3.3.2. Carbonyl Groups

Carbonyl groups (aldehydes and ketones) are produced on the side chains of the protein when they are oxidized [49]. The carbonyl contents of WPI, SC and SPI were 1.32, 1.63 and 2.26 nmol/mg (Figure 8), respectively. Due to the preparation process of SC, it contains about 6% of small ions in addition to the pure casein, mainly calcium, phosphate, magnesium and citric acid [15], leading to the worst oxidation stability. The extraction of SPI from soy flour may accelerate its carbonylation [50], since soy protein is extremely vulnerable to the attack of peroxyl radicals, and its degree of oxidation is related to the residual lipid content and LOX activity during the preparation process [27,51]. The carbonyl content of WPI increased during storage, reaching 2.7 nmol/mg after 30 days, which was invariable with the addition of resveratrol (Figure 8). The carbonyl content of SPI and SC increased as the concentration of resveratrol increased after 10 days. When the resveratrol concentrations were 0, 25, 50 and 100 μM, the carbonyl content of SPI increased from 3.95 to 5.11 nmol/mg, while the carbonyl content of SC increased from 2.77 to 3.29 nmol/mg after 30 days. The increase in carbonyl content may be related to the formation of peroxides in the system, which is generated by oxygen molecules attacking free radicals. The formation of peroxides on the α-carbon or other carbons of protein amino acid residues will result in an increase in the carbonyl content [27]. From Figure 8, it indicated that the peroxide content in the three protein solutions was in the order of SPI > SC > WPI, and the addition of resveratrol to SC and SPI solutions produced more peroxides. Together with the sulfhydryl contents in Figure 7, these results indicated that the SPI was more labile to oxidation than SC in the presence of resveratrol.

**Figure 6.** ABTS· <sup>+</sup> scavenging capacity of resveratrol, WPI (black), SC (red), SPI (blue) and WPIresveratrol, SC-resveratrol and SPI-resveratrol complex nanoparticles. The concentrations of proteins were 0.01%, 0.1% and 1%, while the concentrations of resveratrol were 25 (**A**), 50 (**B**) and 100 (**C**) μM.

**Figure 7.** Surface (**A**) and total (**B**) sulfhydryl content of WPI-resveratrol and SPI-resveratrol complex particles before (no pattern) and after (sparse pattern) storage at 45 ◦C for 30 days. The concentration of proteins was 1%.

#### 3.3.3. Amino Acid Composition

The oxidative attack of proteins modifies the side-chain groups of amino acid residues [52]. Tables 1–3 show the amino acid composition of WPI, SC and SPI in the absence and presence of resveratrol before and after storage for 30 days. The addition of resveratrol had no significant effect on the amino acid composition of the proteins before storage. The content of Cys ranked in order WPI > SPI > SC, and the surface and total sulfhydryl contents of SC were too low to be detected by the method of sulfhydryl analysis with DTNB (Figure 7). In the case of WPI alone, the content of Trp, Tyr, Thr, Lys, Met and Phe reduced after storage (Table 1), consistent with the indirect oxidation of WPI caused by the photodecomposition of folic acid [6]. Resveratrol had no effect on the change in the amino acid contents of WPI (Table 1). As for SC alone, the content of Trp, Tyr, Thr, Lys, Met, Asp and Arg reduced after storage and was more pronounced in the presence of resveratrol (Table 2). In addition, the content of Glu, Ser, Gly also reduced in the presence of resveratrol. The losses of Trp were about 11% for WPI and 79% and 87% for SC in the absence and presence of resveratrol, respectively. These results are consistent with a previous study that the tryptophan oxidation product, kynurenine, was higher in casein than β-LG upon photo-oxidation induced by

riboflavin [4]. In the case of SPI alone, Asp, Ser, His, Gly, Thr, Tyr, Cys, Val, Met, Lys, Trp reduced after storage and was more pronounced in the presence of resveratrol (Table 3). In addition, the content of Glu also reduced in the presence of resveratrol. The reduction in the kinds and contents of total amino acids ranked in the order of SPI > SC > WPI (Tables 1–3).

**Figure 8.** Carbonyl content of proteins in WPI-resveratrol (**A**), SC-resveratrol (**B**) and SPI-resveratrol (**C**) complex nanoparticles with various resveratrol concentrations during storage at 45 ◦C for 30 days. The concentration of proteins was 1%.
