*2.5. Thermal Properties*

The thermal stability of *T. flavidus* collagen was characterized by denaturation temperature (Td) and maximum transition temperature (Tmax). Td refers to the temperature at which the triple-helical collagen structure in solution is disintegrated into random coil [16]. The viscosity of *T. flavidus* collagen (Td = 28.4 ± 2.5 ◦C, Figure 3A) was found to decrease modestly slower than that of Alaska pollack (Td = 16.8 ◦C) [37], indicating a milder denaturing process for *T. flavidus* collagen. Furthermore, Tmax refers to the temperature at which collagen fiber shrinks to one third of its length. The phase transition involving the conversion of a crystalline triple-helical collagen structure to an amorphous random coil form occurs during the shrinkage process [38]. Differential scanning calorimetry (DSC) analysis of *T. flavidus* collagen confirmed this observation with a Tmax value of 41.8 ± 0.35 ◦C (Figure 3B). The difference between the Tmax and Td values of *T. flavidus* collagen was about 13 ◦C. The Tmax value of *T. flavidus* collagen was higher than those of bighead carp, bigeye snapper, and grass carp (24.6–33.3 ◦C) [14,16,24]. It is therefore reasonable to speculate that *T. flavidus* collagen tri-helices are more stable at higher temperature than other fishes. More interestingly, the Tmax value of *T. flavidus* collagen was similar to calf skin collagen (40.8 ◦C) [39]. Considering the importance of thermal stability for collagen applications in foods [5] and the aquacultural potential of *T. flavidus* [40], we postulate that collagen from *T. flavidus* could potentially be used as an alternative source of collagen.

The superior thermal stability of *T. flavidus* collagen was likely due to its imino acid content. Imino acids, especially Hyp, have been known for their ability to stabilize collagen tri-helices via intermolecular hydrogen bonds [41]. Our study showed that *T. flavidus* collagen (Table 2, 246 ± 0.04 residues/1000 amino acid residues) had significantly higher imino acid content than the skin collagen of bighead carp, bigeye snapper, grass carp, and *T. rubripes* (*p* < 0.05, 165–193 residues/1000 amino acid residues) [14,16,23,24]. The additional imino acids might form extra hydrogen bonds within *T. flavidus* collagen tri-helices, therefore increasing the molecular stability through rising entropy [42].

In addition, a secondary exothermal peak (32.9 ± 0.31 ◦C) was identified in the DSC thermogram (Figure 3B). It was consistent with our observation on the viscosity changes in *T. flavidus* collagen (Figure 3A), indicating a partial denaturation of collagen supramolecular structure due to defibration of thermally unstable hydroxyproline-free sequence in collagen triple helices [28].

**Figure 3.** Fractional viscosity (**A**) and Differential scanning calorimetry (DSC) (**B**) of *T. flavidus* collagen extract.

#### *2.6. Relative Solubility*

In the absence of NaCl, 0.5 mol/L acetic acid was able to fully dissolve *T. flavidus* collagen at a concentration of 0.16 mg/mL. Increments in NaCl level (≤3 g/100 mL) appeared to have a modest impact on the collagen solution, leading to a minor reduction in the relative solubility (99.5%–89.8%, Figure 4A). Higher levels of NaCl (≥4 g/100 mL), however, significantly decreased the relative solubility (11.0%–11.7%, *p* < 0.05). This phenomenon is in agreement with an earlier study on catfish skin collagen, which significantly precipitates at high NaCl levels (≥4 g/100 mL) [43]. Considering that Na+ and Cl− are the major forces depriving water molecules from hydrophilic amino acid residues of collagen during NaCl-mediated salting-out process [44], it is reasonable to postulate that collagen solubility is affected by the ionic strength of solutions. To avoid collagen aggregation due to potential salting-out events, the ionic strength of 1.01 (equivalent to the solution containing 3 g/100 mL NaCl

and 0.5 mol/L acetic acid) could serve as a threshold value for preparing *T. flavidus* collagen in other salt solutions.

The protein content of *T. flavidus* collagen at pH 3.0 (0.04 mg BSAE/mL) was used to determine the relative solubility of the collagen among all pH conditions (Figure 4B). While adjusting pH values within acidic environments could modestly reduce relative solubility to 82.1 ± 0.1% (1 ≤ pH ≤ 3.0, *p* < 0.05), sharper reductions were observed between pH 3.0 and 5.0, with the lowest relative solubility being 37.9 ± 0.01%. This event was also reported by Singh and colleagues in a research involving catfish skin collagen [43]. As ionic strengths of *T. flavidus* collagen solutions (0.50–0.65) were much lower than the threshold value (1.01) during pH adjustments, it might have had a minor impact on the relative solubility of the collagen.

The reduction in relative solubility of *T. flavidus* collagen between pH 3.0 and 5.0 was most likely due to deprotonation of the charged amino acid residues. In acidic environments (pH ≤ 3.0), collagen behaved as a positively charged particle, with most charged amino acid protonated. The net positive charges of collagen assisted in forming hydrogen bonds with water molecules and preventing aggregation with surrounding proteins [45]. However, when the solution pH was increased from 3.0 to 5.0, the collagen lost a large fraction of net positive charges due to deprotonation of the side chains within Asp (pK 3.86) and Glu (pKa 4.25), which accounted for 49.2% of the charged amino acid residues of the collagen (Table 2). Considering the increasing hydrophobic interactions among neutralized collagens [44], it is reasonable to postulate a sharp reduction in relative solubility.

**Figure 4.** Solubility of *T. flavidus* collagen extract in the presence of varied NaCl concentration (**A**) or pH condition (**B**).

In neutral or basic environments (pH ≥ 6), the relative solubility of *T. flavidus* collagen continuously decreased, with the lowest value found at pH 7.0 (22.7 ± 0.02%, Figure 4B). This observation is in accordance with previous studies on type I collagen [12]. Changes in relative solubility were partially due to the elevated ability of imino acids (Hyp in particular) to facilitate intermolecular cross-linking within collagen tri-helical structures [41,42] as imino acids contributed to 24.6% of the total amino acid compositions of *T. flavidus* collagen (Table 2). It could also be attributed to alterations in total net charges of collagen molecules as electrostatic expulsion is too weak to shield proteins from aggregation around the isoelectric point (pI ≈ 7 for type I collagen) [45].
