*3.6. Statistical Analysis*

The experiments (carried out at least in triplicate) were presented as mean ± standard deviation. Statistical interpretation of the results was performed by One way ANOVA and LSD (least significant differences) tests (using *p* < 0.05 level of significance to compare mean values) using the software SPSS 22.0 (SPSS 22.0 for Windows, SPSS Inc., Chicago, IL, USA).

#### **4. Conclusions**

In a first step the type of collagen extracted from the hound-smooth skin was identified. Thus, electrophoretic patterns revealed that both extracted ASC and PSC are mostly composed of α (α1, α2) and β-chains with low content of γ-chain suggesting that hound-smooth skin collagen should most likely be classified as type I collagen.

Fourier transform infrared investigations showed that the secondary structure and the triple helical structure of ASC and PSC were both maintained intact even after the enzymatic hydrolysis with pepsin. Similar denaturation temperatures were found for ASC and PSC (26.68 ◦C and 26.66 ◦C respectively).

In a second step, collagen was used to elaborate film but with the incorporation of chitosan. The addition of chitosan increased the films mechanical strength and reduced its water solubility. The FTIR spectra clearly indicated that interactions between both polymers occurred and the secondary structure of collagen triple helix have been changed by the addition of chitosan.

The addition of 50% chitosan into collagen films was sufficient to obtain an edible film with good mechanical properties, suitable solubility and antioxidant activity. Owing to its anti-UV properties, such collagen-chitosan film could be used as a protective material to preserve nutraceutical products. **Author Contributions:** E.B.S., and S.S. conceived and designed the experiments; E.B.S. conducted the experimental analysis and wrote the manuscript; S.S. supervised, validate the data analyses and co-wrote the manuscript. All authors reviewed the manuscript.

**Acknowledgments:** This work was conducted within the project "Biotechnologie Marine Vecteur d'Innovation et de Qualité-BIOVecQ PS1.3\_08" co-financed by the cross-border IEVP Italy-Tunisia program and the ministry of Higher Education and Scientific Research-Tunisia.

**Conflicts of Interest:** The authors declare no conflict of interest.
