## 3 Results and Discussions
### Mechanistic Investigation of Hempseed Protein AE-IEP Extraction
#### 3.1.1 The Effect of pH and Temperature on Protein Extraction Kinetics  
It has been reported in other plant protein systems that high extraction pH and temperature can increase protein yield [49], and it can be hypothesised that the hempseed protein system will behave similarly. The effect of pH on the protein extraction rate was first carried out at a fixed temperature (37 \({}^{\circ}\)C). As shown in Figure 3A, the rate of extraction was significantly enhanced as the extraction pH increased. For instance, after a 2 h extraction, the protein concentration obtained at pH 12 (3.6 \(\pm\) 0.1 mg/mL) was more than twice that at pH 9 (1.5 \(\pm\) 0.2 mg/mL). This can be explained by the fact that, at higher extraction pH, more carboxyl groups will be deprotonated, leading to an increase in hydrophilicity, hence the increased protein extractability. Another contributing factor could be that the hempseed cell wall might be disrupted under highly alkaline conditions due to alterations of the hemicellulose structure and partial lignin removal [50; 51].  
The observed differences in extraction kinetics are also reflected in the final HPI yield. As shown in Table 2, increasing extraction pH can enhance the HPI yields. This finding accords with many previous investigations on the extraction of various plant proteins [52; 53; 54; 55]. In this study, the extraction yield increased from 17.2 \(\pm\) 0.3% to 22.4 \(\pm\) 0.4% as the pH increased from 9.0 to 12.0. By using a similar AE-IEP protocol to extract protein from non-dehumed hempesed raw material, [PERSON] et al. [10] reported an HPI extraction yield of 16.67 \(\pm\) 0.05%, [PERSON] et al. [6] acquired 24.24 \(\pm\) 0.22%, while [PERSON] et al. [2] obtained 21.52 \(\pm\) 0.36%, respectively. The slight difference in the value could be attributed to minor modifications in the HPI extraction protocol and variations in the raw materials. The protein recovery yield based on the initial protein content in the material also varied amongst different studies, having been reported as 37.9. [4], 46.9% [2], 50.6% [6], 58.1% [56], and 73% [15]. The variance in protein recovery yield might also be due to variations in the raw materials and treatments, such as the defatting and dehulling process [2; 6].  
Figure 3: Impacts of extraction conditions on the kinetics of hempesed protein extraction. The effects of pH on (**A**) protein concentration and (**B**) free -SH content at a fixed temperature (37 \({}^{\circ}\)C) as a function of extraction time. The impact of temperature on (**C**) protein concentration (**D**) and free -SH content at fixed pH (10.0). Experiments were carried out as triplicates.  
To investigate the impact of temperature on protein extraction, experiments were carried out at a fixed extraction pH (pH = 10.0). As depicted in Figure 3C, the rate of extraction was much higher at 37 \({}^{\circ}\)C than at 24 \({}^{\circ}\)C. Interestingly however, there was very little effect of temperature between 37 and 70 \({}^{\circ}\)C. Importantly, these effects were similar for both the rate of protein recovery (Figure 3C) and decrease in -SH groups (Figure 3D). This suggests that mildly elevated temperature can help accelerate the extraction process but cannot be used to decouple yield and quality, and that high temperatures provide little benefit.