**3. Materials and Methods**

### *3.1. Preparation of Codfish Skin*

Codfish skins were removed from cold preserved fish body parts in an industrial plant and kindly provided by Frigoríficos de Ermida, Lda. (Gafanha, Nazaré, Portugal). Frozen skin was thawed at 4 ◦C for 24 h, with the scales, remaining flesh and bones then manually removed, and the skin cut into small pieces (1.0 cm × 1.0 cm) and washed with ice-cold deionized water. To remove non-collagenous proteins, the skin was treated with 10 volumes of 0.1 M NaOH solution, changed each 24 h, for 72 h at 4 ◦C under magnetic stirring. The skin was then rinsed with ice-cold deionized water until reaching pH 7. All procedures were carried out at 4 ◦C to minimize collagen denaturation.

#### *3.2. Extraction of Acid-Soluble Collagen from Codfish Skin*

All the procedures were carried out at 4 ◦C and collagen was extracted using an acid-base procedure. The skin was soaked in 0.5 M acetic acid with a sample:solution ratio of 1:10 (*w*/*v*) for 72 h with continuous stirring. The solution was then strained and centrifuged (Eppendorf 5810R, Hamburg, Germany) at 20,000× *g* during 1 h. An accurate volume of 50 mM Tris-HCl containing 2.6 M of NaCl at pH 7.4 buffer was added to the supernatant to reach a final concentration of 0.9 M of NaCl to precipitate collagen. The precipitated collagen was removed from solution by centrifugation at 20,000× *g* for 30 min. A minimal volume of 0.5 M acetic acid was added to the pellet and then dialyzed for 48 h against 10 volumes of 0.1 M acetic acid, 48 h against 0.02 M acetic acid and 48 h against ultrapure water, with the solutions being changed every 24 h. The resulted acid-soluble collagen was freeze-dried for 1 week and stored at −80 ◦C until further use.
