*2.1. Materials*

Raw goa<sup>t</sup> milk (≥8.15% nonfat solids, 3.86% protein, and 4.02% fat, *w*/*v*) was purchased from a local market (Feihe Dairy Industry Co., Ltd., Harbin, China). Soy isoflavones (SIF, ≥80% purity) were provided by Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Deionized water was prepared by a water filtration device (Millipore Corp., Bedford, MA, USA).

#### *2.2. Nanoparticle Preparation*

#### 2.2.1. Preparation of Goat Milk Whey Protein Concentrate

Raw goa<sup>t</sup> milk was heated to 55 ◦C and skimmed with a separator (SA 10-T, Frautech SRL, Thiene, Italy) to obtain skimmed goa<sup>t</sup> milk and cream. The skimmed goa<sup>t</sup> milk was filtered by microfiltration (MF, 0.1 μm, 50 ◦C). MF permeate was ultrafiltrated (UF) by a cut-off 10 kDa spiral-wound membrane to 10-fold, and the UF retentate was electrodialyzed (ED) to remove 85% of salt [13]. Subsequently, the concentrated goa<sup>t</sup> milk whey protein was freeze dried in a freeze dryer (Alpha 1-2, Marin Christ Inc., Osterode, Germany) to obtain goa<sup>t</sup> milk whey protein powder (80.99% protein, 18.67% lactose, and 0.34% ash, *w*/*w*).

#### 2.2.2. Preparation of Polymerized Goat Milk Whey Protein

The goa<sup>t</sup> milk whey protein powder was dissolved in deionized water to obtain a 10% (*w*/*v*) goa<sup>t</sup> milk whey protein solution and stored at 4 ◦C for 12 h to complete hydration. The solution was adjusted to pH 7.7 with 1 M sodium hydroxide, heated to 75 ◦C for 25 min with continuous stirring, and was then quickly cooled to room temperature and marked as polymerized goa<sup>t</sup> milk whey protein (PGWP).

#### 2.2.3. Preparation of Soy Isoflavones Solution

The soy isoflavones (SIF) were dissolved in 70% ethanol (5 mg/mL) by stirring using a magnetic stirrer (IKA, Staufen, Germany), and then the solution was heated to 50 ◦C for 1 h to obtain a clear solution. The container was wrapped with aluminum foil.

#### 2.2.4. PGWP-SIF Nanoparticle Preparation

The PGWP-SIF solutions were prepared by combining PGWP solution, SIF solution, and deionized water to ge<sup>t</sup> different concentrations of SIF (2.1, 2.4, 2.7, and 3.0 mg/mL), while the content of PGWP remained at 40 mg/mL. All samples were stirred for 2 h to obtain a stable system. The mixed solution was treated to remove ethanol by nitrogen gas, and deionized water was added to maintain the original volume, and it was stored in darkness. In this study, samples of SIF-loaded PGWP nanoparticles with different SIF concentrations were termed as PGWP-SIF-A (2.1 mg/mL), PGWP-SIF-B (2.4 mg/mL), PGWP-SIF-C (2.7 mg/mL), and PGWP-SIF-D (3.0 mg/mL), respectively. PGWP (40 mg/mL) was set as a control.

#### *2.3. Encapsulation E*ffi*ciency Determination*

The encapsulation efficiency of SIF within PGWP was measured using a previous method with some modifications [14]. The PGWP-SIF solutions were centrifuged at 5500× *g* for 20 min (25 ◦C), and the concentration of SIF in the supernatant was measured at 261 nm using a UV-visible spectrophotometer (UV-2550, Shimadzu, Tokyo, Japan). The linear regression of absorption versus concentration was conducted, and the regression equation was calculated. The equation is y = 0.1079x − 0.0223, R<sup>2</sup> = 0.9991. The encapsulation efficiency was calculated as follows in Equation (1):

$$\text{Encapsulation efficiency} \left(\%\right) = \mathbb{C}\_0 \% \times 100 \tag{1}$$

where C0 is the concentration of SIF in the supernatant after centrifugation, and C is the concentration of SIF in the nanoparticle.

