## 3 Results and Discussions
### The Influence of Disulphide Bond Formation on Protein Functionality
#### 3.3.2 Emulsifying Properties  
The emulsifying properties of a protein can significantly impact the texture, sensory quality, and stability of a food matrix [65]. Proteins are amphiphilic and function as an emulsifier by creating a film over oil droplets dispersed in water. The emulsifying capacity and stability are two key parameters that determine the ability of proteins to produce and stabilise emulsions [66]. The emulsifying ability (EAI), stability (creaming index), and emulsion droplet size for emulsions made from HPI extracted for different times at 37 \({}^{\circ}\)C and pH = 10.0 are summarised in Table 3. Unsurprisingly given the observed intramolecular disulphide bonding, the emulsifying properties of hempesed protein declined as the extraction proceeded. The best emulsifying properties of HPI were observed at the shortest 5 min extraction time, which had the highest free -SH content among all the samples. The decreased emulsifying properties as the extraction proceeds can be attributed to more aggregation between the protein molecules, as evidenced by the decrease in free -SH content. Our results were slightly higher than the other reported hempesed protein investigation by [PERSON] et al. (5.6 \(\pm\) 0.2 m\({}^{2}\)/g) [18]. The difference can be ascribed to different raw materials and extraction conditions. A lower temperature (24 \({}^{\circ}\)C) but longer extraction time (120 min) was selected in their study [18]. Moreover, the greater emulsifying ability found in the 5 min sample can also be linked to the emulsion droplet size, where the droplets in the 60 min sample (39.0 \(\pm\) 9.4 \(\upmu\)m) were almost four-fold larger than those in the 5 min sample (11.0 \(\pm\) 0.9 \(\upmu\)m). This suggested that the capacity of the hempesed protein to absorb at the water-oil interface gradually declined as the extraction time increased.  
The emulsion stability was assessed by gravitational phase separation, with poorly stabilised emulsion droplets agglomerating into a top layer after some time. The creaming index of the 60 min sample (91.6%) was significantly higher than that of the 5 min sample (14.2%), suggesting a substantial decline in hempesed protein emulsifying stability properties occurred during prolonged extraction. Similarly, the investigation of [PERSON] et al. [67] demonstrated that SS formation contributed to the aggregation of kidney bean protein, which adversely affected the kidney bean protein emulsion stabilising properties.  
Figure 6: (**A**) pH–solubility profiles and (**B**) \(\zeta\)–potential profiles of HPI samples extracted at pH 9.0, 10.0, 11.0, and 12.0. The other extraction parameters were set at a fixed temperature (37 \({}^{\circ}\)C) and time (2 h).  
To better illustrate our emulsifying results, the hempesed-protein-stabilised emulsions were imaged by optical microscopy (Figure 7). It is evident that the droplets in the freshly made emulsion were progressively larger when made from protein obtained from longer extraction times (Figure 7A1-A5). Consistent with the particle sizing results, the smallest emulsion droplets were present in the emulsion formed using proteins at an extraction time of 5 min. In addition, very large droplets are visible in the background of images of the 7-day emulsions from longer extraction times (Figure 7B3-B5), indicative of re-coalescence occurring in these emulsions. As can be seen in the macroscopic images (inserts), after 7 days of storage, only the emulsion made from the 5 min-extracted hempesed protein had not separated. Based on these results, it could be proposed that hempesed protein should exhibit at least 65 \(\upmu\)mol/g of free -SH content to be suitable for emulsifying applications (Section 3.5).