Complexation with Alginate in Pumpkin Leaf Protein Solutions for the Encapsulation of Folic Acid: The Effect of Extraction Protocols

This study aimed to assess pumpkin leaves as a protein source and determine the feasibility of these proteins to form complexes with alginate for the encapsulation of folic acid. Different isolation protocols, two based on isoelectric precipitation (one with thermal pretreatment and the other with alkali pre-extraction) and one based on stepwise precipitation with ammonium sulfate, were compared regarding the yield and structural properties of the obtained leaf protein concentrates (LPC). The highest purity of protein was achieved using the thermal-acid protocol and the salting-out protocol at 40% saturation. RuBisCO protein was detected by SDS-PAGE in all LPCs, except for the fractions obtained through salting-out at saturation level ≥ 60%. Complexation of the LPC solutions (1 mg/mL) and sodium alginate solution (10 mg/mL) was monitored as a function of LPC:alginate ratio (2:1, 5:1, and 10:1) and pH (2-8) by zeta-potential measurements and confirmed by FT-IR analysis. Based on the results, the strongest interaction between LPCs and alginate occurred at a pH between 2.20 and 2.80 and an LPC:alginate ratio of 10:1. Complexation resulted in particle yields of 42-71% and folic acid entrapment of 46-92%. The LPC-folic acid interactions elucidated by computational protein-ligand docking demonstrated the high potential of RuBisCO as a biocarrier material for folic acid. The in vitro release study in the simulated gastrointestinal fluids indicated that complexes would be stable in gastric conditions, while folic acid would be gradually released in the intestinal fluids.