Formation and Underlying Mechanism of Soy Protein Nanoparticles via Controlled Alcalase Hydrolysis

In the present study, soybean protein isolate (SPI) was hydrolyzed by alcalase for up to 24 h. It was found that controlled enzymatic hydrolysis could induce the self-assembly of SPI to form a series of homogeneous spherical soy protein nanoparticles (SPNs) with different surface properties that were uniformly distributed (polydispersity index (PDI) < 0.3) ranging from 90 to 200 nm, and that the degree of hydrolysis (DH) and the disassociation/degradation of subunits were the key factors affecting the assembly process. At the initial stage of enzymatic hydrolysis (10–30 min, DH~3%), the α and α’ subunits of β-conglycinin (7S) were partially degraded, thereby being beneficial to release the amphipathic structure, improve the protein surface hydrophobicity (H₀) and reduce the critical aggregation concentration and ultimately resulting in the formation of type Ⅰ nanoparticles (SPNs-DH 3%) containing relatively complete 7S and glycinin (11S) subunits. As the hydrolysis time increased from 1 to 2 h, the α and α’ subunits were further degraded, promoting the exposure of the hydrophobic β subunits and the B subunits, enhancing the hydrophobic interactions and consequently the system’s turbidity, and ultimately leading to the formation of soluble aggregates. The soluble aggregates could transform into insoluble hydrophobic ones, leading to a sharp decrease in the protein surface hydrophobicity and the formation of type Ⅱ hydrophilic nanoparticles (SPNs-DH 5%) dominated by the A subunits and part of the β subunits. At the late stage of hydrolysis (4–24 h), the A subunits were further degraded to produce more hydrophilic peptides, which was not conducive to the formation of nanoparticles. The circular dichroism spectrum implied that transformation from α-helix and random coil to β-sheet might facilitate the formation of SPNs. Moreover, both type Ⅰ and Ⅱ SPNs were mainly maintained by hydrophobic interactions, while hydrogen and disulfide bonds were responsible for the surface and internal structure of the particles, respectively. More folded structures were stabilized by disulfide and hydrogen bonds formed in SPNs-DH 5% compared with SPNs-DH 3%. In addition, due to the continuous release of antioxidant peptides during enzymatic hydrolysis, the antioxidant activity of the formed SPNs was higher than that of SPI.