# A Walkway from Crayfish to Oligochitosan
## 3 Results and Discussion  
### Crawfish Waste Treatment and Separation of Cittin  
In the present study, the main attention was devoted to the elaboration of a laboratory protocol for the preparation of oligochitosan hydrochloride directly from the crayfish _Acta cus leptodactylus_ aqua cultured in an artificial lake, with the purpose of determining how the level of protein and heavy metals in the crayfish chitosan, as a precursor of oligochitosan, met the requirements of USP2013 to become pharmaceutical-grade chitosan. As was shown earlier, commercial chitosan batches contained excessive concentrations of residual heavy metals (mainly Fe, Cr, and Ni) originated from the leaking of these metal ions from stainless steel-made reactors during the process of deacetylation at harsh conditions (high temperature and NaOH concentration) [35]. A lack of data on the applicability of aqua-cultured crayfishes as a source of preparation for pharmaceutically pure oligochitosan hydrochloride moved us to evaluate how _A. leptodactylus_ suits to the preparation of oligochitosan hydrochloride suitable to biomedical testing, at least on a laboratory scale.  
For this purpose, crayfish shell waste of whole crayfish was separated from the edible crayfish meat. The crude shells, which were disintegrated to tiny pieces, were thoroughly washed to separate most of the unbound proteins. Residual proteins were extracted from the crude shells by 1 M NaOH solution at heating. As shown, the extraction led to a transparent crude extract (CS), brown in color (Figure 1, sample 1). After the deproteination, demineralization, and decolorization of the shells, the residual proteins in the deproteinated shells (DPS) and deproteinated/demineralized/decolorized shells (DDS) were additionally extracted using 1 M NaOH solution to evaluate the content of residual proteins after these stages. As shown in Figure 1 (samples 2 and 3), alkaline DPS extract and DDS extract were yellow in color. Since [PERSON]'s reagent is an acidic solution of CBB-250, all alkaline extracts were acidified to pH-7. After the acidification, the CS extract remained brown but become turbid at further acidification to pH \(<\) 5 due to precipitation of excessive proteins from the solution (Figure 1, sample 5). In contrast, alkaline DPC and DDC extracts were transparent and light yellow in color (Figure 1, samples 2 and 3) and remained as transparent and almost colorless solutions even after further acidification since they contained less proteins (Figure 1, samples 6 and 7).  
The concentration of protein in the extracts determined by [PERSON]'s method is shown in Table 1. At this stage, the importance of the glass flask chosen for deproteination and demineralization should be mentioned, namely the glass composition. Preliminary ICP-MS analysis showed the presence of Fe, Cr, and Ni in the crude waste, and the absence of any other heavy metals (Cd, Pb, and Hg) including arsenic (Table 1). After washing off the waste, deproteination, demineralization, and decolorization stages, most of proteins as well as the main part of Fe and all residual Cr and Ni were separated from crayfish chitin (Table 1). Nevertheless, semi quantitative EDX analysis revealed a presence of Al (0.7%) and Si (0.3%) in the chitin (Chitin-AlSiG) collected after the stage when the processes of were carried out in an alumosilicate glass (AlSiG) flask (Figure 2a). In contrast, the use of a borosilicate glass (BSiG) flask, which was more resistant to the alkaline and acidic media, led to the chitin Chitin-BSiG, which contained a low concentration of Fe (Table 1) and residual Si (0.2%) with an absence of aluminum (Figure 2b).  
Figure 1: Alkaline (1–4) and acidified (5–8) extracts: SC extract (1,5), DPS extract (2,6), DDS extract (3,7), and chitosan extract (4,8).