# A Walkway from Crayfish to Oligochitosan
## 3 Results and Discussion
### Molecular Characterization of Chitosan and Oligochitosan Hydrochloride  
Chitosan is mainly characterized by molecular weight and degree of deacetylation (DD); i.e., describing chitosan requires that its main characteristics are mentioned. In addition, the term \"chitosan\" describes a family of polysaccharides that consist of glucosamine and N-acetylglucosamine, and differ by composition and molecular weight distribution. Polydispersity index \(\mathrm{D_{m}}=\mathrm{M_{w}}/\mathrm{M_{n}}\), which is equal to the ratio of the weight average molecular weight (\(\mathrm{M_{w}}\)) and the number average molecular weight (\(\mathrm{M_{n}}\)), is usually used as a measure of molecular weight inhomogeneity of macromolecules in a polymer sample. All these characteristics determine the physicochemical, antimicrobial, and biological properties of chitosan, as reviewed in [57]. After the separation of chitosan and the preparation of oligochitosan hydrochloride, their molecular characteristics were analyzed by the use of size exclusion chromatography (Figure 4). The calculated values are shown in Table 2.  
Figure 3: (**a**)—EDX analysis of chitosan; (**b**)—EDX spectrum of oligochitosan. As shown, the molecular characteristics of chitosan (M\({}_{\rm w}\) 370 kDa; D\({}_{\rm m}\) 2.88) and oligochitosan (M\({}_{\rm w}\) 11.3 kDa; D\({}_{\rm m}\) 2.88) correspond to the values of chitosan prepared from other crayfish species at similar conditions [31] and with the required characteristics of oligochitosan. The degrees of deacetylation of chitosan and oligochitosan were determined by a simple and fast \({}^{1}\)HNMR method using the relationship of the integral intensities of the protons belonging to residual acetyl-groups and protons of saccharide subunits. As it was determined, deacetylation of chitin in harsh conditions (40% NaOH solution, 120 \({}^{\circ}\)C) led to the product having a high degree of deacetylation of 98% (Figure 5); i.e., the value usually occurred when the deacetylation of crustacean chitin was subjected to deacetylation at harsh conditions [31]. Furthermore, the hydrolytic cleavage of chitosan macromolecules accomponed by additional acid deacetylation of chitosan [54] and its product led to oligochitosan with an increased degree of deacetylation of 98% (Figure 5). As shown in Figure 5, the spectra contains a low signal of residual acetyl-group protons at 1.92 ppm, the signal of proton at C2 position of the saccharide ring at 3.04 ppm, and a complex of signals of C2-C6 protons belonging to glucosamine and N-acetylglucosamine fragments in the range of 3.7-4.0 ppm. Molecular characteristics of oligochitosan determined by HP-SEC and NMR studies showed that these values were characteristic of oligochitosan and can provide a higher antimicrobial activity of oligochitosan, which grows with increasing DD [20,57]. Just as with the concentration of residual heavy metals and proteins, all other characteristics of chitosan and oligochitosan hydrochloride have been found to be in the range required by the United States Pharmacopeia (2013) for \"chitosan\" and the European Pharmacopeia (2004) for \"chitosan hydrochloride\" (Table 3). \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Sample** & **M\({}_{\rm w}\) (kDa)** & **M\({}_{\rm n}\) (kDa)** & **M\({}_{\rm p}\) (kDa)** & **D\({}_{\rm m}\)** \\ \hline Chitosan & 370 & 128 & 295 & 2.88 \\ \hline Oligochitosan & 11.3 & 6.0 & 8.6 & 1.89 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Molecular characteristics of chitosan and oligochitosan. Figure 4: HP-SEC elution profiles of chitosan (1) and oligochitosan (2). As it has been shown earlier, both chitosan and oligochitosan show both antimicrobial activity and synergistic/additive effects in combination with antibiotics that are promising with regards to their potential as antimicrobial activity-enhancing agents in pharmaceutical compositions [58; 59; 60; 61]. These properties of crayfish chitosan and oligochitosan make them promising for the application in pharmaceutical and cosmetic compositions.