# Shellfish Chitosan Potential in Wine Clarification
## 1 Introduction
The second step, called \"deacetylation\", is performed with a low amount of substrate (1:30-1:40 _w/w_) at a high alkali concentration (NaOH 1-4 M), high temperature (80-121 \({}^{\circ}\)C), and for short contact times (15 min-3 h) in order to remove the acetyl group from the chitin chains. The third step, called \"post-treatment\", generally occurs as a low concentration acid washing (frequently acetic acid at a concentration of 0.5-2% _v/v_ for fungi and 2-10% _v/v_ for crustaceans) that permits the recovery of deacetylated chitin (chitosan), leaving behind insoluble chitin. In fungi, the residual chitin is typically associated with the \(\beta\)-glucans through covalent bonds that make its recovery difficult without degradation. The amount of insoluble chitin-glucan complex could easily reach 16% of the total \(\beta\)-glucans [7]. After the first washing, the chitosan-acid solution is brought to pH 10 to precipitate the CTS. Finally, the precipitate is washed, commonly with  
Figure 1: General structure of fungal cell wall. a mixture of water, ethanol, or acetone, and dried. However, several variants of this general protocol could be found in the literature [2,3] according to the producers manufacturing process. Chitosan demonstrates high plasticity, and, thus, it can be prepared in different forms, namely as films, gels, beads, and nanoparticles [1,8,9]. CTS could be used in several sectors, such as medicine, cosmetics, agriculture, and food [1,10] in light of the high number of its valuable properties, such as its biodegradability, biocompatibility, and low toxicity. CTS also exhibits high potential as antimicrobial and antioxidant agents; it could be used in the preparation of films that act as a barrier against chemical-physical changes, and the properties that it possesses by itself could be further enhanced through the combination with other useful compounds (i.e., silver, catechins, or organic acids) [10]. In winemaking, a pioneering work [11] demonstrated the possibility of using chitosan to remove phenolic compounds and increase the oxidative stability of white wines. A few years later, chitin and its derivatives were suggested to remove specific wine proteins (i.e., class IV grape chitinases) [12]. The authors found that the addition of chitin reduced the wine haze of 50% even at 1 g/L and that 20 g/L was sufficient to achieve 80% of potential haze removal. Chitosan has been admitted by the International Organisation of Vine and Wine (OIV) and European Commission since 2009 and 2011, respectively [13, 14, 15]. Since then, it spread as a fining agent for different purposes, i.e., regulation of iron and copper excess; reduction of heavy metals or possible contaminants (for example, ochratoxin); and inhibition of unwanted microbial growth, especially _Brettanomyces_ spp. Today, OIV only permits the use of fungal chitosan (from _A. niger_), in order to avoid allergenic reaction due to the crustacean material, even if the functionality and the structure of the chitosan derived from crustaceans and fungi are declared identical by the producers. Several studies tried to define the details for the optimization of chitosan extraction [4,16,17]. The most determinant chemical characters for chitosan are the deacetylation degree and the molecular weight. Previous studies discovered that the acid (organic or inorganic) used for chitosan dissolution manifests an effect on chitosan properties, such as the viscosity, mainly through the interaction with -NH\({}_{2}\) charged residue [18]. The acids used for dissolution were supposed to contribute in a different way to other chitosan properties, enhancing its antifungal activity [19] or the interaction with other compounds [20], for example. In this work, the efficiency of chitosan on wine clarification was evaluated, comparing animal and fungal chitosan. At first, an overall of 10 commercial samples were characterized for the degree of deacetylation and chitosan purity.