*2.2. Enzymatic Hydrolysis of PP*

The hydrolysates of PP with different enzymatic hydrolysis degrees (*DH*) were prepared based on the study by Phoon et al. with some modification [16]. The specific steps were as follows: 10 g PP was dispersed in distilled water according to the ratio of protein to distilled water 1:15 (*w*/*w*), and stirred overnight at room temperature to make the protein fully hydrated. The pH of the aqueous solution of protein was adjusted to the optimum pH of the enzyme (alcalase and trypsin were pH 8; neutrase and flavourzyme were pH 7), and kept in a water bath (50 ◦C) for 15 min. Then, 1% enzyme (the ratio of enzyme to substrate was 1:100) was added for enzymolysis. In the process of enzymatic hydrolysis, 1 mol/L NaOH solution was added continuously to maintain the pH. After the enzymatic hydrolysis, the enzyme suspension was quickly moved into a water bath at 95 ◦C and heated for 15 min to inactivate the used enzyme. After cooling to ambient temperature, the pH adjusted to neutral.

#### *2.3. Structure Properties*

#### 2.3.1. Degree of Enzymolysis (*DH*)

The degree of enzymolysis (*DH*) is defined as the number of hydrolyzed peptide bonds relative to the number of peptide bonds per unit weight expressed as a percentage. The *DH* of PP was calculated using the pH-stat method [17]. In the enzymatic hydrolysis process, the *DH* of PP was calculated in real time by representing the volume of NaOH consumed by the sample. When the *DH* reached 2, 4, 6, and 8%, the enzymatic hydrolysis process was stopped and samples for subsequent experiments were obtained, Equation (1) is as follows:

$$DH = \frac{B \times N\_b}{a \times h\_{hot} \times M\_p} \times 100\% \tag{1}$$

where *B* represents the volume of NaOH consumed by the sample; *Nb* refers to the concentration of the calibrated NaOH; *α* is the degree of amino dissociation, which has different values under different enzymolysis conditions; *Mp* refers to the net protein content of PP; and *hhot* is the number of millimoles of peptide bonds per gram of protein, which is usually an empirical value. The *hhot* of PP is 7.55 mep/g.

#### 2.3.2. Intrinsic Fluorescence Spectroscopy

The intrinsic fluorescence spectra of PP and PP with different *DH* samples were determined according to previous papers by using an fluorescence spectrophotometer (F-7000, Hitachi, Tokyo, Japan) equipped with a 10 mm square quartz cell [18]. The emission spectra of PP samples were collected to investigate the effect of hydrolysis on the intrinsic fluorescence of PP. The critical parameters used in fluorescence were: excitation wavelength 295 nm, excitation slit 2.5 nm, emission slit 2.5 nm, and scan rate 1200 nm/min. Then, the emission spectra of PP samples were collected to investigate the effect of the enzyme on the intrinsic fluorescence of PP.

#### 2.3.3. Surface Hydrophobicity Measurements

The surface hydrophobicity of PP and PP with different *DH* samples was determined according to Li et al. [19]. ANS was used as a fluorescence probe; the excitation wavelength was set at 380 nm and emission spectra were collected from 390 to 660 nm. Both excitation and emission slits widths were fixed at 5 nm. Briefly, 20 μL 8 mM ANS was added into 3 mL samples with different protein concentrations (0.004%, 0.008%, 0.012%, 0.016%, and 0.020%). Under conditions of fluorescent probes present in excess, protein relative fluorescence intensity *F* was plotted against protein concentration *C*, and the slope of the line was represented as Equation (2):

$$S\_0 = \frac{\Delta F}{\Delta \mathcal{C}} \tag{2}$$

#### 2.3.4. Circular Dichroism (CD) Spectroscopy Studies

The secondary structure of PP and PP with different *DH* samples were measured utilizing a MOS-450 CD spectrometer (Claix, France) as in a previous study described by Li et al. [20]. The spectra of samples were collected from 180 to 260 nm. The PBS was measured as a background to correct the CD spectra. The secondary structure contents including α-helix, β-sheet, β-turn, and random coil of PP samples were calculated by the analytical approach of previous research using online CONTIN program [19].

