## 2 Chitooligosaccharides Production  
Several technological approaches employed for the preparation of COS are summarized in Figure 2. The production of COS can be achieved by physical, chemical and enzymatic methods or a combination of them. Figure 1: Types of chitooligosaccharides (COS): fully deacetylated (_fd_COS), partially acetylated (_pa_COS) and fully acetylated (_fa_COS). Physical methods for chitin or chitosan hydrolysis include ultrasonication, hydrothermal treatment, hydrodynamic cavitation, and electromagnetic, gamma rays or microwave irradiation [21; 22; 23]. However, these methods have been scarcely investigated and are not well-suited for producing well-defined COS mixtures. Chemical procedures for COS preparation comprise degradative methods, such as chitin or chitosan depolymerization by acid hydrolysis or oxidation, and chemical synthesis strategies. Acid hydrolysis is the most employed method for COS production at an industrial scale and involves the use of hydrochloric acid, nitrous acid, phosphoric acid or hydrogen fluoride [24]. The oxidative-reductive depolymerization is also widely applied and generally relies on the use of hydrogen peroxide, persulfate or sodium nitrite [25]. Both alternatives give rise to heterogeneous COS mixtures with typically low degrees of polymerization. In addition, they are environmentally risky and generate detrimental byproducts that make COS not suitable for human consumption due to possible contamination and toxicity. Chemical methods for the synthesis of COS have progressed substantially in the last decades, but they usually result in low yields and may require protection/deprotection steps and the use of hazardous chemicals [26]. Enzymatic production of COS is very attractive since it requires milder conditions (relatively low temperatures and slightly acidic pH) and does not generate harmful byproducts or wastes [27], which makes it safer and more environmentally friendly approach [28]. In addition, strategies based on biocatalysis present a higher efficiency and allow easier control of the product composition due to inherent enzyme specificity. However, the physicochemical properties of the substrate substantially affect the chemical composition of the final product. Enzymatic depolymerization of chitin or chitosan can be catalyzed by several glycoside hydrolases (GHs), including specific enzymes, such as chitinases [29; 30] and chitosanases [31; 32], as well as nonspecific enzymes like other glycosidases (cellulaases, glycanases, pectinases, muramidase, etc. ), proteases, and lipases [16; 33; 34]. Figure 2: Methodologies for the preparation of chitooligosaccharides. Chitinases (EC 3.2.1.14) occur in families GH18, GH19 and GH20 [35] and are able to hydrolyze GlcNAc-GlcNAc linkages [36; 37]. Family GH18 comprises bacterial and fungal chitinases [38], while family GH19 mainly contains plant chitinases. Chitinases can be classified, according to their mode of action, into three major groups: (1) endo-chitinases, (2) exo-chitinases and (3) \(\beta\)-N-acetylglucosamine. Glycos belongs to families GH18 and GH19 are considered endo-chitinases since they catalyze the depolymerization of chitin. Family GH20 only includes exo-chitinases and \(\beta\)-N-acetylglucosamine. COS production requires the action of endo-chitinases, whereas N-acetyl-glucosamine preparation entails the use of exo-chitinases and \(\beta\)-N-acetylglucosamineaminidases. Chitinases are also capable of hydrolyzing both GlcNAc-GlcNAcN and GlcN-GlcNAc linkages, enabling chitosan depolymerization, although to a lesser extent than chitosanases. GH18 chitinases require a GlcNAc moiety at subsite -1 [39], whereas GH19 chitinases have no necessity for GlcNAc at this subsite. For this reason, chitinases belonging to family GH18 give rise to COS with GlcNAc residues at their reducing ends [40]. On the other hand, oligomers produced by GH19 chitinases can present N-acetylated or deacetylated reducing ends.