*2.3. Preparation of Covalent HPI-EGCG Complexes*

Covalent HPI-EGCG complexes were prepared using a previously described method [17], with slight modifications. HPI (0.5 g) was dissolved in 50 mL of distilled water. Different concentrations of the EGCG mother liquor were prepared, and the HPI and EGCG solution was mixed in a volume ratio of 1:1 to ensure that the concentration of EGCG in the mixed solution was 0, 1, 2, 3, 4, or 5 mM. The solution was adjusted to a pH of 9.0 and magnetically stirred for 24 h under an aerobic condition at 25 ◦C and then dialyzed. The HPI-EGCG conjugate was obtained by freeze-drying.

#### *2.4. Determination of Sulfhydryl Content*

The determination of sulfhydryl content was based on the method of previous researchers [18], with slight modifications. Ellman reagent was prepared with 4 mg of DTNB and 1 mL of Tris-glycine. Tris-glycine (contained 8 M of urea) buffer was used to prepare an HPI solution at a concentration of 3 mg/mL. Fifty microliters of Ellman reagent was then added to 5 mL of the HPI solution and reacted for 1 h in a dark environment. The absorbance value under ultraviolet light was 412 nm, and the sulfhydryl content was calculated using the following formula:

$$\text{sulfyday content (\mu mol/g protein)} = (73.53 \times A\_{412} \times D)/C \tag{1}$$

where A412 is the absorbance at 412 nm, D is the dilution coefficient, and C is the protein concentration of the HPI sample (mg/mL).

#### *2.5. Determination of Free Amino Group Content*

The free amino group content of HPI and the HPI-EGCG complexes was determined using the *o*-phthaldialdehyde (OPA) method [19]; 80 mg of OPA powder was accurately weighted, added to 4 mL of methanol solution, and mixed to prepare OPA reagent. Then, 50 mL was added to 0.1 M of borax solution, 200 μL of β-mercaptoethanol, and 5 mL of sodium lauryl sulfate solution (20%, *w*/*w*). The resulting solution was transferred to a volumetric flask and made up to 100 mL with distilled water. Two hundred microliters of the sample solution, at a concentration of 1 mg/mL, was mixed with 4 mL of the OPA reagent and vortexed to obtain a homogeneous solution. After protecting from light for 2 h, the spectrophotometer was set to 340 nm and the absorbance of the solution was measured. L-leucine solutions of different concentrations were used to construct a standard curve of the absorbance value vs. the L-leucine concentration. The content of free amino groups was calculated with the obtained absorbance value through the standard curve.

#### *2.6. Determination of EGCG Content*

Total phenol equivalents were determined in the HPI and HPI-EGCG complex samples [20]. Folin–Ciocalteu reagent (2.5 mL, 0.2 N) was added to 0.5 mL of HPI-EGCG complex solution and mixed thoroughly using a wandering oscillator. After reacting for

5 min, 2 mL of an Na2CO3 solution (7.5%, *w*/*v*) was added, and the mixture was shaken for 30 s and reacted for 2 h in the dark. Absorbance was then measured at 760 nm, usually with distilled water as a blank. EGCG standard solutions of different concentrations were used to construct a standard curve of absorbance vs. EGCG concentration. The EGCG content of the sample was then calculated according to the standard curve, and the final result was expressed as μmol EGCG/g protein.

#### *2.7. Zeta Potential and Particle Size Determination*

According to the method used by previous researchers [21], the zeta potential and particle size of the covalent complex were measured, with some modifications.

The average particle size distribution of each sample was determined, and a sample solution was prepared with a concentration of 0.2 mg/mL using a Malvern particle size analyzer (Malvern Panalytical, Malvern, UK). The samples were centrifuged at 4000× *g* for 15 min and passed through a 0.45 μm cellulose acetate filter membrane, and the particle size was then measured at room temperature.

A Nano ZS90 zetasizer was used to determine the zeta potential, according to the method used by previous researchers [22], with slight modifications. Each sample was measured six times, and the average value was calculated as the final result.

#### *2.8. Circular Dichroism Analysis*

Far-ultraviolet circular dichroism (CD) can analyze the changes in the secondary structure of the HPI-EGCG complex [23]. A sample solution with a protein concentration of 0.5 mg/mL was prepared in distilled water. Far-ultraviolet CD measurements were performed in a 1 mm diameter quartz cuvette using an MOS-450 spectrophotometer (French Bio-Logic SAS, Seyssinet-Pariset, France). The following parameters were used: spectral scan range, 190–250 nm; scan resolution, 1 nm; scan rate, 100 nm/min; and general band width, 2.0 nm. Samples were scanned nine times and the average value was calculated.

## *2.9. Three-Dimensional Fluorescence Spectroscopy*

The change in fluorescence intensity of the samples was measured using an F-6000 fluorometer spectrophotometer (Hitachi Ltd., Tokyo, Japan) [24]. A 1 mg/mL sample was prepared, and the samples were placed in a quartz cuvette for measurement. The excitation wavelength (λEx) was set at 200–350 nm. Continuous scanning of the three-dimensional fluorescence spectrum was performed at an emission wavelength (λEm) ranging from 200 to 500 nm, with an initial λEx of 200 nm, a scanning speed of 3000 nm/min, and a slit width of 5 nm.
