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.

To clarify the formation mechanism of soybean protein nanofibrils and broaden the industrial application of iron fortifiers, nanofibrils were prepared by heating soy protein isolate (SPI) for five hours under acidic conditions in the present study. Changes in protein structure before and after fibrillation were systematically investigated. Furthermore, iron nanoparticles (Fe NPs) were prepared and the stabilizing effect of SPI nanofibrils on them was evaluated. The results showed that during the formation of SPI nanofibrils, a considerable amount of β-sheet structure was produced and combined with thioflavin T, increasing the fluorescence intensity. In addition, the degradation of 7S fraction was observed at the early stage, being conducive to the formation of fibril nucleation, and then 11S was gradually hydrolyzed, promoting fibril growth. Meanwhile, a large number of small peptides were produced, improving the reducing power of the hydrolysate. SPI nanofibrils were used to deliver Fe NPs. It was found that Fe NPs and SPI nanofibrils could form in situ a complex with good colloid stability, existing as Fe (Ⅱ) and having less influence on the color and stability of emulsions compared with ferrous sulfate and ferric chloride. The results of the present study should guide the construction of a new plant-based iron delivery system.

In the present study, in order to broaden the application of iron fortifiers in the food industry, alkali-heat treated soybean protein isolate (HSPI) was reacted with FeSO₄ to fabricate stable iron-containing nanocomplexes. The results showed that the reaction of HSPI with an appropriate concentration of FeSO₄, which was pH dependent, could form stably dispersed nanocomplexes (Fe-HSPI). The size of Fe-HSPI changed with increasing reaction pH, and the color became darker. Fe-HSPI was stable against in vitro digestion, and the binding between protein and Fe²⁺ was maintained during simulated gastrointestinal digestion, which could be beneficial to prevent free iron stimulating the gastrointestinal tract. Herein, Fe-HSPI prepared at pH 7 (Fe-HSPI_{pH 7}) had good homogeneity (polydispersity index, PDI < 0.2) and the content of soluble iron after digestion was more than 80%, showing a remarkable potential for absorption and utilization. The Fe²⁺in Fe-HSPI_{pH 7} mainly combined with the nitrogen atom of the amino group and the oxygen atom of the carboxyl group in HSPI, thereby facilitating the folding of the peptide chain and resulting in the formation of a structure stabilized by disulfide bonds. Compared with FeSO₄, Fe-HSPI_{pH 7} induced a significantly lower degree of lipid oxidation when applied in oil-in-water emulsions, thus exhibiting lower reactivity and having better potential for application as an iron fortifier.

In the present study, a new type of selenium nanoparticles (SeNPs) was synthesized using oyster protein hydrolysates (OPH) as both a stabilizer and capping agent upon ultrasonication. OPH with alcohol dehydrogenase activation activity was prepared and used to fabricate OPH-SeNPs, where monodisperse SeNPs with a transparent orange appearance, ranging from 140 to 310 nm, were obtained. Physicochemical analysis further suggested a weak interaction between SeNPs and the –NH, C=O, COO–, and C–N groups of the OPH, and the formed nanocomposite with improved stability against aggregation was in an amorphous state. The hepatoprotective effect of OPH-SeNPs on HepG2 cells showed that pre-incubation with OPH-SeNPs significantly decreased cell apoptosis induced by hydrogen peroxide and could well maintain cell integrity. A reduction in intracellular reactive oxygen species was found, along with ameliorated activity of internal antioxidant enzymes. The observed upregulation of key antioxidant-related components, i.e., glutathione peroxidase, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1, suggests activation of the Nrf2-antioxidant response element signaling pathway, which mechanistically explains the hepatoprotective effect of OPH-SeNPs against hydrogen peroxide-induced cytotoxicity in HepG2 cells.