, Bruker Corporation, Billerica, MA, USA) was carried-out with collagen samples by following the standard operating procedure [41]. SNL-series silicon 1 cantilevers with a normal spring cantilever of 0.35 N/m were used. The nominal AFM tip radius is 2 nm and the maximized tip radius is 12 nm. 10 μL collagen aqueous solution was cast on freshly cleaved muscovite mica and was allowed to dry in ambient air for 60 min. Images were obtained using a multimode scanning probe microscope with NanoScope Analysis software (version 1.80, Bruker Corporation, Billerica, MA, USA) operating in the tapping mode, in air, at room temperature. Surface images were acquired at fixed resolution (512 × 512 data points) with a scan rate of 1.0 Hz.

### *4.12. Statistical Analysis*

Each experiment was replicated three times. Data were expressed as mean ± standard deviation and mentioned in the figure legends. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Inc, San Diego, CA, USA) using two-way ANOVA with Fisher's LSD test multiple comparison analysis. The values identified as outliers were excluded from statistical analysis. Results were considered statistically significant if the *p*-value < 0.05.
