*2.4. Thermal Denaturation Temperatures of ASC and PSC*

The tripeptide chains are bound by non-covalent bonds such as hydrogen bonds, which is the basis of the stability of collagen. When the collagen molecules absorbed enough heat from the outside, these non-covalent bonds were destroyed, causing the triple-helix structure to become a random coil structure and destroying the biological properties of collagen. The thermal stability of ASC and PSC were studied by viscosity measurement [62]. According to Figure 2, ASC and PSC have similar curves, and their denaturation temperatures (Td) were 36.1 ◦C and 34.4 ◦C, respectively. The Td values can be regarded as the temperature at which the triple-helix structure of collagen is deformed into a random coil structure. The Td values of ASC and PSC from tilapia skin are similar to the collagen extracted from fish living in warm tropical climates such as salmon (29.3 ◦C) and bigeye snapper

(30.4 ◦C) [49], and higher than the cold-water fish, such as Baltic herring (15.0 ◦C) and Argentine salmon (10.0 ◦C) [63], but lower than terrestrial animals such as bovine (39.7 ◦C) or porcine (37 ◦C) [64]. The difference in amino acid composition was the primary cause of the different thermostability of collagen. The loss of the PSC telopeptides has a certain influence on the stability of the triple-helix structure, resulting in lower thermal stability than ASC, which is consistent with the results of amino acid composition analysis and FTIR analysis. Although the thermal stability of tilapia skin collagen is lower than that of terrestrial organisms, it is higher than that of common aquatic organisms, which is an advantage for its application in the field of biomedical materials.

**Figure 2.** Thermal denaturation curves of ASC and PSC from Nile tilapia skin. The denaturation temperature was determined as the mid-point temperature where viscosity changes reach 0.5.