#### *2.4. Particle Size and Zeta Potential Analysis*

Particle size and zeta potential were carried out using a Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK). The PGWP and PGWP-SIF solutions were diluted to a protein concentration of 0.1% with deionized water [15]. The refractive indexes for protein and water were 1.450 and 1.333, respectively. All measurements were performed in triplicate.

#### *2.5. Rheological Properties Measurement*

Rheological properties of the PGWP and PGWP-SIF solutions were analyzed by a rheometer (Thermo Rheometer, San Jose, CA, USA) equipped with a diameter of 35 mm plate at 25 ◦C, according to Khan et al. [11]. Flow ramp measurement was carried out using shear rate from 0.1 to 300 s<sup>−</sup>1. For peak hold analysis, apparent viscosity data was recorded by keeping shear rate at 200 s<sup>−</sup><sup>1</sup> for 60 s.

#### *2.6. Fourier Transform Infrared (FT-IR) Spectroscopy*

Fourier Transform Infrared (FT-IR) spectra of samples were obtained using a FTIR spectrometer (Thermo Electron Scientific Instruments Corporation, San Jose, CA, USA) with a pressurized tablet method. The SIF, PGWP, and PGWP-SIF solutions were pre-frozen at −80 ◦C for 4 h and freeze dried at 4 ◦C for 12 h. Solid samples were mixed with potassium bromide (KBr) and ground into fine powder. The wavenumber ranged from 4000 to 400 cm<sup>−</sup><sup>1</sup> with a resolution of 4 cm<sup>−</sup><sup>1</sup> and 32 scans [16]. The spectral region ranges of 1600–1700 cm<sup>−</sup><sup>1</sup> were applied to calculate the secondary structure of protein using Peak FIT software. Bands between 1610–1637 cm<sup>−</sup><sup>1</sup> and 1680–1692 cm<sup>−</sup><sup>1</sup> belong to β-sheet; bands between 1638–1648 cm<sup>−</sup><sup>1</sup> belong to random coil; bands between 1649–1660 cm<sup>−</sup><sup>1</sup> belong to α-helix; and bands between 1660–1680 cm<sup>−</sup><sup>1</sup> belong to β-turn. The band area was calculated using the Gaussian function [17].

#### *2.7. Fluorescence Spectroscopy*

Fluorescence measurement was performed using an F-7000 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Japan). The excitation wavelength was set at 280 nm, and the emission was collected from 300 to 500 nm with both slit width at 2.5 nm. The emission spectra were collected at a photomultiplier tube voltage of 500 V with scan rate at 240 nm/min. The PGWP and PGWP-SIF solutions were incubated in water baths at 298, 303, and 308 K for 30 min to achieve equilibrium before measuring [18]. Synchronous fluorescence spectra were recorded at 260–320 nm (Δλ = 15 nm) and 240–320 nm (Δλ = 60 nm) [19].

#### *2.8. Di*ff*erential Scanning Calorimetry (DSC)*

Thermal properties of the samples were analyzed using Di fferential Scanning Calorimetry (Mettler Toledo, DSC 3, Zurich, Switzerland) according to method by Khan et al. [11] with some modifications. All solid samples (about 5 mg) were sealed in aluminum pans and heated from 30 to 300 ◦C at 10 ◦C/min under a nitrogen flow rate of 50 mL/min. An empty sealed aluminum pan was used as a control.

#### *2.9. Transmission Electron Microscopy (TEM)*

Images of samples were obtained using transmission electron microscopy (H-7650, Hitachi High-Technologies, Tokyo, Japan) as described by Ghorbani Gorji et al. [20]. The PGWP and PGWP-SIF solutions were diluted to the appropriate concentration, and 10 μL of the sample was placed on a carbon copper and dyed with a negative staining method. The sample was air dried before imaging.

#### *2.10. Statistical Analysis*

Data of triplicate experiments were statistically analyzed and expressed as mean ± standard deviation. Analysis of variance ( *P* < 0.05) and Tukey's test were carried out using SPSS 20 software (SPSS Inc., Chicago, IL, USA).
