# Shellfish Chitosan Potential in Wine Clarification
## 3 Results and Discussion
### Wine Clarification Performance  
Several works explored the effects that different solvents have on chitosan properties, testing, for example, antimicrobial activities against bacteria and mold [19; 32], CTS membrane properties and hydrophobicity [33], CTS film water vapor permeability [17], resistance, and elasticity [34]. However, no studies explored whether and how the choice of the acid used for the CTS dissolution influences wine clarification. Clarification is a process that occurs in nature and is linked to the flocculation and precipitation of suspended colloids, and chitosan is known to enhance this process by the instability generated by the interaction between colloids and NH\({}_{2}\) residues of chitosan [35]. Chitosan physiochemical characteristics, such as degree of deacetylation and molecular mass, affect the clarification results [36]. Studied samples evidenced heterogeneities for both DDA and molecular mass, and the viscosity test indicated that dissolution acid could affect chitosan molecular conformation. Therefore, a clarification test was performed comparing fungal- and animal-derived chitosan dissolved into the four acids. Turbidity (3593 NTU at the beginning) was recorded at 30 min, 2 h, 4 h, and 24 h. After 24 h, all of the samples demonstrated a very low turbidity, on average 112 NTU in the control, that makes the comparison difficult. Therefore, that point was excluded from further considerations. Even though chitosan has been studied as wine fining in previous works [37; 38], only the effects on the final wine composition were analyzed, without deepening its flocculation and clarification capacity. In addition, this is the first time that animal and fungal chitosan are compared in the wine clarification: the results here reported evidence that fungal CTSs efficiently remove the colloids during the treatment; however, the data showed a clear distinction between chitosan categories (\(\chi^{2}_{(2)}=49.83\), p \(<0.01\)), with fungal CTS that already reduces the wine turbidity after 30 min, by about 25%, and keeps on lowering it in the successive hours (Figure 6), and animal CTS showing a surprising clarification capacity by dropping the NTU value by about 60%. Even though the need of an acidic environment for chitosan dissolution is well known, the first chitosan-based products proposed for the enology sector were supplied as a powder to be prepared in water or directly in wine. Only recently has the market started to propose \"soluble\" chitosan, which already contain the acidic component needed for their dissolution. In most cases, hydrochloric acid is used (chitosan hydrochloride, CAS 70694-72-3), but other inorganic and organic acids can be used for the same scope. For this reason, the effect of the four dissolving acids, choosing among those compatible with the wine environment, on the clarification capacity was also studied. The relationship between CTS and the dissolving acid was evaluated more specifically at two time points, namely after 4 h for fungal CTS and after 2 h for animal CTS, according to the significant statistical difference detected between the two successive time points (F\({}_{(2,\,141)}\)= 15.3, p \(=\)\(\leq\)0.001 and F\({}_{(2,\,213)}\) = 12.76, p \(=\)\(\leq\)0.001, respectively). Figure 7 reports the turbidity values achieved from the samples 4 h after the treatment with MC. Statistical analyses revealed a significant difference among samples that is independent from the dissolving acid, expressed by the different numbers above the groups. In more detail, the sample outcomes depended--only in two out of four cases--on the interaction between the sample and the dissolving acid, namely when the higher ionic strength acids (SA, HCI) were used. These findings suggest that the variations depend on the mechanism of the primary amines protonation and probably on the CTS charge density [34].