#### 2.3.5. Morphology Observation

The microstructure of PP and PP with different *DH* samples was imaged utilizing a scanning electron microscope (Quanta-200, FEI Company, Eindhoven, The Netherlands). The freeze-dried protein powders were stuck onto one side of double adhesive tape attached to a circular specimen stub and then sputter-coated with a thin film of gold. Then, the microstructural images of the samples were captured under an accelerating voltage of 5.0 kV with magnification at 300-fold.

## 2.3.6. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE under nonreducing conditions was performed to determine the protein fractions patterns of PP and PP with different *DH* samples according to He et al. [5]. Briefly, samples were dispersed in 10 mM phosphate buffer (pH 7) to obtain a 1 mg/mL protein sample. A 60 μL protein solution was mixed with 20 μL 4× loading buffer, followed by heating at 95 ◦C for 8 min. An aliquot containing 10 μg protein and the Thermo Scientific Page Ruler Prestained Protein Ladder (ranging from 11–245 kDa) were loaded to the specific cell. Electrophoresis was run for 15 min at 80 V for stacking gel (5%) and 55 min at 120 V for separating gel (12%). After electrophoresis, the gel was stained using 0.25% Coomassie Brilliant Blue in 50% methanol and 10% acetic acid for at least 50 min. Then, the destaining processing was performed using a water solution of 5% methanol and 7.5% acetic acid.

#### 2.3.7. Determination of Sulfhydryl Groups (SH)

The total sulfhydryl content of PP and PP with different *DH* samples was detected using a micro total mercapto assay kit (Sigma-Aldrich Co., Ltd., Shanghai, China) according to the method reported by Yang et al. [21]. Briefly, the sample of 0.1 g was weighed, and 1 mL of extract was added to prepare 10% homogenate. The sample was centrifuged at room temperature for 10 min at 8000× *g*, and the supernatant was taken to be measured. A certain amount of the samples and reagents was added according to the requirements of the kit, and the absorbance value was measured at a 412 nm.

#### 2.3.8. Amino Acid Composition Analysis

Amino acid composition was analyzed according to Xie et al. [22]. Briefly, 1.0 g samples, 10 mL hydrochloric acid (6 mol/L), and 1.0 g phenol were added to a sealed tube and the solution was treated with nitrogen for 15 min. The above samples were hydrolyzed for 24 h at 110 ◦C. The hydrolyzed samples were transferred to a 50 mL volumetric flask for constant volume and were then filtrated. The filtrated samples (1.0 mL) were evaporated to dryness at 60 ◦C in a water bath. Subsequently, the dried samples were diluted by sample diluent (3–5 mL, pH 2.2, 0.02 mol/L Sodium citrate buffer solution). The mixed samples were filtrated by a 0.22 μm filter membrane and then analyzed utilizing an automatic amino acid analyzer (S-433D, Sykam, Munich, German). The amino acid compositions for PP with different *DH* were presented as g/100 g protein.

### 2.3.9. Hydrophobicity Analysis Based on Amino Acid Composition

The hydrophobicity of PP with different *DH* samples was determined according to previous research [17], Equations (3) and (4) are as follows:

$$Q = \sum \Delta Q i\tag{3}$$

$$
\Delta Q = \left[ (AAi \;/\ M i\;) / \left( \sum AAi \;/\ M i\;\right) \right] \times \Delta f \tag{4}
$$

where *AAi* refers to the amount of various amino acids in 100 mL of protein; *Mi* represents the molar mass of various amino acids; ∑*AAi/Mi* is the total number of moles of amino acids; Δ*f* refers to the free energy values of amino acid side chains; and Δ*Q* is the free energy for the transfer of an amino acid side chain from ethanol to water.

