4.4.4. XRD

The diffractograms of the samples were recorded by X-ray diffractometer (X'Pert Pro XRD, PANalytical, The Netherlands), which was operated at 40 kV and 40 mA with CuK α radiation (λ = 1.5406 Å). The data were collected at scanning speed of 4.5◦·min−<sup>1</sup> and 2θ range of 5–50◦. Bragg equation was used to calculate the d values of collagen:

$$\mathrm{d}\left(\stackrel{\circ}{\mathrm{A}}\right) = \frac{\lambda}{2\sin\theta} \tag{2}$$

where λ is the X-ray wavelength (1.54◦) and θ is the Bragg diffraction angle.

### *4.5. Amino Acid Analysis*

The samples were hydrolyzed in 6 M HCl at 110 ◦C for 8 h. After being vaporized, the residue was dissolved in 100 mL of 0.1 M HCl [22]. Then 50 μL of the sample solution was analyzed using high-performance liquid chromatography (HPLC-MS/MS, Ultimate 3000-API 4000 Q TRAP, Thermo Fisher Scientific, Dreieich, Germany).

### *4.6. Microscopy Characterisation*

The collagen solution (5–10 μL) without acetic acid was poured into a 12-cm-diameter lyophilization dish and then freeze-dried. The morphology of the sample was imaged using SEM (S-4800, HITACHI, Tokyo, Japan), with an accelerating voltage of 5 kV. After being coated with Pd, the samples were observed at 400× and 800× magnifications.

### *4.7. Thermal Stability*

The thermal stability of the samples was measured using a differential scanning calorimeter (DSC2, Mettler-Toledo corp., Zurich, Switzerland) under a nitrogen atmosphere with a flow rate of 100 mL min−1. The samples were dissolved in 0.4 M acetic acid at the ratio of 1:40 ( *w*/*v*) for 48 h at 4 ◦C. The solution (5 mL–10 mL) was placed into aluminium crucible, and then scanned over the range of 20–70 ◦C at a heating rate of 1 ◦C/min. The empty aluminium crucible was used for reference. The maximum transition temperature (Tmax) was obtained from the DSC thermogram, and the enthalpy of denaturation ( ΔH) was calculated from the area of the corresponding endothermic peak.
