**2. Results**

### *2.1. Collagen Yield*

The collagen from lizardfish scales was prepared using acid extraction and enzymatic extraction separately. The yield of ASC and PSC was 4.2 ± 0.2% (based on a dry weight basis) and 4.7 ± 0.1% (dry weight), respectively.

### *2.2. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)*

The electrophoretic patterns of ASC and PSC from lizardfish scales are illustrated in Figure 1. It is clear that ASC and PSC show similar electrophoretic patterns as both consist mainly of two different types of α-chains (α1 and α2) and dimeric β-chains. The molecular weight of collagen was analyzed using Quantity One 4.6.0 software (Bio-Rad Laboratories, Hercules, CA, USA); we found that the molecular weight of ASC (α1-MW, 137 kDa; α2-MW, 127 kDa) was slightly higher than that of PSC (α1-MW, 135 kDa; α2-MW, 123 kDa), which can be attributed to the removal of telopeptide regions of the PSC [21]. The protein patterns of ASC and PSC were similar to those of the collagen obtained from tilapia skin [18] and Pacific cod skin [22]. Although pepsin removed the cross-link-containing telopeptide, the electrophoresis patterns showed that PSC contained a higher intensity of β-chains than ASC, indicating that PSC has high molecular cross-linkages [23,24]. Moreover, the ratio of α1 and α2 was calculated by Image J software (VERSION 1.8.0, National Institute of Mental Health, Bethesda, MD, USA); specifically, the ratios of α1 and α2 for ASC and PSC were 1.86 and 2.23, respectively, both close to 2:1, implying that ASC and PSC extracted from lizardfish scales are type I collagen ([α1]2α2) [25].

**Figure 1.** SDS-PAGE patterns of ASC and PSC from lizardfish scales. Lane 1: Marker standard; Lane 2: PSC; Lane 3: ASC. The experiment was conducted only once (n = 1).

### *2.3. Spectroscopy Characterization*

### 2.3.1. UV Absorption Spectrum

Generally, collagen has a maximum absorption peak in 210–240 nm range, which is attributed to the presence of C=O, –COOH, and CONH2 groups in the polypeptide chains of collagen [23]. The UV absorption spectra of lizardfish scales collagen are shown in Figure 2a, namely, ASC and PSC showed sharp and intense maximum absorption peaks at 235 nm and 236 nm, respectively, which is consistent with the UV absorption characteristics of type I collagen [25]. The aromatic residues, including tyrosine and phenylalanine, have a maximum absorption peak at 280 nm. As shown in Figure 2a, ASC and PSC did not demonstrate a significant absorption peak at 280 nm.

**Figure 2.** Spectroscopy properties of ASC and PSC. (**a**) UV absorption spectra, (**b**) Fourier transform infrared spectroscopy, (**c**) circular dichroism, and (**d**) X-ray diffraction. The experiment was conducted only once (n = 1).

### 2.3.2. Fourier-Transform Infrared (FTIR) Spectrum

FTIR spectra of collagen from lizardfish scales are displayed in Figure 2b. ASC and PSC from lizardfish scales contained five major characteristic absorption bands, including Amide A, Amide B, Amide I, Amide II, and Amide III. The Amide A band (3400–3440 cm<sup>−</sup>1) is mainly associated with the stretching vibration of N–H [18]. However, the hydrogen bond formation leads to a change in wavenumber to a lower frequency [18]. The Amide A absorption bands of ASC and PSC were found at 3307 cm<sup>−</sup><sup>1</sup> and 3324 cm<sup>−</sup>1, respectively, indicating that N–H groups were involved in the formation of hydrogen bonds, which resulted in a shift of the Amide A band to the lower frequency. The Amide B band (3080 cm<sup>−</sup>1) is linked to the asymmetrical stretch of –CH2. We showed that the Amide B bands of ASC and PSC were located at 3080 cm<sup>−</sup>1. In the present study, the positions of Amide I bands of ASC and PSC were found at wavenumbers of 1653 cm<sup>−</sup><sup>1</sup> and 1654 cm<sup>−</sup>1, respectively; Amide II bands of both ASC and PSC were located at 1542 cm<sup>−</sup>1; and Amide III bands of ASC and PSC were observed at 1240 cm<sup>−</sup><sup>1</sup> and 1241 cm<sup>−</sup>1, respectively. Moreover, the ratios of absorption intensities between the Amide III band and 1450 cm<sup>−</sup><sup>1</sup> band were approximately 1.0, confirming that the triple helical structures of ASC and PSC were well maintained [6].

