## 2 Chitosan in Brief  
### Extraction and Structure of Chitosan from Natural Sources  
Although chitin and CS have been known since the nineteenth century and the work of [PERSON] (1811) [15], research on these compounds really started around 1930 and was intensified after 1970. The major obstacle to their use lied in the difficulty of solubilizing them. However, research was encouraged by the fact that resources were abundant. Indeed, chitin is one of the most abundant polysaccharides on Earth, second only to cellulose [16; 17; 18]. It plays an essential structural role in the cell wall of fungi and yeasts, and in cuticles of arthropods and insects. chitin is a natural linear cationic polysaccharide consisting of \(\beta\)-(1,4) linked \(N\)-acetyl-d-glucosamine (GlcNAcNac) (Figure 1). CS is obtained by the deacetylation of chitin with concentrated NaOH solution, and consists of a heteropolysaccharideaccharide of \(\beta\)-1,4 linked d-glucosamineamine and \(N\)-acetyl-d-glucosamine (Figure 1). chitin and CS are characterized by the degree of acetamidation, denoted DA, and expressed as a percentage of acetamide groups present: it is greater than 50% in chitin and less than 50% in CS [18; 19]. In the case of CS, it is often preferred to mention the rate (%) of deacetylation, called DD, which corresponds to the relative amount of acetyl groups removed from chitin during the preparation of CS. Another definition considers that it is the solubility of the material in a solution of acetic acid, which defines the polymer as chitin or CS. In insects, fungi, diatoms, or marine animals, chitin is synthesized by chitin synthase (E.C. 2.4.1.16) [20]. In these organisms, chitin assembles in three distinct polymorphic forms named \(\alpha\), \(\beta\), and \(\gamma\) (parallel, antiparallels, or mixture of both) [1; 21]. The form of the chains is found to depend on the origin, and \(\alpha\)-chitin is the most abundant form. Chitin deacetylase (E.C. 3.5.1.41) partially removes acetyl substituents and defines the acetylation degree of the final chitin [22]. CS is rarely found in nature, contrarily to chitin. Extraction of chitin (Figure 2) from fishery wastes (carapace of crustaceans and shellfish) requires strong chemical treatments, such as deproteinization with hot alkali (NaOH 1 N, at 60-100 \({}^{\circ}\)C for several hours) and demineralization with acid (HCl 0.3-2 N at about 100 \({}^{\circ}\)C for one or two days) to eliminate calcium carbonate, and discoloration [17]. Regarding fungal biomasses, chitin is covalently linked with glucan. The extraction process of the chitin-glucan is more recent (Figure 2) [23; 24]. The extraction method includes hydrolysis steps, to separate the chitin-glucan from the rest of the mycelium, and lipid elimination by washing and drying. Then, CS is generally produced by partial deacetylation of chitin-glucan in a concentrated sodium hydroxide solution, for several hours at 110-115 \({}^{\circ}\)C, under inert atmosphere (N\({}_{2}\)), in the presence of a reducing agent (NaBH\({}_{4}\)). The deacetylation reaction is rarely complete, to avoid a sharp reduction in the molecular weight of the polymer. The use of high temperatures generally improves the reaction rates and yields [25]. Ultrasound and microwave technologies were also proposed to enhance the extraction and deacetylation steps [26; 27; 28; 29; 30; 31]. Furthermore, biological treatments offer an alternative to such hard chemical reactions: lactic acid bacteria and bacterial protease can be used to remove proteins and deacetylation can also be performed with enzymes [32; 33]. This produces higher quality products (better control of Mw and DA), but requires longer processes. The product is then dried and re-dissolved in an organic acid solution, in order to purify it. The CS obtained is in the form of an amorphous solid.