**1. Introduction**

During the last decades, the increased consumer awareness of the nutritional value of fish and seafood and the shift towards more processed fishery products in convenient form; has generated larger quantities of by-products accounting for up to 70% of the volume of fish and shellfish [1]. In most cases, such biomasses, which include skin, head, bones and viscera, cause serious economic and ecological issues. However, such biomass is currently of high interest to researchers and industry as it represents a valuable source of compounds with high added value such as proteins, lipids, enzymes, and polysaccharides.

Animal body contains high amount of collagen constituting around 30% of the total amount of protein in vertebrates [2]. Actually, 27 types of collagens have been identified and collagen type I is the most frequent one and is known as fibrillar collagen and plays a structural role by contributing to the molecular architecture, shape and mechanical properties of skin tissues [2,3]. Due to its excellent properties (non-toxicity, low antigenicity and allergenicity, biocompatibility, the ability of film-forming and biodegradability), collagens are utilised in various fields such as medical, pharmaceutical and cosmetics industries, and also as materials for food packaging [4–6].

At the start of its use, collagen was mainly extracted from porcine and bovine sources. Later, people started to show reticence toward this practice due to religious background, beside the proliferation of bovine spongiform encephalopathy (BSE) [7] which represented a source of hazard contamination for the extracted protein [8,9]. As a consequence, several researchers have been interested in marine collagen as an alternative because of the absence of disease transmission and dietary restriction [10].

Collagen has been studied and characterised from various marine sources, mainly from marine invertebrates such as cuttlefish [11], octopus [12], squid [13], jellyfish [14], starfish [15], sea urchin [16], sea cucumber [17] and also of sponges which represent the key of their complex structure and integrity [18]. In marine vertebrate organisms, such interest was rather oriented to fish by-products including scales [19], skin [20,21], swim bladder [22], bone [23] and cartilage [24].

For their extractions, collagens are commonly solubilized in organic acid, generally acetic acid which causes the protonation of collagen polypeptides and consequently the repulsion between the tropo-collagen leading to enhanced collagen solubility [25]. However, such procedure referred to Acid Solubilised Collagen (ASC) gives generally low collagen yield. Therefore, research was oriented to enzymatic extraction to increase collagen solubilisation with pepsin being among the most efficient enzyme. Thus, pepsin provokes not only the cleavage of the collagens teleopeptide region maximising their solubility, but also the hydrolysis of non-collagenous proteins increasing collagen purity. In this case, pepsin maximise the extraction yield of collagens while reducing their antigenicity [26,27].

The common coastal smooth-hound *Mustelus mustelus,* is an abundant species of the genus *Mustelus* in the Mediterranean Sea where it is regularly caught all over the year either as by-catch or as targeted species [28,29]. The consumption of such species generates significant amounts of waste that may be used as source to extract substance of interest such as collagen.

To our knowledge, the extraction of collagen from the skin of *Mustelus mustelus* and its valorisation has never been reported. Therefore, the aim of this work was to extract and characterize collagen using two methods. In a first step, the isolation of collagen was elaborated using acetic acid, which allows a better solubilisation of the molecule followed in a second step, by an enzymatic extraction using pepsin.

Our second objective was to elaborate a biodegradable film using collagen, however following the extraction process (alkali then acid process), the collagen molecule loses its strong mechanical strength compared to the native form [30]. To overcome such issue, we blended collagen with another natural polymer such as chitosan derived from chitin known as the second most abundant polysaccharide after cellulose. Thus, chitosan has attracted much attention for its biodegradability, biocompatibility, bacteriostatic and fungistatic activities as well as for its texturizing properties and its ability to film forming [31]. Therefore, we used chitosan as an adjuvant to elaborate composite film.
