**4. Discussion**

Resveratrol self-aggregates at a concentration higher than 40 μM, due to the hydrophobic stacking of aromatic phenol rings [55]. The aggregation of resveratrol reduces its contact

with the external environment and affects its antioxidant activity with the highest value observed at a concentration of 30 μM [56]. Therefore, the retention of resveratrol increased from 68% to 74% as its concentration increased from 25 to 100 μM (Figure 9). α-Tocopherol (Log P ~ 8.84, https://go.drugbank.com/drugs/DB00163/ accessed on 8 July 2021), a hydrophobic vitamin E, was reported both bound in the molecular level and encapsulated as the aggregate in WPI particles, while naringenin (LogP ~ 2.84, https://go.drugbank. com/drugs/DB03467/ accessed on 8 July 2021), a polyhydroxy flavonoid, was bound in the molecular level [34]. Resveratrol (LogP ~ 3.4, https://go.drugbank.com/drugs/DB02709/ accessed on 8 July 2021) is more hydrophobic than naringenin but more hydrophilic than α-tocopherol. When calculated, based on the loading efficiency of resveratrol in Figure 5, the encapsulated amount of resveratrol in protein particles increased as the polyphenol concentration increased (Figure S3). It is thus possible that the aggregated resveratrol in protein particles increased as its concentration increased, which was supported by the transfer from the additive to the masking effect of total antioxidant activity (Figure 6). Therefore, the polyphenol retention increased with its concentration in protein particles (Figure 9).

For WPI, the solvent-accessible (bounded in the molecular level and in free state) resveratrol can scavenge and control the free radicals in the system within a certain range. Its oxidation was the least and not affected by resveratrol during storage for 30 days (Figures 7 and 8 and Table 1). At the same time, the stability of resveratrol was improved by WPI, with a retention of above 74% after 30 days (Figure 9). It is thus speculated that there is no reciprocal oxidation between WPI and resveratrol during storage. As the concentration of WPI increased, the loading efficiency of resveratrol increased (Figure 5), but the polyphenol stability decreased (Figure 9). These results suggest that the loaded microenvironment is not conducive to the polyphenol stability, compared to the free part in the WPI solution. The protective effect of WPI on resveratrol stability might not be attributed to the complex property of the protein.

For SPI, the encapsulated resveratrol located in the hydrophobic core could not exert its antioxidant capacity. Thus its oxidation was the most at the beginning and accelerated by resveratrol during storage after 10 days (Figures 7 and 8 and Table 3). At the same time, the stability of resveratrol decreased upon loading in SPI particles (Figure 9). These results suggest the occurrence of reciprocal oxidation between SPI and resveratrol. The co-oxidation has been reported for whey protein and Antarctic krill oil in oil-in-water emulsion [57]. The initial state of the SPI system contained more peroxides than SC and WPI (Figures 7 and 8), free radicals and hydroperoxides generated during protein oxidation may accelerate the degradation of resveratrol [58] (Figure 9). It has also been reported that ascorbic acid acted as a co-oxidant by generating superoxide anions in the presence of air and extracting hydrogen from the carrier [59]. Resveratrol is oxidized to generate H2O2 [60]. When the retention of resveratrol was between 59 and 73% after 10 days (Figure 9), the polyphenol may act as a co-oxidant to accelerate the oxidation of SPI (Figure 8).

However, most of the resveratrol in the SC system was encapsulated in the hydrophobic core of the protein, but also partially bounded with submicelles in the molecular level, which can play their antioxidant effect to a certain extent. The oxidation of SC was more pronounced than that of WPI but less than that of SPI at the beginning and during storage in the absence and presence of resveratrol (Figures 7 and 8 and Table 2). At the same time, the impact of SC on resveratrol stability basically changed from a protective to a harmful effect during storage (Figure 9). The antioxidant activity of SC was greater than that of WPI and SPI (Figure 6), and the loading efficiencies of resveratrol in SC particles were greater than those in SPI and WPI particles at protein concentrations of 0.1% and 1% (Figure 5). Therefore, the stability of resveratrol was initially improved by SC (Figure 9). A stable protein carrier can maintain the stability of polyphenols through scavenging free radicals and isolating the interference of external unfavorable factors [61]. Then, with the increasing oxidation of SC, the ability to scavenge free radicals was not enough to resist the auto-oxidation of SC. The system was out of balance and the protein changed from antioxidant to pro-oxidant to cause the co-oxidation with resveratrol (Figures 8 and 9).

According to the molecular mechanism of the protein–polyphenol interaction, the di-phenol part of polyphenol is easily oxidized by molecular oxygen and side-chain amino groups under certain conditions to form quinine, which can form a dimer in a side reaction and interact with the amino group of polypeptide or the irreversible reaction of the sulfhydryl side chain leads to the formation of protein cross-links. The closer the distance between the formed oxidation product and the α-carbon or other carbons of protein amino acid residues, the more easily the reaction occurs (Figure 4). Meanwhile, quinine can undergo condensation reactions to form high molecular weight, highly reactive brown tannins [17], which is verified in Table 4 and Figure S1. The formation of a covalent EGCGprotein complex involved the reaction of dimer quinone with protein nucleophilic side chains, such as lysine and cysteine residues, which is consistent with the results of amino acid composition in SC/SPI-resveratrol complex particles after storage (Tables 1–3). It has been assumed that the structure of SC and SPI gradually became flexible during storage and the exposed active groups benefited from the covalent interactions of protein-resveratrol complexations [62].
