*3.3. Codfish Extract Purity*

Protein content was evaluated by Micro BCA assay (Fisher Scientific, Rockford, IL, USA). Briefly, 1 mg/mL of collagen solution in 20 mM acetic acid was neutralized with 25 mM of Tris-base containing 3% SDS and denatured at 65 ◦C during 1 h. After equilibration at room temperature the sample was analysed. Sircol assay (Biocolor, County Antrim, UK) was performed according to the manufacturer's instructions. Collagen type I from rat tail was used as control in Micro BCA and Sircol assays. Extract purity was accessed comparing the protein/collagen content with the initial extract concentration.

#### *3.4. Sodium Dozdecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)*

Codfish skin extract was first dissolved at 1 mg/mL in 20 mM acetic acid at room temperature and then mixed with three-fold-concentrated loading buffer containing 0.1 M 1,4-dithiothreitol (DTT) to a final protein mass of 10 μg. Protein samples were heat-denatured at 65 ◦C for 30 min and analysed by SDS-PAGE according to Laemmli [67] using 4% stacking and 7.5% resolving gels in a Mini Protean III unit (Bio-Rad Laboratories, Hercules, CA, USA) at 27 mA/gel. Protein bands were stained with Coomassie brilliant Blue R250 and destained with 32% (*v*/*v* %) methanol 5.6% (*v*/*v* %) acetic acid solution (destaining solution I), and 5% (*v*/*v* %) methanol and 7% (*v*/*v* %) acetic acid solution (destaining solution II). Rat and bovine collagen were used as controls.

#### *3.5. Fourier Transformed Infrared (FTIR) Spectroscopy*

Freeze-dried products were individually mixed with vacuum dried potassium bromide (KBr) and pressed into pellets with a hydraulic press. The infrared spectra were obtained in the wavenumber range between 4400 and 400 cm−<sup>1</sup> at a resolution of 5 cm<sup>−</sup>1, using the infrared spectrometer IRPrestige 21 (Shimadzu, Kyoto, Japan). Each spectrum resulted from the average of 50 scans. Extracted collagen was compared with freeze-dried commercially available rat and bovine collagens.
