Hemp protein functionality affected by protein extraction method: Comparative study between divalent salt, deep eutectic solvent, and alkaline extraction

Research into sustainable protein ingredient extraction from agricultural byproducts is growing. This study aimed to assess the hemp protein isolate (HPI) extracted from cold-pressed meals using three methods: divalent salt, deep eutectic solvent (DES), and alkaline extraction. Initially, varying monovalent and divalent cation ratios (NaCl: MgCl2) of 1:0, 0.75:0.25, 0.5:0.5, 0.25:0.75, and 0:1 were used in a one-way ANOVA design to optimize salt-based extraction. Then, the optimized salt extraction, choline chloride-glycerol-based DES, and alkaline extraction methods were used for HPI extraction. Finally, the extracted HPIs were compared for their functional, chemical, and structural properties. The 0.25:0.75 ratio of NaCl: MgCl2 showed the best results in salt-based optimization. In terms of protein purity (conversion factor=5.37), salt-extracted protein (HP-SALT) and DES-extracted protein (HP-DES) demonstrated the highest purity of 93.67 ± 0.35% and 92.55 ± 0.94%, respectively, compared to 87.13 ± 1.65% for alkaline-extracted protein (HP-ALK). HP-SALT also exhibited the highest extraction yield (23.29 ± 0.81%) and protein recovery rate (45.04 ± 1.57%). Protein secondary structure analysis of HPIs indicated minimal differences between extraction methods, with approximately 60-62% β sheets, 15-18% α helix, 11-13% coils, and 10-11% turns. All three protein isolates exhibited the highest emulsifying activity at pH 9 (HP-ALK=10.83 ± 0.10, HP-DES=10.18 ± 0.24, and HP-SALT=10.11 ± 0.36 m2/g). HP-ALK displayed the highest foaming capacity of 54.63 ± 4.24% at pH 7 and demonstrated the highest values for water and oil holding capacities, likely due to increased exposure of functional groups during extraction. At pH 7, HP-SALT achieved the highest solubility of 7.15 ± 0.45%. Concerning gelation capacity, only 8% HP-DES formed a gel, while the other isolates did not gel even at 10%. In summary, no single extraction method excels universally for all functionalities. The choice should align with the target application and the required functional properties.