**3. Discussion**

Collagen is an important and diverse biopolymer that has seen a significant increase in applications in food, medicine, cosmetics, and tissue engineering [35] with the highest structural order and the greatest stiffness, is widely used in materials for biomedical applications [36]. Marine collagen has been successfully isolated from marine by-products [1,37,38].There are no reports on the use of lizardfish scales for collagen preparation.

In the present study, we isolated type I collagen from lizardfish scales by using acid and enzymatic extraction methods. It was found that the use of pepsin increased the yield of the collagen extraction, and this could be attributed to the fact that pepsin cleaves the crosslinked molecules in the telopeptide region, leading to further extraction with increased yield. This made the extraction yield of PSC higher than that of ASC. These results agreed with those of Keawdang et al. (2014), who reported that ASC and PSC from yellowfin tuna swim bladders were extracted with yields of 1.07% and 12.10%, respectively [38], and Matmaroh et al. (2011), who reported that ASC and PSC from spotted golden goatfish were extracted with yields of 0.46% and 1.20%, respectively [39]. The difference in the extraction yields could be attributed to the varying cross-linking of collagen fibrils in the different raw materials. In this study, the collagen yields from lizardfish scales were higher than that from bighead carp scales (2.7%) and spotted golden goatfish scales (ASC 0.46% and PSC 1.20%). Both the ASC and PSC had similar UV absorption spectra to those of soft-shelled turtle collagen [23], carp scales collagen [37], and red stingray skin collagen [25]. In addition, we also studied the infrared spectra of the ASC and PSC, and the infrared spectra of the ASC and PSC were similar to the spectra of type I collagen from tilapia skin and scales [18], giant salamander skin [33], and silver carp skin [6], where the Amide I band (1600–1700 cm<sup>−</sup>1) typically corresponds to the stretching vibration of C=O along the protein polypeptide backbone. This can be used as a positive marker for peptide secondary structure, and therefore it is often used in the analysis [37]. The Amide II band (1500–1600 cm<sup>−</sup>1) commonly arises from N–H bending coupled with C–N stretching vibrations [40]. The Amide III band (1200–1300 cm<sup>−</sup>1) arises due to C–N stretching and

N–H in-plane bending from amide linkages, and this is the standard confirming presence of the triple-helical structures of collagen [17]. The absorption peaks of the Amide A band of PSC showed a higher wavenumber than those of the ASC, suggesting that fewer N–H groups in PSC were involved in hydrogen bonding in the polypeptide chain. Similar results were found in chicken feet collagen [41]. It has also been reported in the literature that the hydrolysis of telopeptide by pepsin might increase the free amino group, and this may have led to the higher wavenumber of PSC [40,42]. Moreover, the results of the CD spectrum obtained from the ASC and PSC was similar to the CD spectrum of gulf corvina collagen (positive absorption peaks at 221 nm and negative absorption peaks at 198 nm) [10], *Perinereis nuntia* cuticle collagen (positive absorption peaks at 221 nm and negative absorption peaks at 199 nm) [28], and Nile tilapia skin collagen (positive absorption peaks at 221 nm and negative absorption peaks at 197 nm) [16]. In addition, the XRD spectrum analysis showed that the distance between the molecular chains and the distance between their skeletons of the ASC and PSC were similar to the Pacific cod skin collagen [22] and the cuticle of the *Perinereis nuntia* collagen [28]. The results of the FTIR, CD, and XRD indicated that both the ASC and PSC had a native triple helix conformation, and that the acid and enzymatic extraction methods of collagen had no adverse effects on the molecular integrity of the collagen. The highly porous structure is an important feature of biomedical materials that can influence cell seeding, migration, growth, and other physiological activities [28]. The morphology results suggested that ASC and PSC from lizardfish scales have the potential for biomedical materials [41].

The pyrrolidone ring formed by the amino acids facilitates the strengthening of the triple helix structure of collagen, and this is directly linked to thermal stability and is one feature that determines the potential use of collagen. An analysis of the amino acid content showed that the ASC and PSC had higher amino acid contents than that of the grass carp skin collagen (186 residues/1000) [43], the spotted golden goatfish collagen (ASC 186 residues/1000 and PSC 189 residues/1000, respectively) [39], and the calf skin collagen (221 residues/1000) [44]. Therefore, collagen extracted from the lizardfish scales may have high thermal stability based on the amino acid analysis. Thus, we further characterized the thermal stability of the collagen. In general, collagen obtained from fish species that live in cold environments is often less thermal stable than collagen from fish species that live in warmer environments [39]. The lizardfish (*S. macrops*) is widely distributed in tropical and subtropical waters [45], and the Tmax of lizardfish scale collagen is similar to that of spotted golden goatfish scale collagen (ASC 41.58 ◦C, PSC 41.01 ◦C), a common and abundant species in tropical and sub-tropical regions [39]. In addition, it is higher than cold-water species arabesque greenling skin collagen (ASC 15.7 ◦C and PSC 15.4 ◦C, respectively) [46] and temperate-water fishes grass carp skin collagen (28.4 ◦C) [43]. These results were consistent with the results previously reported, indicating that the collagen obtained from the fish species living in cold environments often had lower hydroxyproline contents exhibited less thermal stability than collagen from fish species living in warmer environments [39]. These results were consistent with the amino acid composition of the above studies, with lizardfish scales collagen containing a higher total amino acid content (ASC 237 residues/1000 residues and 236 residues/1000 residues) than arabesque greenling skin collagen (ASC 159 residues/1000 and PSC 157 residues/1000, respectively) [46] and grass carp skin collagen (186 residues/1000 residues) [43]. Thermal stability is one of the most important properties that determine the potential applications of collagen, and it is related to the total amino acid content, habitat temperature, and body temperature [1]. In addition, the Tmax of lizardfish scale collagen was also higher than calf skin collagen (35.0 ◦C) [44], a collagen from terrestrial sources, and this indicated that collagen from lizardfish scales has the potential for use as an alternative source of terrestrial collagen.