#### 2.3.10. Sensory Evaluation

The color, taste, beany flavor, and bitterness of PP and PP with different *DH* samples were evaluated by a slightly modified method described by Garcia-Arteaga et al. [12] In brief, 10 mL sample solution was used in this test and served at random to the panelists. The samples were tested at 25 ◦C, in a uniformly illuminated room, by a five-member panel selected from a pool of students and staff members of our research team. The evaluation

criteria were as follows: color (light to dark), taste (rough to fine), and beany flavor (serious to slight). Those sensory properties' intensity was estimated on a five-point scale, and bitterness (light to heavy) was also evaluated on a ten-point scale. Water was provided for rinsing between samples.

#### *2.4. Functional Properties*

#### 2.4.1. Solubility

The solubility of PP and PP with different *DH* samples was determined by the Lowry method [23]. The specific methods are as follows: the lyophilized samples were dissolved in distilled water; then, pH was adjusted to neutral. After that, the suspension was centrifuged at 4500× *g* for 15 min. The protein content of the supernatant was determined. Bovine serum albumin (BSA) was used as the standard. Solubility was calculated according to Equation (5).

$$\text{Solubility } (\%) = \frac{\text{protein content in supernatant}}{\text{total protein content}} \times 100\% \tag{5}$$

#### 2.4.2. Foaming Performance

The foaming properties of PP and PP with different *DH* samples were characterized at room temperature using a method described previously with some modification [24]. A fixed volume (18 mL) of protein solution was subjected to mechanical shearing (10,000 r/min, 60 s) using a high-shear mixer to generate foam (Ultra TURRAX homogenizer, T18digital, IKA, Staufen, Germany). The foamability and foam stability of each test sample were then calculated using the following Equations (6) and (7):

$$\text{Foamability} \left( \% \right) = 100 \times \frac{V\_0}{18} \tag{6}$$

$$\text{Foam stability } (\%) = 100 \times \frac{V\_{20}}{V\_0} \tag{7}$$

where 18 is the volume of the test sample before shearing (18 mL), *V*<sup>0</sup> refers to the volume of the foam (mL) immediately after shearing, and *V*<sup>20</sup> is the volume of the foam (mL) at 20 min after shearing.

#### 2.4.3. Emulsifying Performance

The emulsifying ability index (*EAI*) and emulsifying stability index (*ESI*) of PP and PP with different *DH* samples were measured according to previous papers with some modifications [25]. Then, 16 mL of protein solution was mixed with 4 mL of soybean oil, and followed by stirring at 10,000 rpm for 1 min using the high-shear mixer mentioned above. Then, 50 μL of emulsion was dispersed into 5 mL of 0.1% SDS solution at 0.5 cm at the bottom of the container, vortexed, and mixed. The absorbance values were measured at 500 nm for 0 and 10 min. *EAI* and *ESI* were calculated using the following Equations (8) and (9):

$$EAI\,\left(\text{m}^2/\text{g}\right) = \frac{2 \times 2.303 \times A\_0 \times DF}{\text{C} \times (1 - \rho) \times \theta \times 10,000} \tag{8}$$

$$ESI \text{ (min)} = \frac{A\_0 \times \Delta t}{A\_0 - A\_{10}} \tag{9}$$

where *A*<sup>0</sup> refers to the absorbance at 500 nm of the sample solution, *DF* represents the dilution factor, *C* is the protein concentration, *ϕ* is the oil volume fraction, and *θ* refers to the path length. In Equation (9), Δ*t* is the time difference (10 min) and *A*<sup>10</sup> represents the absorbance at 500 nm of sample solution after 10 min.

#### *2.5. Statistical Analyses*

The experiments were conducted in triplicate. The statistical analyses were performed by SPSS 26.0 (IBM Inc., Chicago, IL, USA). Significant differences (*p* < 0.05) between the means of parameters were analyzed with one-way ANOVA test followed by Duncan's LSD test.

#### **3. Results and Discussion**
