## 3 Results and Discussions
### The Influence of Disulphide Bond Formation on Protein Functionality
#### 3.3.3 Surface Tension (\(\gamma\))  
The capacity and stability of the protein-stabilised emulsions are primarily associated with the protein surface activity at the water-oil interfaces [68]. Differences in the flexibility of protein molecules can cause them to either unfold or maintain their tertiary structure. For instance, lysozyme contains excessive disulphide bonds and retains its structure when adsorbed at an interface and is therefore not recommended to be used as an emulsifier [69]. In the current study, the adsorption kinetics of protein was investigated to help explain observed changes in emulsifying ability based on protein adsorption capacity at an interface. The interfacial surface tension of different hempesed proteins was measured for 20 min, as shown in Figure 8A. Among the hempesed proteins, the larger reduction in surface tension was observed for the proteins extracted for the shortest time of 5 min, with a value of \(\sim\)10 mN/m after 20 min of adsorption. According to penetration theory, the cube root of the molecular weight showed an inverse proportionality with the diffusion coefficient [70], for samples extracted for a longer time which exhibited more SS bonds, leading to larger particle size. Therefore, the lower adsorption kinetics in the longer-time-extracted protein samples might be attributed to their larger particle size, reducing their susceptibility to conformational changes. \begin{table}
\begin{tabular}{c c c c c c} \hline
**Extraction Time** & **Free -SH Content** & **0-Day** & **7 th-Day** & **EAI** & **Creaming Index** \\
**(min)** & **(\(\upmu\)mol/g Soluble** & **Droplet Size D\({}_{4,3}\)** & **Droplet Size D\({}_{4,3}\)** & **(\(\upmu\)m)** & **(\(\upmu\)**)** \\ \hline
5 & 65.0 \(\pm\) 0.6 a & 11.0 \(\pm\) 0.9 c & 11.6 \(\pm\) 0.3 d & 7.1 \(\pm\) 0.5 a & 14.2 \\
10 & 51.7 \(\pm\) 1.3 b & 12.5 \(\pm\) 0.8 c & 13.6 \(\pm\) 0.3 c & 6.4 \(\pm\) 0.1 b & 67.3 \\
20 & 37.6 \(\pm\) 0.7 c & 20.0 \(\pm\) 4.0 b & 22.6 \(\pm\) 1.9 b & 6.0 \(\pm\) 0.1 c & 69.6 \\
40 & 32.1 \(\pm\) 2.4 d & 27.8 \(\pm\) 1.8 ab & 31.6 \(\pm\) 1.1 b & 5.9 \(\pm\) 0.2 cd & 90.2 \\
60 & 28.0 \(\pm\) 0.9 d & 39.0 \(\pm\) 9.4 a & 43.6 \(\pm\) 7.2 a & 5.8 \(\pm\) 0.1 d & 91.6 \\ \hline \end{tabular}
\end{table} 
Table 3: The free sulfhydryl group (-SH) content, volume-mean emulsion droplet size D\({}_{4,3}\), emulsifying ability index (EAI), and creaming index of hempesed-protein-solubilised emulsion. The extraction was conducted at fixed temperature (37 \({}^{\circ}\)C) and pH (10.0). The droplet sizes were measured immediately following emulsion formation (0 days) and after 7 days of storage. Means denoted by a different letter indicate significant differences at \(p<0.05\) probability level. Figure 7: Microstructure of hempseed-protein-stabilised emulsions as imaged by an optical microscope. The freshly-made emulsions stabilised with hempseed protein from specific extraction times of 5, 10, 20, 40, and 60 min are shown in images (**A1–A5**). In addition, their corresponding emulsions kept for 7 days are shown in images at (**B1–B5**). The macroscopic images of protein-solubilised emulsions were taken by the camera and are presented as the insert. The scale bar in optical microscopy images is 20 \(\upmu\)m.  
The dynamic surface tension changes were also probed and shown on a logarithm timescale, with two different regimes (I-II) identified, as marked in the Figure 8B. A similar investigation into protein adsorption at the interface was carried out by [PERSON] et al. [71]. As demonstrated by them, three different regimes could be observed when probing for more extended time scales. In this study, the main concern is the induction time in Regime I. Typically, in Regime I, the interfacial tension remains essentially constant for a short period until the surface-active proteins diffuse to the interface [46].