2.2.4. Fourier Transform Infrared Spectra of Composite Films

For the composite biofilms, FTIR was used to detect the new interactions between collagen and chitosan and to identify the nature of the new linkages between both molecules.

The FTIR spectra revealed the characteristics of the specific bands corresponding to functional groups in all films (Figure 8).

**Figure 8.** Fourier transform infrared spectra of different collagen films, CO: pure collagen film; CH: pure chitosan film; C50: Collagen-chitosan film 50%:50%, C75: Collagen-chitosan film 25%:75%.

The CO, C50, and C75 composite films; displayed amide I bands at the wavenumbers of 1641.2, 1645.5 and 1642 cm−<sup>1</sup> respectively. The shift to higher wavenumber was a result of a structural rearrangements occurring in the film structure with a strong affinity between the collagen and chitosan.

The amide I peak in CH film at the wavenumber of 1642.6 cm−<sup>1</sup> was assigned to C=O stretching of N-acetyl group [76]. The amide II band was found at approximately the same wavenumber in CO film (1543.9 cm−1) and C50 film (1543.6 cm−1) but at lower peak compared to C75 (1550.61 cm−1) and CH films (1550.8 cm<sup>−</sup>1).

At amide III region, the addition of the chitosan had as result a significant decrease in the wavenumber from 1239.9 for CO film to 1016.9 and 1022.7 cm−<sup>1</sup> for C50 and C75 films respectively. This shift induced by chitosan addition suggested some interaction between the CH2 side chains of collagen molecule with that of chitosan molecules [67].

As shown in Figure 8, amide A peak of collagen film decreased after incorporation of chitosan from 3294.5 to 3250.4 cm−<sup>1</sup> for C50 film and to 3288.5 cm−<sup>1</sup> for C75 film, this suggests the loss of hydrogen bonding between water and collagen by chitosan interaction [77].

Similarly, a shift to lower frequency was noticed for amide B from 2941.9 for collagen film to 2932.2 and 2925.42 cm−<sup>1</sup> for collagen-chitosan films C50 and C75. As previously reported, when the CH group of a peptide was involved in a hydrogen bond with other polymer, the position of amide B moved to lower frequency.

The FTIR spectra clearly indicated that interactions between the two polymers have taken place and the secondary structure of collagen had been changed by chitosan incorporation.

#### 2.2.5. Radical Scavenging Activity of Films

The radical scavenging activities, DPPH (1,1-diphenyl-2-picrylhydrazyl) of the different type of films are showed in Table 4.


**Table 4.** Radical scavenging activity DPPH (1,1-diphenyl-2-picrylhydrazyl)of the different biofilms.

All values are mean ± standard deviation; a,b different superscripts in the same column indicate significant differences (*p* < 0.05). C50: Collagen-chitosan film 50%:50%, C75: Collagen-chitosan film 25%:75%.

Pure collagen film exhibited the highest radical scavenger with 30.8%. However, the addition of chitosan into the collagen solution, induced a significant decrease (*p* < 0.05) of the DPPH radical-scavenging ability of the composite films to values of 23.91% and 19.77% for C50, C75, respectively, when compared to pure collagen film. This result might be explained by the reaction that took place between residual free amino groups of chitosan and free radicals which may form stable macromolecular radicals and ammonium groups [81].

The scarcity of data on the DPPH- scavenging activity of collagen-based films, did not allow a comparative study. However, when comparing with other work such as cuttlefish gelatin-based films, CO film exhibited similar DPPH- scavenging values (Table 4). Regarding the composite films (C50, C75), scavenging activity was less or similar to other composite films (Table 4).

#### **3. Experimental Section**

#### *3.1. Raw Materials*

Smooth-hound (*Mustelus mustelus*) were purchased from a local market in La Goulette, Tunisia and brought immediately to the laboratory. Fish were thoroughly washed with cold tap water and manually de-skinned as occurs in the marked. The cleaned fish skins were cut into pieces (approximately 1 cm × 1 cm) and subjected to a pre-treatment for collagen extraction.