The study of the effect of the NaCl concentration and pH on the relative solubility of collagen can provide useful information for collagen preparation as well as for processing and application. When collagen is used as a source in production in moisturizing cosmetics, solubility is a major determinant. This is because the hydrolyzed substances are used for

cosmetic and medical cream formulations in this industry [47]. The ASC and PSC solutions exhibited the lowest solubility at pH 7 and pH 8, respectively, and this was attributed to the pI of protein with the total net charge of protein molecules being zero when the pH of the solution is equal to the pI [6,48]. In this case, the hydrophobic interaction between collagen molecules is enhanced, leading to aggregation and precipitation of the protein, thereby leading to the low solubility of the solution [21,37]. In contrast, as the solution pH increases above the pI, the net negatively charged residues of the protein increase, causing the ASC and PSC to display a slight increase in solubility at pH levels above 7 and 8, respectively. The differences in the relative solubility of collagen at varying pH levels are related to the molecular properties and conformation of collagen [38]. Kaewdang et al. (2014) [38] reported that the difference in the relative solubility of ASC and PSC at different pHs may be due to the removal of telopeptide regions that affect the protonation or deprotonation of charged amino and carboxyl groups, and this may affect the repulsion of molecules associated with different solubilities. Moreover, the effect of the NaCl concentration on the solubility of collagen solutions showed that the relative solubility of the PSC solutions decreased sharply above a 6% NaCl concentration, while the ASC solutions maintained a high relative solubility (greater than 80%). The relative solubility of the ASC solutions decreased sharply until the NaCl concentration was greater than 10%. The relative solubility of the collagen solutions decreased as the concentration of NaCl increased, and this may have been due to the protein precipitation and salting-out effect [21]. Jongjareonrak et al. (2005) [49] explained that the addition of salt increases the ionic strength and enhances the hydrophobic interaction between protein chains, resulting in a decrease in the solubility of collagen solutions. Thus, the ASC might be less susceptible to the "salting out" effect compared to the PSC [50]. A similar phenomenon has been found in giant croaker swim bladder collagen [48] and silver carp skin [6].

The results of the dynamic frequency scan test revealed that the preparation method markedly affects the rheological parameters, G and G, of ASC and PSC extracted from lizardfish scales. An analysis of the frequency dependence of G and G suggested that the elasticity of the PSC had a greater dependence on frequency than that of the ASC, while the viscosity of the ASC had a greater dependence on frequency than that of the PSC. Moreover, it was noted that the G and G values of PSC were higher than the corresponding G and G" values of ASC between 0.01 and 10 Hz (Figure 6), and these were similar to the collagen from chicken feet. In addition, the G and G of PSC were higher than those of ASC at a scan frequency range of 0.2–10 Hz [41], suggesting that the PSC exhibited good viscoelasticity. It was also observed that G was higher than G for all of the collagen, indicating a greater contribution of viscosity than elasticity in the ASC and PSC from lizardfish scales.

The CCK-8 assay was used to determine the viability of live cells. The relative viability of the HaCaT and MC3T3-E1 cells on the ASC and PSC were greater than 70% during the 48 h of cell culture, indicating that the ASC and PSC from lizardfish scales are not toxic to HaCaT and MC3T3-E1 cells [6]. However, the relative viability of the HaCaT and MC3T3-E1 cells increased during the 48 h of cell culture, suggesting that the lizardfish scales collagen had the ability to promote cell proliferation. And the relative viability of the HaCaT and MC3T3-E1 cells were both higher on ASC than PSC (*p* < 0.05). These results suggested that the ASC was associated with higher cell viability than PSC. Moreover, a morphological examination of the cells showed that both the HaCaT and MC3T3-E1 cells had similar cell growth patterns as the control groups over the culture period (Figure 8). Thus, the results suggested that lizardfish scales ASC and PSC can be used as non-toxic materials in the biomedical field.

### **4. Materials and Methods**
