## 3 Results and Discussions
### The Influence of Disulphide Bond Formation on Protein Functionality
#### 3.3.1 Protein Solubility and \(\zeta\)-Potential  
Protein solubility is an essential index that impacts many functional properties, such as foaming, gelling, and emulsifying capacities [62; 63; 64]. It is also a prerequisite for protein to be further applied in the food industry. Protein solubility (PS) is highly related to the carried charge of amino acids displayed on the protein surface [24]. Zeta potential is indicative of protein surface charges in the solvent and can help to explain its behavior. Generally, for commercial food applications, the pH of the food matrix should be slightly acidic (pH 4.0-6.0) up to neutral (pH 7.0) [28]. In this study, the hempseed protein pH-solubility profile (dispersing range pH 3-11) and its corresponding zeta potentials were determined, with the focus on the behavior in neutral pH and acidic range. As shown in Figure 6B, the \(\zeta\)-potential of HPI solution ranged from positive (+11.2 mV) at pH 3.0 to highly negative (\(-\)36.2 mV) at pH 11.0, with a similar profile to a previous investigation [2]. The positive charge is owing to the protonation of carboxyl groups by H\({}^{+}\). When the solution is more basic, the carboxyl groups are deprotonated, and the amino groups on the side chain are neutralised by OH\({}^{-}\), leading to a net negative charge. As the pH is decreased, the carboxyl groups become protonated and the pH neutralised. The zero charge, or isoelectric point for hempseed protein, can be observed between pH 4.0-5.0. The isoelectric point difference among these four samples was negligible, and the minor difference could be attributed to slight changes in amino acid content.  
As visualised in Figure 6A, HPI samples produced from different extraction pH values (pH 9-12) all displayed a typical U-shape pH-solubility profile, which was consistent with the previous studies [2; 15]. In all HPI samples, there was higher protein solubility in the alkaline range (pH > 8.0) than in the neutral and acidic range. The largest difference between the samples extracted at different pH occurred at a neutral and alkaline dispersion pH. For instance, the solubility of the HPI extracted at pH 12 and pH 9 differed by approximately 50% (32.4 \(\pm\) 2.6% and 22.7 \(\pm\) 1.1%, respectively) when dispersed at pH 7, but only differed by around 10% (89.1 \(\pm\) 4.8% compared to 81.6 \(\pm\) 1.4%) when dispersed at pH 11.0. As mentioned before, enhanced intermolecular SS bond formation incurred more protein aggregation at more alkaline extraction pH. In addition, after a 2 h extraction, a great extent of aggregation might exist in the final products, which further causes the poor solubility of HPI. The larger protein aggregates are presumably linked to the poor protein solubility. Strikingly, the protein solubility of all HPI samples in the food-processing range (pH 4-6) was extremely low. However, this is not surprising as the dispersing pH is around the protein isoelectric point of the protein (pH \(\sim\)5). This again affirms that the currently used plant protein extraction protocol is not appropriate for the hempseed system. This is likely due to the unique characteristics of hempseed protein, which has a high content of sulfhydryl groups that distinguish it from other typical plant protein systems. The mechanistic understanding developed in this work can subsequently be used to develop improved extraction protocols able to produce more functional HPI.