2.5.4. Surface Hydrophobicity (H0)

Surface hydrophobicity of the complex particles was conducted on the basis of the description of Kato et al. [31]. The samples were diluted into a series of concentrations (0.2, 0.4, 0.6, 0.8, and 1 mg/mL) with deionized water with pH adjusted to 4.0 using 0.1 M HCl or NaOH solutions. Then, 50 μL of 1-anilino-8-naphthalene-sulfonate (ANS) solution (8 mM in deionized water of pH 4.0) was mixed with 5 mL of the diluted sample incubated in the darkroom (25 ◦C) for 15 min. The relative fluorescence intensity of the sample was determined by a fluorescence spectrophotometer (F-380, Tianjin GangDong scientific and technology Co., Ltd., China) at 390 nm (excitation wavelength, slit width 5 nm) and 468 nm (emission wavelength, slit width 5 nm). The initial slope of fluorescence intensity versus protein concentration (%, *w/v*) was calculated by linear regression analysis and used as an index of H0.

#### 2.5.5. Determination of Emulsifying Properties

The determination of emulsifying property of complex particles was carried out according to the method of Li et al. [32], slightly changed. A total of 30 mL of samples were homogenized at room temperature at 20,000 r/min for 1 min after adding 10 mL of oil. After homogenization, 50 μL of the sample was taken from the bottom immediately and added into 5 mL 0.1% sodium dodecyl sulfate (SDS). The absorbance of the diluted solution was measured at 500 nm, and 0.1% SDS was used as blank. The absorbance measured immediately (A0) and standing for 10 min (A10) after emulsion formation were used to calculate the emulsifying activity index (EAI) and emulsion stability index (ESI) as follows.

$$\text{EAI} = 2 \times 2.303 \times \frac{\text{A}\_0 \times \text{DF}}{\text{C} \times \text{ }\text{ }\text{ }\text{ }\text{ }\text{ }\text{ }\text{ }\text{ }\text{ }^\text{ }10000\text{ }\tag{1}$$

$$\text{ESI} = \frac{\text{A}\_0}{\text{A}\_0 - \text{A}\_{10}} \times \Delta \text{t} \tag{2}$$

where A0 is the absorbance at 0 min; DF is the dilution factor (100); θ is the proportion of the oil phase (0.25); ϕ is the optical path of the cuvette (1 cm); C is the concentration of complex particles (g/mL); A10 is the absorbance at 10 min; Δt is 10 (min).

#### *2.6. Determination of Stability*
