**1. Introduction**

The small-spotted catshark (*Scyliorhinus canicula*) is one of the most abundant elasmobranchs in the Northeastern Atlantic Ocean [1]. Although its landings are sometimes devoted for human consumption (rendering 10% and 16% of fish weight in the form of skin and viscera by-products respectively), it has low commercial value and very often is captured as by-catch resulting in a very high discard rate reaching 100% in some European fisheries. The reduction of post-harvest fish losses (discards and by-products) by the promotion of a full use of fishing captures is one of the main purposes of EU fishing policies [2]. The full use of fishing captures includes the transformation of raw materials for the isolation/production of molecules that could be used in a wide variety of applications, which is indeed one of the approaches included in the "blue growth" strategy of the European Commission. One interesting bioactive compound which could be obtained from fish discards is collagen. Collagen is the main protein present in animal connective tissue and although there are several types of collagens,

type I is the most abundant in the skin and bone of teleost fish [3]. Type I collagen, which is a fibrillar collagen, is a heterotrimeric molecule composed of two α1-chains and one α-2 chain with a similar molecular weight of about 100 kDa [4].

Collagens obtained from marine sources include several *Osteichthyes* and *Chondrichthyes* species, jellyfish, mollusks, sponges and sea cucumbers, among others [5–10]. As collagens are being used increasingly as alternatives to mammalian collagens for cosmetics, tissue engineering and other biomedical and pharmaceutical uses, due to safety reasons and ethical or religious constraints, fish skins from discards or by-products represent an excellent and abundant source for obtaining this biomolecule [5,8]. The main difference between marine and mammalian collagen includes a lower content of imino acids (proline and hydroxyproline) in marine collagen, which also influences the lower thermal stability shown by marine collagens [4,6]. In the literature, there is abundant information regarding the extraction of collagen from the skin of different marine species [4,6], however there are only few publications regarding the optimization of the key parameters influencing the process of extraction (temperature, concentration of NaOH and acetic acid and also time of incubation) [11–13]. Thus, having in mind the importance of extraction conditions to achieve a higher collagen yield, and although acid-soluble collagen (ASC) has been obtained previously from the skin of the small-spotted catshark [6], the yield was 52%. It is necessary to study the effects of extraction conditions on trying to obtain a higher recovery of collagen from this species.

The collagen extraction process comprises two main steps: the first step consists of the removal of non-collagen proteins and other impurities such as lipids, calcium etc. from the skin, with the aim of increasing the purity of the collagen extracted. To achieve this objective, 0.1 M NaOH is generally used, with different stirring incubation times [14–16]. The de-proteinized skin is then washed with cold water until it reaches a neutral pH and filtered. The second step in the extraction process consists of an acidic extraction of the previously NaOH treated skin, commonly using 0.5 M AcOH with different incubation times (48 h, 72 h, etc.) [4,5,17]. All procedures are usually done at 4 ◦C. After centrifugation, supernatant containing the acid-soluble collagen (ASC) is dialyzed and freeze-dried.

As there are several factors influencing the two-step collagen extraction process (time, temperature, NaOH concentration and AcOH concentration) and there is a need to study the optimal conditions of each variable and also the interactions between them, response surface methodology (RSM) has been employed to predict the optimal experimental conditions. RSM is a tool that has been previously used for the optimization of collagen extraction conditions from the skin of different fish species [12,13,18,19], however none of those studies included all the key optimization parameters influencing the two main steps of the extraction process. Thus, this is the first study optimizing the complete process for the extraction of acid-soluble collagen by means of three variables in each optimization stage: temperature, time and chemical treatment (NaOH or AcOH) concentration, from the skin of the small-spotted catshark.
