*3.2. Pretreatment of Fish Skin*

To remove pigments and non-collagenous proteins; the fish skins pieces (FSPs) were immersed into a solution of NaOH (0.1 M, ratio skin: solution 1:10) during 48 h with a daily solution changing. To reach neutral pH, the FSPs were washed with cold distilled water, then soaked for 24 h in butanol solution (10%, ratio 1:10) to eliminate fat and then thoroughly washed with cold water.

#### *3.3. Collagen Extraction*

Acid solubilised collagen and pepsin solubilised collagen were isolated from hound-smooth skin following the method proposed by Nagai and Suzuki [23] to which we introduced some modification as detailed in the following paragraph. All preparations were conducted in a cold room at 4 ◦C.

#### 3.3.1. Acid Extraction

To extract collagen, the pre-treated FSPs were suspended in a solution of acetic acid (0.5 M, ratio 1:10) for 3 days with a continuous stirring, filtered and the residue was subjected to a second extraction under the same conditions. Following this step NaCl was added to both supernatants to a final concentration of 2.0 M to precipitate collagen. The pellets were recovered by centrifugation (7000× *g*, during 1 h) then re-dissolved in acetic acid (0.5 M).

For purification, the resulting acetic acid solution was dialysed (bag cut-off of 14 kDa) against an acetic acid (0.1 M) solution, then against distilled water during 48 h in each case. The purified extract was thereafter lyophilized (Christ, Alpha 2–4 LD plus, Osterode am Harz, Germany), and the resulting collagen was referred as acetic ASC.

#### 3.3.2. Enzymatic Extraction

Un-dissolved materials (residue 2) resulting from the previous steps (Figure 9) were washed with cold distilled water and re-suspended in acetic acid (0.5 M) containing 1% pepsin (*w*/*w*) at a ratio of 1:10 (*w*/*v*) then incubated for 72 h at 4 ◦C. The filtrate was recovered in two steps for precipitation, dialysis and freeze drying as explained in ASC purification. The resulting collagen was called PSC. The collagens were stored at −20 ◦C until analyses.

#### *3.4. Extracted Collagen Characterisation*
