Improving the Quality of Canola Meal and Protein Isolates Using Natural Deep Eutectic Solvents, Ultrasound Treatment, and Seed Dehulling

Canola is a crop of exceptional strategic importance to Canada and contains high content of nutritious and sustainable protein. However, protein production from canola has faced several challenges, including the lack of green and efficient extraction techniques that can maximize protein extraction while reducing the co-extraction of canola’s abundant anti-nutritional components. Additionally, the harsh steps used for canola oil extraction often leave the proteins in the meal, the starting material for protein isolate production, highly denatured and unsuitable for human consumption. To address these challenges, this dissertation focused on the improvement of the quality of canola protein isolates through extraction using natural deep eutectic solvents (NDESs) and ultrasound treatment, as well as the enhancement of the quality of canola meal through the use of seed dehulling and moderate pressing temperatures. The first research chapter compared the capacity of four NDES formulations (choline chloride/d-sorbitol, glycerol, d-glucose, or urea/water at a ratio of 1:2:1) to conventional alkaline extractions at pH 12 and pH 9. All NDESs extracted less protein than pH 12 (68.34%, dark-green isolate) but more than pH 9 (39.14%, yellow isolate). Among NDESs, the urea formulation extracted a bright-yellow isolate with an efficiency of 52.89%, while other NDESs produced light-green isolates with efficiencies of ~44%. Additionally, NDESs extracted higher napin contents, caused less protein structural alterations, and produced isolates with comparable or improved functionalities compared to alkaline isolates. While the previous chapter highlighted the promising potential of NDESs for canola protein extraction, limitations such as high solvent production costs and insufficient mechanistic understanding still remained. Therefore, the next chapter was systematically designed to address these shortcomings and included 18 extraction treatments, half paired with sonication, to extract canola protein isolates using two groups of NDESs: choline chloride-glucose-water (CG) and choline chloride-water (C), with water contents ranging from 30-90%. The highest extraction efficiencies were observed at 60% water content for CG group (50.28%) and 90% for C group (45.48%), which increased with sonication to 60.68% and 58.11%, respectively. These results, along with the reduced levels of anti-nutritional components achieved through higher solvent water content and sonication, demonstrated the role of NDES hydration and ultrasound treatment in improving extraction efficiency and isolate quality, while simultaneously enhancing the solvent’s cost-effectiveness. Additionally, despite increasing the solvents’ viscosity and density, the combination of glucose and water as hydrogen bond donors proved more effective for protein extraction than water alone, especially for larger molecular fractions such as cruciferins and aggregates, underscoring the importance of hydrogen bond donor selection when designing NDES formulations. Furthermore, both variations in water content and the use of sonication affected the proteins’ tertiary structure and particle size distribution, providing insights into how the strength and flexibility of NDES nanostructures influence protein properties. The final chapter shifted the focus to improving the quality of canola meal by evaluating the effects of dehulling and pressing temperature (40, 80, and 120 ℃) on the meals and their derived protein isolates. The removal of 80-85% of hulls reduced heat-induced aggregation and preserved more of the meal’s native microstructure during pressing, especially at 120 ℃. Moreover, the secondary and tertiary structure of meal proteins were better preserved, particularly at higher treatment temperatures. These improvements were attributed to the reduction of both friction-induced effects and covalent protein modifications caused by hull components during pressing, as a result of dehulling. These observations directly corresponded with protein extraction efficiencies under alkaline (pH 11) and salt (0.8 M NaCl) conditions. Dehulling significantly increased the extraction efficiency of alkaline and salt isolates by an average of 10.49% and 8.12%, respectively, with differences between non-dehulled and dehulled meals becoming more pronounced as the pressing temperature increased. These improvements were associated with higher extraction of unfolded cruciferins and aggregates from dehulled meals, particularly under alkaline conditions. Overall, this dissertation introduced NDESs as green and efficient alternatives for extracting high-quality canola protein, provided simple and inexpensive strategies for tailoring their properties to maximize protein extraction while minimizing the co-extraction of anti-nutritional components, and presented dehulling as an effective technique for preserving protein quality during seed processing. Together, these findings significantly contribute to the improvement of the quality of canola meal and protein isolates, and advance the field of sustainable protein extraction using NDESs.