Fabrication of polyphenol-pumpkin seed protein isolate (PSPI) covalent conjugate microparticles to protect free radical scavenging activity of polyphenol

Covalent conjugates of pumpkin seed protein isolate (PSPI) and different polyphenols (epigallocatechin gallate (EGCG), chlorogenic acid (CA) and gallic acid (GA)) were prepared at pH 9.0 to develop covalent microparticles to protect the stability of polyphenols under storage and digestive conditions. The results for polyphenol-bound equivalents, free amino group contents, sulfhydryl contents and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) indicated that polyphenols were covalently bonded with PSPI, while the secondary structure of conjugates became more regular and stable compared with PSPI alone due to the covalent interactions. EGCG-PSPI conjugates displayed the highest polyphenol-bound equivalents (204.75 nmol/mg) and most stable structure compared with CA-PSPI and GA-PSPI because of the eight active phenolic hydroxyl groups within EGCG, which provided more sites for covalent and non-covalent interactions. Accordingly, EGCG-PSPI microparticles (5–8 μm) were fabricated by cluster aggregation of nano- and micro-sized particles through pH shifting treatment. Compared with the EGCG control, EGCG-PSPI microparticles efficiently increased the retention rate of free radical scavenging activity to 47.47%, 22.04% and 39.94% under light, freeze-thaw cycle storage conditions and simulated intestinal fluids, respectively. Therefore, polyphenol-PSPI covalent conjugate microparticles had great potential to be used as efficient carriers for stabilizing the DPPH radical scavenging ability of polyphenols under illumination storage, freeze-thaw and digestive conditions.