*2.7. Solubility*

2.7.1. The Influence of pH on the Solubility of Collagen Solutions

The relative solubility of ASC and PSC extracted from the scale of lizardfish at different pH showed similar trends, as shown in Figure 5a. ASC and PSC exhibited higher relative solubility in the very acidic pH range (1–4), and both ASC and PSC showed the maximum relative solubility at pH 2. The relative solubility of ASC and PSC decreased with increasing pH, and a sharp decrease in relative solubility of ASC and PSC occurred at pH above 5 and 4, and the minimum relative solubility was 11.09% and 7.70%, respectively. The isoelectric points (pI) of ASC and PSC were approximately around 7 and 8, respectively [33].

**Figure 5.** Relative solubility of ASC and PSC. (**a**) Effect of pH; (**b**) effect of NaCl concentration. Values represent the means ± standard deviations (SD) of duplicate assays (n = 3). Different letters indicated significant differences between the samples.

### 2.7.2. The Influence of NaCl Concentration on the Solubility of Collagen Solutions

The effect of NaCl concentration on the relative solubility of ASC and PSC from lizardfish scales is shown in Figure 5b. ASC and PSC showed high relative solubility at low NaCl concentrations, both above 80%. The relative solubility of ASC and PSC from lizardfish scales decreased with increasing NaCl concentrations, with the lowest values at 14% and 10% and 11.42% and 13.64% NaCl concentrations, respectively. Subsequently, as the NaCl concentration increased, the relative solubility of collagen remained relatively stable but very low (around 20%).

### *2.8. Rheological Properties*

The frequency dependence of the rheological parameters elastic modulus (G) and viscous modulus (G) from lizardfish scales ASC and PSC was assessed using dynamic frequency scan tests. The G was defined as the elasticity of protein, and G was defined as the viscous behavior of the protein [34]. Figure 6 exhibits the dynamic frequency sweep tests of ASC and PSC, and the G and G values of ASC and PSC showed an increasing trend as the frequency increased from 0.01 to 10 Hz. The G and G values of PSC are higher than the corresponding G and G values of ASC between 0.01 and 10 Hz. As shown in Figure 6a, the increase in the G value of PSC was higher than that of ASC in the test frequency range 0.01–10 Hz.

**Figure 6.** Rheological behavior of collagens solution (**a**) storage modulus (G); (**b**) loss modulus (G). The experiment was performed only once (n = 1).

### *2.9. Cell Compatibility*

The cytotoxicity of the HaCaT (Cat No. CBP60331) and MC3T3-E1 (Cat No. CBP60946) cells lines on lizardfish scales collagen after 24 h and 48 h was investigated using a CCK-8 assay. The results of relative cell viability are shown in Figure 7. After 24 h of cell culture, the

relative viability of the HaCaT cells on ASC and PSC were 107.18 ± 1.78% and 101.44 ± 3.62%, respectively, and for the MC3T3-E1 cells, 113.43 ± 2.40% and 105.95 ± 1.90%, respectively. Moreover, the relative viability of HaCaT cells on ASC and PSC were 111.78 ± 1.74% and 106.45 ± 1.89%, respectively, and for the MC3T3-E1 cells, 117.80 ± 1.65% and 110.64 ± 2.70%, respectively, after 48 h. The relative viability of the HaCaT and MC3T3-E1 cells on ASC and PSC increased during 48 h of cell culture. Moreover, the morphology of cells was observed under an inverted microscope, and there were no observable changes in the HaCaT and MC3T3-E1 cells compared to the control group (Figure 8).

**Figure 7.** Relative cell viability of HaCaT and MC3T3-E1 cells after 24 h and 48 h of incubation in ASC and PSC. (**a**) is HaCaT cells; (**b**) is MC3T3-E1 cells. Values represent the means ± standard deviations (SD) of duplicate assays (n = 6). \* *p* < 0.5, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

(**b3**)

**Figure 8.** *Cont.*

**Figure 8.** Morphology of HaCaT and MC3T3-E1 cells on lizardfish scales collagens (40× magnifications). (**a1**–**a3**) HaCaT cells for 24 h cell culture, (**a1**) is control, (**a2**) is ASC, and (**a3**) is PSC; (**b1**–**b3**) MC3T3-E1 cells for 24 h cell culture, (**b1**) is control, (**b2**) is ASC, (**b3**) is PSC; (**c1**–**c3**) HaCaT cells for 48 h cell culture, (**c1**) is control, (**c2**) is ASC, and (**c3**) is PSC; (**d1**–**d3**) MC3T3-E1 cells for 48 h cell culture, (**d1**) is control, (**d2**) is ASC, and (**d3**) is PSC.
