2.1.4. Ultraviolet Spectrophotometric Analysis

The ShS-ASC and PSC collagens exhibited maxima absorbencies at 235 nm and 240 nm respectively (Figure 4), which is in agreement with the maximum absorption of the collagen molecule (230 nm) [39]; principally due to the *n* → π\* transitions of the peptide band C=O [40].

Unlike other protein types no peak was detected at 280 nm, suggesting that ASC and PSC collagens have low amount of aromatic residues such as tyrosine and phenylalanine [41]. These results are in line with those previously found in fish skin collagens [34,42–44] and confirm the effectiveness of the alkaline treatment for the removal of non-collagenous proteins.

**Figure 4.** Ultraviloet-Visible spectra of acid soluble (ShS-ASC) and pepsin soluble collagen (ShS-PSC) from the skin of hound smooth.

#### 2.1.5. Fourier Transform Infrared Spectra of Collagens

In order to determine the isolated collagens type; Fourier transform infrared technique was used to detect the vibrational modes of bands and individual chemical groups in the extracted collagens [45].

The ShS-collagen's Fourier Transform Infrared (FTIR) spectra represented in Figure 5, showed that the amide A band of ASC and PSC originated from the stretching vibrations of N–H group were found at 3293.57 and 3296.16 cm−<sup>1</sup> respectively, although it commonly appears in the range of 3400–3440 cm−<sup>1</sup> [19,46]. These shifts to lower frequencies means that the collagen NH groups of the samples were involved in hydrogen bonding, which help to stabilize the collagen triple helix structure. The absorption peak of amide B, related to asymmetrical stretch of CH2 [47], appeared at 2942 cm−<sup>1</sup> for ASC and at 3092.05 cm−<sup>1</sup> for PSC. Such results are concordant with that reported for the collagen extracted from the skin of splendid squid [13].

The amide I band mainly associated with stretching vibrations of carbonyl groups (C=O bond) along the polypeptide backbone [48], was depicted at 1629.6 cm−<sup>1</sup> and 1629.5 cm−<sup>1</sup> for ASC and PSC respectively, this amide is actually a sensitive marker for peptide secondary structure [49].

**Figure 5.** Fourier Transform Infrared spectra of ASC (acid soluble collagen) and PSC (pepsin soluble collagen) from the skin of hound smooth fish.

The PSC and ASC amide II bands were situated at wavenumbers of 1548.95 and 1545.17 cm−<sup>1</sup> respectively; while the ASC and PSC-amide III bands were located at wavenumbers of 1237.94 and 1239.84 cm−1, respectively. The amide II band represent N=H bending vibrations coupled with C=N stretching vibration [50], and amide III peak reflects intermolecular interactions in collagen, including peaks from C–N stretching and N–H deformation from amide linkages. It is also related to absorptions resulting from wagging vibrations from CH2 groups from the glycine backbone and proline side-chains [51].

The Infra-Red absorption (IR) ratios between amide III and 1454 cm−<sup>1</sup> band for the ASC and PSC fractions were found around 1 (0.95 and 1.08 respectively); indicating the persistence of the triple helix structure within the extracted collagen [52].

Such detailed description allowed to conclude that the slight differences observed between the ASC and PSC structure may be caused by pepsin treatment which has the effect to remove the telopeptide region, whereas the similarity of IR ratios may suggest that pepsin had no influence on the structure of the collagen triple helix.

#### *2.2. Biofilms Mechanical and Functional Properties*

#### 2.2.1. Mechanical Properties

One of the laboratory objectives was to elaborate green edible biofilm using collagen from seafood by-products without any chemical addition and at low collagen percentage taking in consideration the cost of its production. However, when using skin *M. mustelus* collagen solution at 0.1%; the film was too fragile to allow any mechanical properties analysis (Figure 6A). Therefore, blending collagen with another polymer such as chitosan known for its high film-forming ability and lower cost was necessary to enhance the biofilm compactness (Figure 6B). Beside the resulting film showed akin aspect to the pure chitosan film (Figure 6C). The results suggest that the aggregation occurring between the collagen molecules of the film matrix was filled by the dispersed chitosan enhancing the cohesion between the various complexes within the adsorbed layer as shown in other study [53].

**Figure 6.** Pictures of (**A**) pure collagen film CO, (**B**) bi-composite Collagen-chitosan film and (**C**) pure chitosan film CH.

Such assumption is reflected by the thickness of the elaborated biofilms which showed different values as summarized in Table 1. The highest thickness value was noted in the pure chitosan film (17.15 μm) and decreased with an increased collagen ratio into the chitosan solution. Similar result were reported for the composite films using chitosan and collagen from the unicorn leatherjacket skin [54]. In the present study, higher chitosan ratio (75%) had no significant effect (*p* > 0.05) on film's thickness (Table 1) which showed similar smoothness and compactness. Such results suggest that the chitosan charge density was sufficiently high at the 50% ratio to assure the complexation between protein and polysaccharide knowing that the degree of compactness of the gel network is regulated by the polysaccharide charge density [55].


**Table 1.** Thickness and mechanical properties (TS: Tensile strength and EAB: elongation at break) of CH and composite films C50 and C75.

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

C75 66.28 <sup>±</sup> 2.7 a,b 4.49 <sup>±</sup> 0.23 <sup>b</sup> 16.07 <sup>±</sup> 1.10 <sup>b</sup>

The tensile strength (TS) was also affected by degree of protein/polysaccharide ratio. Thus, the pure chitosane film value was 70.52 MPa and decreased with the addition of collagen. The increased chitosan proportion in composite films increased significantly (*p* < 0.05) the tensile strength from 55.42 MPa for C75 to 66.28 MPa for C50. This is not only due to the interactions between chitosan and collagen molecules by electrostatic force but also by hydrogen bonding [56,57]. Similarly, the percentage of elongation at break (EAB) increased significantly (*p* < 0.05) with the incorporation of collagen in composite films from 4.25% in pure chitosane films to 4.49–5.67% in C75 and C50 respectively. This phenomenon is attributed to the hydrophilic properties of collagen which provides a certain increase in the hydration degree of the film giving an upper elongation at break value [58].

The comparison of the mechanical properties of the obtained composite films with those found in literature gives contradictory interpretations (Table 2) due to several factors including species origin (habitat, diet), the collagen amino acid composition [59], the protocol of extraction and the polymers ratio (collagen/chitosan).

Similarly, the mechanical properties of chitosan-based films are affected by various parameters such as the chitosan deacetylation degree, their molecular weight, as well as the conditions of film preparation (pH of the film-forming solution, the water content, and the drying conditions) [60–63].

In this study, composite films (C50, C75) exhibited tensile strength of similar or higher values than commercial films (LDPE 13%, HDPE 26%, Hydroxypropyl cellulose 15%) and collagen-chitosan-based films reported in other studies (Table 2). However, the elongations at break values of the composite films (C50, C75) were much lower than those of commercial films, since there is an inverse relationship between TS and EAB [64].


**Table 2.** Table summarising tensile strength (TS) and elongation at break (EAB) values of biofilms reported in other works and commercial films.
