*3.7. Relative Solubility*

The effects of pH and NaCl on sample solubility were characterized as described in our earlier study [46].

The relative solubility of the *T. flavidus* collagen extract was determined in the presence of 0–6 g/100 mL NaCl. Briefly, 8 mL of 3 mg/mL collagen extract was mixed with 5 mL of NaCl solution containing 0.5 M acetic acid. The mixture was centrifuged at 20,000× *g* and 4 ◦C for 30 min. The protein concentration of the supernatant was measured according to the calibration curve for bovine serum albumin by Folin assay [50]. The absolute protein concentration of the collagen extract was expressed as mg bovine serum albumin equivalent/mL (mg BSAE/mL). The relative solubility was calculated with Equation (2) below by normalizing the protein concentrations collected at all NaCl levels with values obtained from collagen extracts that received no NaCl treatment.

Relative solubility (%) = (Protein concentration of supernatant with NaCl treatment)/ (Protein concentration of supernatant without NaCl treatment) <sup>×</sup> <sup>100</sup> (2)

The relative solubility of the *T. flavidus* collagen extract was also determined at varied pH values (1–10). The collagen extract (8 mL, 3 mg/mL) was adjusted to designed pH condition by 6.0 M HCl (or NaOH) with a final volume of 10 mL. The resulting solution was centrifuged at 20,000× *g* and 4 ◦C for 30 min. The protein concentration of the supernatant was determined by Folin assay as described above. The relative solubility was calculated with Equation (3) below by normalizing the protein concentrations collected at pH conditions with values obtained at pH 3.0.

$$\begin{aligned} \text{Relative solubility (\%)} &= \text{(Protein concentration of supernantant at varied pH)} / \\ \text{(Protein concentration of supernantant at pH 3.0)} &\times 100 \end{aligned} \tag{3}$$

#### *3.8. DSC*

The thermal properties of the sample were characterized by an established DSC method [14] with slight modification. Lyophilized collagen extract was hydrated with 0.05 M acetic acid (1:40 *w*/*v*), incubated at 4 ◦C for 2 days and then characterized using a DSC 2 calorimeter (Mettler-Toledo, Zurich, Switzerland). Heat flow of the hydrated samples was measured between 5 and 75 ◦C at 1 ◦C/min using an empty aluminum pan as reference. Tmax was defined as the peak of the transition curve.

#### *3.9. Viscosity*

The viscosity of the sample was characterized based on a method described by Kittiphattanabawon et al. [24] with slight modification. Briefly, protein dispersion (0.1% *w*/*v*) was prepared by hydrating the collagen sample in 0.05 M acetic acid at 4 ◦C for 2 days. An Ostwald's viscometer (Kusano Inc., Tokyo, Japan) loaded with collagen dispersion was incubated in a 15 ◦C water bath for an extended period. Small temperature increments (2 ◦C) were then applied to the viscometer at 30-min intervals in a stepwise manner. Viscosity was recorded between 15 and 50 ◦C prior to each temperature change. Fractional viscosity was calculated with Equation (4). Td was defined as the temperature for fractional viscosity to reach 0.50.

$$\begin{aligned} \text{Fractional viscosity} &= \text{(Measured viscosity } - \text{minimum viscosity)} / \\ &\quad \text{(Maximum viscosity } - \text{minimum viscosity)} \end{aligned} \tag{4}$$