### 2.3.3. Circular Dichroism (CD) Spectrum

CD is a simple and effective technique to identify whether the triple helical structure is intact [22]. The CD spectrum of native collagen with a triple-helix structure shows a positive peak at 221 nm (maximum positive cotton effect), a negative peak at 198 nm (maximum negative cotton effect), and a crossover point (zero rotation) at approximately 213 nm [10,22]. As shown in Figure 2c, the CD spectrum of lizardfish scales ASC and PSC exhibited weak positive absorption peaks at 221 nm and 220 nm, respectively, and negative absorption peaks were observed at 198 nm and 197 nm, respectively, both with a crossover point at 213 nm. Moreover, the Rpn values (the ratio of the positive to negative) of ASC and PSC were 0.12 and 0.14, respectively, indicating that the collagen extracted from lizardfish scales possess a triple-helix conformation [26,27].

### 2.3.4. X-ray Diffraction (XRD) Spectrum

The XRD patterns of ASC and PSC are shown in Figure 2d. We found that ASC and PSC consisted of two peaks, a sharp and a broad peak. The diffraction angles (2θ) of ASC were 7.86◦ and 21.25◦, and those of PSC were 7.58◦ and 21.02◦, which are consistent with the characteristic diffraction peaks of collagen [28]. The d value of the first sharp peak of ASC was 11.25 Å, and that of PSC was 11.66 Å, and this reflects the distance between the molecular chains [28]. The distance between the molecular chains of PSC was greater than that within ASC, indicating weaker molecular interactions in PSC. This may be related to the cleavage of the terminal peptide sequence of collagen [29]. The d value of the second relatively broad peak of ASC was 4.18 Å, and that of PSC was 4.23 Å, and this reflects the distance between their skeletons [22].

### *2.4. Amino Acid Composition*

The amino acid compositions of the lizardfish scales ASC and PSC are shown in Table 1. It can be seen that glycine was the abundant amino acid in collagen, with ASC and PSC containing 35.1% and 34.9% of glycine, respectively. Similar results were found in the giant groaker skin collagen [30] and the Pacific cod skin collagen [22]. The results are consistent with glycine, which is identical in that in the collagen polypeptide chain, the repeating (Gly-X-Y)n assembles into a triple helix structure [30]. Alanine and proline accounted for 161 residues/1000 residues and 159 residues/1000 residues, and 158 residues/1000 residues and 157 residues/1000 residues in ASC and PSC, respectively. In addition, both the ASC and PSC were devoid of cysteine and tryptophan. Further, the amino acid (proline and hydroxyproline) contents of the ASC and PSC were 237 residues/1000 residues and 236 residues/1000 residues, respectively.


**Table 1.** Amino acid composition of the ASC and PSC from lizardfish scales. The results are expressed as residues/1000 total amino acid residues. Values represent the means ± standard deviations (SD) of duplicate assays (n = 3).

### *2.5. Morphology Characterization*

The morphology of collagen is vital for assessing its potential application in biomedicine [31]. The collagen solution obtained from lizardfish scales was lyophilized, and the morphology of collagen sponges was observed by scanning electron microscopy (SEM) (Figure 3). As shown in Figure 3a,a', ASC and PSC were observed as white sponges with loose, uniform, and porous structures observed by the naked eye. ASC and PSC surfaces under SEM were partially wrinkled, which may be attributed to water being sublimated during the freezedrying process [32]. The SEM images showed that ASC and PSC had a similar multilayer overlapping and porous microstructure. However, there were some differences in the structure between ASC and PSC under SEM observation. As observed at a magnification of 400×, ASC exhibited a compact sheet and porous structure (Figure 3b), while PSC had a loose and large sheet structure (Figure 3b'); ASC exhibited a more porous structure than PSC. It was clearly visible at higher magnifications (800×) that ASC had considerable fibrillary structure and a small number of sheet structures (Figure 3c), while PSC had large sheet-like film structures (Figure 3c').

**Figure 3.** *Cont.*

**Figure 3.** SEM images of ASC and PSC. (**a**) ASC, (**b**) ASC at 400× magnification, (**c**) ASC at 800× magnification, (**a'**) PSC, (**b'**) PSC at 400× magnification, and (**c'**) PSC at 800× magnification. The experiment was done only once (n = 1).

### *2.6. Thermal Stability*

Differential scanning calorimetry (DSC) was used to measure the maximum transition temperature (Tmax) of collagen. The DSC curves of collagen from the lizardfish scales are shown in Figure 4. It was observed that the Tmax of collagen from lizardfish scales was higher than that of rat tail collagen. The Tmax values of rat tail collagen, ASC, and PSC were 39.4 ◦C, 43.2 ◦C, and 42.5 ◦C, respectively. The Tmax of ASC was higher than PSC and the rat tail collagen, and the ΔH of ASC (0.981 J/g) was also higher than the PSC (0.711 J/g) and the rat tail collagen (0.680 J/g).

**Figure 4.** The DSC curve of ASC and PSC. The experiment was performed only once (n = 1).
