## 4 Discussion  
In this study, chitin extracted from cicada sloughs harvested in two different years (2019 and 2020) has been characterized from a physicochemical point of view and compared to the chitin extracted from cicada sloughs harvested in 2017, the results of which are reported in a previous study [9]. The main objective of this comparison was to assess the stability of cicada sloughs as a source of chitin and to evaluate the efficiency of the extraction process. For that, we evaluated two of the parameters that mostly influence the chemical, physical and biological behavior of chitin and its derivative chitosan: the degree of acetylation/deacetylation (\(DA/DD\)) and the index of crystallinity (\(Crl\)) using simple techniques such as FTIR and XRD. Our results show that chitin extracted from cicada sloughs harvested in 2020 has a significantly lower degree of acetylation compared to chitin extracted from cicada sloughs collected in 2017 and 2019. Other authors reported variabilities within the source harvested in different years or seasons. For example, [PERSON] et al. [8] reported that the degree of deacetylation of chitosan obtained from the transformation of chitin extracted from shells of crabs harvested in three different seasons and years (July 2004, January 2005, and September 2007) was slightly different: 89.01%, 84.11% and 82.49% for the three seasons, respectively. However, in addition to having been harvested in three different years and seasons, samples (crab shells) were stored for different time before being characterized, and in particular for 3.5 years, 3 years and 3 months, respectively for shells harvested in July 2004, January 2005, and September 2007. The results showed that chitosan having longer storage time presented higher \(DD\). However, the authors of the study were unable to conclude if the differences were due to the storage time or to the season or year of collection of crab shells.In another study, [PERSON] et al. [7], reported that within the same source (that in their case was crustacean shells), the content in minerals, proteins and chitin and the physicochemical properties of the latter can differ based on the season when the shells are collected. However, again, there is no information about how the different compositions in \begin{table}
\begin{tabular}{c c c c} \hline
**Extracted Chitosan** & **CS-80** & **CS-90** & **CS-95** \\ \hline \(Crl\) (\%) & 53.7 & 42.8 & 49.4 & 49.4 \\ \hline \end{tabular}
\end{table} 
Table 5: Crystallinity index (\(Crl\)) % of the extracted chitosan and CS-80, CS-90 and CS-95. Figure 7: SEM images of: (**a**) extracted chitosan, (**b**) CS-80,(**c**) CS-90 and (**d**) CS-95. minerals, proteins and chitin would influence the characteristics of the final product chitin, in terms of degree of acetylation. In order to better analyze the differences that we found in the degree of acetylation of chitin extracted from cicada sloughs in different years, we compared the FTIR spectra of the raw materials (i.e., cicada sloughs). We found that two specific peaks (at 1420 and 1375 cm\({}^{-1}\)) have an opposite conformation in the sample CH-20 as compared to CH-17 and CH-19 which might justify the lower degree of acetylation of CH-20. However, the reasons for the lower degree of acetylation are unknown and further investigations are needed to understand the relation between \(DA/DD\) and, for example, abundance and structural organization of proteins and minerals within the source. Concerning the crystallinity, we found that chitin extracted from cicada sloughs collected in 2017 has a greater crystallinity compared to the ones extracted from cicada sloughs harvested in 2019 and 2020. The evaluation of the \(CrI\) measured for the raw material showed that also the raw material of cicada sloughs harvested in 2017 had a greater crystallinity compared to the raw materials of cicada sloughs collected in 2019 and 2020.