## 3 Results and Discussion
### Analysis of Bio-Functional Characteristics
#### 3.5.2 CD Analysis  
Circular dichroism spectroscopy is an effective method to study the three-dimensional structure of an organic biomacromolecule, which can provide the absolute configuration,  
\begin{table}
\begin{tabular}{c c c c} \hline  & **-OH\&-NH\({}_{\text{2}}\)** & **All Groups** & **PS** \\ \hline CS & 1225.89 & 4360.71 & 0.28 \\ CSC & 1291.36 & 4306.67 & 0.30 \\ CSS & 1306.55 & 4216.13 & 0.31 \\ \hline \end{tabular} PS means primary structure value, which is the ratio of hydroxyl group to amino group in the molecular structure of chitosan.  
\end{table}
Table 4: Primary structure value of CS, CSC and CSS.  
Figure 5: FT-IR spectra of chitosan (**a**) and fitted FT-IR spectra of CS (**b**), CSC (**c**), CSS (**d**).  
conformation and other information of molecules. [PERSON] [45] reported that the absorption peak height was related to the content of acetyl group. [PERSON] [46] deemed that the absorption of CD is independent of the configuration of \(\alpha\) and \(\beta\) heteroephalic carbon, the length of the molecular chain, the ionic strength and the pH.  
The CD spectral of chitosan samples are shown in Figure 6. It can be observed that each sample has a wide negative absorption peak at around 210 nm, indicating that chitosan still maintains a double helix structure, which is caused by the n-\(\pi^{\ast}\) electronic transition of NH-CO chromophore in chitosan molecule. The low CD absorption peak of CSS is attributed to high DD and less carbonyl chromophores, while the absorption peak of CSC and CS are obvious, indicating that the absorption peak decreases with the increase in DD. The positive Cotton effect appeared at 180-200 nm, which is due to the existence of C-O and O-H groups in the polysaccharide molecule, indicating that \(\alpha\)-helix and \(\beta\)-sheet existed in the SS of chitosan. The content of conformation unit was calculated by CD according to [PERSON] et al. [47]. The results in Table 5 showed that the content of the \(\beta\)-sheet in chitosan is higher than that of \(\alpha\)-helix. The spectrum of CSC was similar to that of CS, but the shape and position of the peak changed, and a positive peak appeared at 190 nm and 184 nm, respectively, which indicated that not only the DD of the samples treated by high temperature and high pressure increased, but also the stereoscopic configuration changed. CSS formed a weak positive peak at 196 nm and 183 nm, and a strong negative peak at 190 nm, indicating that it contained \(\beta\)-turn structure. The reason for this difference may be that the \(\alpha\)-helix and \(\beta\)-sheet of \(\alpha\)-chitosan are interphase in space, and most of the folding chains are parallel, while the \(\alpha\)-helix and \(\beta\)-sheet of \(\beta\)-chitosan are separated in space, and most of the folding chains are antiparallel.  
The SS conformation units \(\alpha\)-helix, \(\beta\)-sheet and \(\beta\)-turn have great influence on the biological function of polysaccharides, so the eigenvalue of SS (Table 3) is expressed by the ratio of the total content of \(\alpha\)-helix, \(\beta\)-fold and \(\beta\)-turn to random coil. The effect of the advanced structure on function is more important than that of PS, and the stereo structure of polysaccharide is one of the determinants of its physiological activity. The results laid a foundation for further study on the relationship between the structure-activity relationship and biological activity function of chitosan.  
Figure 6: Circular dichroism spectroscopy (CD) spectra of CS (A), CSC (B), CSS (C).  
\begin{table}
\begin{tabular}{c c c c c} \hline  & \(\alpha\)**-Helix** & \(\beta\)**-Sheet** & \(\beta\)**-Turn** & **Random Coil** \\ \hline CS & 8.5 & 35.1 & 7.1 & 49.3 \\ CSC & 8.3 & 36.6 & 6.9 & 48.2 \\ CSS & 7.7 & 39.5 & 11.5 & 40.3 \\ \hline \end{tabular} Number in the table is the proportion of each secondary structure of chitosan.  
\end{table}
Table 5: Different secondary structure contents of CS, CSC and CSS.