## 4 Conclusions  
This study represents a systematic investigation of hempseed protein extraction. It has been observed that the conventional plant protein extraction processes are not suitable for hempseed. This study reveals that the prolonged extraction times and strongly alkaline conditions provide an ambient environment for intermolecular disulphide bond formation, which are responsible for the detrimental effects on protein functionality. The results show that both the protein solubility and emulsifying properties were reduced due to the formation of intermolecular SS bonds as the extraction proceeded. In terms of the emulsifying properties, the droplet size of emulsions was more than three-fold higher in emulsions made from proteins extracted for 60 min instead of 5 min, and the creaming index was also much higher. While the functionality of hempseed protein extracted after 5 min under pH 10.0 and 37 \({}^{\circ}\)C could be considered acceptable (based on a content of at least 65 umol/g of free -SH content), only a low yield (30.6% of maximum) could be obtained. Subsequently, an optimum set of extraction conditions were determined using an RSM optimization design. However, only a minimal improvement in HPI functionality could be obtained at the optimised extraction conditions, while a three-fold protein yield loss was necessary to compensate for the increased functionality. Importantly, these findings suggest that it is impractical to produce high functionality with an ideal protein yield by only varying the extraction parameters. Additional work is required to investigate the use of other emerging methodologies (e.g., ultrasound and salt concentration) to improve the protein yield without affecting the high-functional properties to make HPI attractive to industrial applications, which will be the focus of future studies.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app13116469/s1](https://www.mdpi.com/article/10.3390/app13116469/s1)]([https://www.mdpi.com/article/10.3390/app13116469/s1](https://www.mdpi.com/article/10.3390/app13116469/s1)), Figure S1: The hempseed protein concentration and free -SH content change after a series of extraction pH adjustments. Figure S2: The impact of antioxidant agent introduction to the system on the protein concentration and free -SH content [77].  
Conceptualization, S.Y.; methodology, S.Y.; software, S.Y.; validation, S.Y., W.L., G.J.O.M. and M.A.; formal analysis, S.Y.; investigation, S.Y.; resources, G.J.O.M. and M.A.; data curation, S.Y.; writing--original draft preparation, S.Y.; writing--review and editing, W.L., G.J.O.M. and M.A.; visualization, S.Y.; supervision, W.L., G.J.O.M. and M.A.; project administration, M.A.; funding acquisition, M.A. All authors have read and agreed to the published version of the manuscript.  
S.Y. acknowledges the University of Melbourne Research Scholarship. The authors acknowledge funding from Maxarham Pty Ltd.  
Not applicable.  
The data shown in this study are available in this article.  
The authors declare no conflict of interest.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Selected Functionality** & **Original HPI** & **Optimised HPI** \\ \hline PS at pH 4.0 (\%) & 11.1 \(\pm\) 0.5 & 18.5 \(\pm\) 3.8 \\ PS at pH 7.0 (\%) & 29.4 \(\pm\) 2.0 & 39.8 \(\pm\) 4.1 \\ EAI (m\({}^{2}\)/g) & 5.8 \(\pm\) 0.1 & 7.4 \(\pm\) 1.2 \\ CI (\%) & 91.6 & 12.9 \\ \hline \hline \end{tabular}
\end{table}
Table 5: A comparison of functionality between the optimised HPI and the hempseed protein extracted by the original protocol. The original HPI extraction conditions were set as pH 10, 37 \({}^{\circ}\)C, and 60 min.