*3.1. Particle Characterization*

Pyrene is often used to investigate the association of macromolecules and the critical micelle concentration (CMC). Its intensity ratio I1/I3 decreased as the hydrophobicity of surrounding microenvironment increased [28]. The I1/I3 ratio of pyrene in water was 1.75 (±0.01). When the concentration of SC was 0.01%, the I1/I3 ratio was 1.67 (Figure 1), and its size distribution had three peaks around 1.5, 25 and 215 nm by intensity (Figure 2A). According to the submicelle model, each casein forms small submicelle units through hydrophobic interactions, and these subunits use calcium phosphate as the cement and further aggregates together to form SC micelles [29]. The relatively low concentration of SC solution is not sufficient to drive the formation of micelles, 0.01% SC mainly dissolved in the molecular level [30]. As the protein concentrations increased above 0.5%, the I1/I3 of SC gradually decreased to about 1.10 (Figure 1). The size distribution of SC at 0.1% showed a major peak around 230 nm and a minor peak around 25 nm, while only a peak at around 380 nm was observed at 1% (Figure 2A). These results indicate that SC aggregates to form micelles at 1% concentration [31]. As for WPI and SPI, the I1/I3 ratios of 0.01% protein were respectively 1.35 and 1.40 (Figure 1). Meanwhile, WPI had two peaks around 220 and 520 nm (Figure 2B), and SPI had two peaks around 110 and 380 nm (Figure 2C). The relatively low I1/I3 and large particle size suggest that WPI and SPI had already aggregated at 0.01%. The size peaks of WPI were not dependent on its concentrations (Figure 2B), while SPI became bigger with increasing concentrations, with two major peaks around 180 and 660 nm at 1% (Figure 2C). This is consistent with the results of the I1/I3 of pyrene. From Figure 1, the I1/I3 ratios of WPI decreased slightly from 1.35 at 0.01% to 1.18 at 0.1%, and then remained unchanged as the protein concentration further increased. The I1/I3 ratios decreased as the concentrations of proteins increased, reaching around 0.80 for 2% SPI (Figure 1). By comparative analysis, these results indicate that SPI particles have the most hydrophobic core, which is consistent with the highest content of hydrophobic amino acids (Tables 1 and 2 vs. Table 3). It has been reported that SPI had lower solubility compared with WPI and SC, while hydrophilic groups and/or water molecules were entrapped in the core of WPI particles [32,33].

**Figure 1.** I1/I3 of whey protein isolate (WPI), sodium caseinate (SC) and soy protein isolate (SPI) solutions at various concentrations.

Since WPI, SC or SPI have an isoelectric point (pI) around pH 4.5~5, the ζ−potential values of their particles are negative at pH 7.0 (Figure 3). ζ-Potential absolute values of the protein particles ranked in order WPI > SC > SPI at the same concentration (Figure 3). This is consistent with their molar ratio of acidic (Asp and Glu) and basic (His, Lys and Arg) amino acids being around 2.38 for WPI, 2.03 for SC, and 1.98 for SPI, calculated from the data in Tables 1–3. Together with the most hydrophobic core of SPI particles in Figure 1, these results indicate that more negatively-charged groups were masked in SPI particles than WPI and SC particles. ζ-Potential absolute values of all complex particles decreased as the protein concentration increased (Figure 3), suggesting that negatively-charged groups were entrapped in the aggregated particle core.

**Figure 2.** Size distribution of SC (**A**), WPI (**B**) and SPI (**C**) particles in the absence and presence of 25, 50 and 100 μM resveratrol. The protein concentrations were 0.01%, 0.1% and 1%.


**Table 1.** Amino acid composition of whey protein isolate (WPI) at 1% in the absence and presence of 100 μM resveratrol (RES) before and after storage for 30 days.

Note: Different lower-case letters in the same row represent significantly different mean values (*p* < 0.05).

**Table 2.** Amino acid composition of sodium caseinate (SC) in the absence and presence of resveratrol (RES) before and after storage for 30 days.


Note: Different lower-case letters in the same row represent significantly different mean values (*p* < 0.05).


**Table 3.** Amino acid composition of soy protein isolate (SPI) in the absence and presence of resveratrol (RES) before and after storage for 30 days.

Note: Different lower-case letters in the same row represent significantly different mean values (*p* < 0.05).

**Figure 3.** ζ−Potential of WPI (black), SC (red) and SPI (blue) particles in the absence and presence of 25, 50 and 100 μM resveratrol. The protein concentrations were 0.01%, 0.1% and 1%.

ζ−Potential absolute values of the protein particles decreased as the concentration of resveratrol increased, which was most pronounced at the protein concentration of 0.01% (Figure 3). The particles of SC, WPI and SPI became more homogeneous upon loading of resveratrol (Figure 2). These results are consistent with the formation of uniform particles of WPI with naringenin, a polyhydroxy flavonoid [34]. Meanwhile, the size distribution of all protein-resveratrol particles increased as the polyphenol concentration increased (Figure 2), which is consistent with the effect of hesperetin or hesperidin concentration on their individual particles with β-conglycinin, one of the major fractions of soy proteins, possibly due to polyphenols acting as bridging agents for protein molecules [35]. SC-resveratrol, WPI-resveratrol and SPI-resveratrol particles had a size distribution around 200–300 nm, 150–250 nm and 100–200 nm, respectively. At 25 μM resveratrol, the size distribution of

SC-resveratrol particles was close to the largest size distribution of SC particles (Figure 2A), while WPI-resveratrol and SPI-resveratrol particles had a size distribution close to the smallest ones of pure protein (Figure 2B,C). These results suggest that the addition of resveratrol favors the aggregation of SC but inhibits the formation of large WPI and SPI aggregates.

#### *3.2. Resveratrol Loading*
