## 3 Results and Discussion
### Effect of Glycerol on the Thermal Denaturation of Collagen and TFE-Mixed Collagen  
We first investigate the effect of glycerol on the thermal denaturation of collagen. As seen in Figure 5, the \(T_{\rm d}\) is increased by increasing glycerol concentration. As for the 3 M glycerol, the \(T_{\rm d}\) is increased to 42.4 \({}^{\circ}\)C and the stabilization of collagen is improved. Figure 6 shows the inhibiting effect of glycerol on the collagen degraded by 10% (\(v\)/\(v\)) TFE. It can be seen that the initial optical rotation is restored and the \(T_{\rm d}\) value is increased to 36.4 \({}^{\circ}\)C as compared to the value of TFE-mixed collagen (\(T_{\rm d}\) = 33.9 \({}^{\circ}\)C). This result provides evidence that glycerol has effective inhibiting abilities on the thermal denaturation of collagen degraded by TFE, even under low glycerol concentration. We further examine the inhibiting effect of glycerol on the high-concentration TFE (20%, \(v\)/\(v\)) mixed collagen, wherein the helical structures have been almost destructed. As shown in Figure 7, 1 M and 2 M glycerol can increase the \(T_{\rm d}\) to 31.3 \({}^{\circ}\)C and 32.8 \({}^{\circ}\)C, respectively. Figure 8 shows the degradation of \(T_{\rm d}\) in the presence of TFE and the inhibiting effect of glycerol under different concentrations. The result indicates that glycerol increases \(T_{\rm d}\) by 2.2 \({}^{\circ}\)C in the absence of TFE, and further increases the \(T_{\rm d}\) of TFE-mixed collagen at high concentration by 4.2 \({}^{\circ}\)C. This improvement is in agreement with the fact that glycerol plays a role of stabilizer in the calfskin collagen solution and increases its \(T_{\rm d}\) by 0.96 \({}^{\circ}\)C [6]. It has been suggested that the inhibiting effect of glycerol on the thermal denaturation is achieved by the incorporation of its hydroxyl groups into the water-collagen molecular structure [7]. In other words, glycerol binds to the surface of collagen molecules through the formation of hydrogen bonding, which competes with the water molecules as a replacement for water-mediated hydrogen bonds. Subsequently, the extensive cross-linking structure inhibits collagen dissociation by glycerol [5]. [PERSON] obtained a negative chemical potential change of collagen binding with glycerol molecules using the Wyman linked function, and reported that the triple helical structure can be preserved due to the fact that glycerol interaction with the surface of native collagen is more energetically favorable than the interaction between water and collagen [10]. With increasing glycerol concentration, a large amount of hydroxyl groups containing additional and stabilizing hydrogen bonds is generated and leads to greater ability in the inhabitation of thermal denaturation. Similar results can be observed for the stabilizing effect of glycerol on the calfskin collagen degraded by urea [7], where about 1 \({}^{\circ}\)C increase of the denaturation temperature per 1 M glycerol was reported.  
Figure 5: _Cont._Figure 5: Effect of glycerol on phase difference of collagen as a function of temperature under (**a**) 1 M; (**b**) 2 M; (**c**) 3 M glycerol concentration (Collagen concentration: 0.341 mg/mL).  
Figure 6: Effect of glycerol on phase difference of TFE (10%, _v/v_)-mixed collagen as a function of temperature under different glycerol concentration: (**a**) 1 M; (**b**) 2 M (Collagen concentration: 0.341 mg/mL). The inset \(T_{\rm d}\) represents the denaturing temperature.  
Figure 7: Effect of glycerol on phase difference of TFE (20%, _v/v_)-mixed collagen as a function of temperature under (**a**) 1 M; (**b**) 2 M. glycerol concentration (Collagen concentration: 0.341 mg/mL). The inset \(T_{\rm d}\) represents the denaturing temperature.