## 4 Discussion
This result suggests that, within the source, chitin might be organized differently, or sloughs have a different composition (in terms of proteins and minerals) giving rise to a more or less crystalline structure [23]. The evaluation of the \(CrI\) and for the intermediary products resulting from the extraction of chitin (i.e., demineralized, deproteinized) gave us some important information about the repeatability of the extraction process. In fact, the trend of crystallinity displayed by these products (i.e., decreasing after demineralization, increasing after deproteinization and being almost stable after decolorization) suggests that the extraction process produces coherent results. We then performed the deacetylation of chitin in order to obtain chitosan using a previously published protocol [10]. We compared the FTIR and XDR spectra and the resulting measured \(DD\) and \(CrI\) values of the extracted chitosan to three commercial chitosan with known \(DD\). We found that the method employed in this study for the calculation of the \(DD\) was efficient for chitosan with high \(DD\) (90 and 95%), while it was less precise for chitosan with lower \(DD\) (80%). Using this method, the \(DD\) that we measured for the extracted chitosan was \(66.2\pm 1.6\%\). Even if the molecule extracted could be considered as chitosan (since the \(DD\) is higher than 50%), this result suggests that the deacetylation process was not the most appropriate for the deacetylation of this source. [PERSON] et al. [10], by using the same protocol for the deacetylation of chitin extracted from cicada sloughs, obtained a chitosan with a \(DD\) of 84.1%. However, in their study, the \(DD\) was calculated using different absorbance peaks of the FTIR spectra, which might lead to different results. Concerning the crystallinity, we found that the extracted chitosan had a higher \(CrI\) compared to the commercial chitosan. Several studies show a tendency of the \(DD\) and \(CrI\) to be indirectly related with higher \(DD\) showing lower \(CrI\)[18, 22]. However, other studies show an opposite trend [20, 21] and this relation is still an object of discussion. The methods employed in this study for the calculation of the \(DA/DD\) and \(CrI\) present some limitations. In fact, more sophisticated techniques such as Nuclear Magnetic Resonance (NMR) spectrometry and X-ray photoelectron spectroscopy (XPS) would allow us to obtain more precise measurements of the degree of acetylation and alternative methods for the calculation of the \(CrI\) have been proposed in the literature (i.e., the use of the peak \(I_{020}\) in the Equation (3) instead of \(I_{110}\)[18]). However, when applied uniformly, the FTIR and XRD methods employed in this study are highly efficient to compare multiple samples among them. The characterization, in particular, of the \(DD\) of chitosan, is essential since it influences its possible applications [24]. In the biomedical field, \(DD\) is one of the most important properties of chitosan since most of its biological properties are attributed to its cationic behavior, which depends on its \(DD\). In fact, in acidic solutions, the amine groups (NH\({}_{2}\)) of chitosan acquire a positive charge (NH\({}_{3}\)\({}^{+}\)) [25]. This property makes chitosan the only naturally cationic polysaccharide, allowing it to form polyelectrolyte complexes with negatively charged polymers [4]. Several studies investigated the effect of different \(DD\) for different applications. Some authors report that chitosan with high \(DD\) is more suitable for tissue engineering [26] because usually constructs made of chitosan with lower \(DD\) degrade faster, leading to less supportive healing. However, other studies reported the higher the \(DD\) of chitosan, the lower was the adhesion of keratinocytes and fibroblasts on chitosan films [27]. Chitosan is also highly used as a vehicle for nucleic acid or drug delivery (in form of nano- or micro-particles). [PERSON] et al.