*2.10. HPI-EGCG Conjugate Properties*

#### 2.10.1. Solubility

The total protein content change of the sample solutions was determined using the Garcia method [25], with slight modifications; 20 mL of the 5.0 mg/mL protein sample was stirred for 1 h at a constant temperature of 25 ◦C. The samples were then centrifuged at 3000× *g* for 15 min, and the amount of soluble protein was determined using the following formula:

$$\text{SI (\%)}=\text{W}\_2/\text{W}\_1 \times 100\tag{2}$$

where SI is the protein solubility, W1 is the original protein quantity (g), and W2 is the soluble protein quantity (g).

#### 2.10.2. Emulsifying Properties

Emulsification and emulsification stability were determined as previously described [26]. Briefly, 5 mL of soybean salad oil were added to 15 mL of a concentration of 3.0 mg/mL HPI-EGCG conjugate solution at a pH of 7.0 and centrifuged at 9000× *g* for 1 min. A 50 μL sample of the emulsion was removed and mixed with 0.1% (*w*/*v*) sodium dodecyl sulfate. The absorbance of the sample at 500 nm at 0 and 30 min was recorded. The following

formulas were used to calculate the Emulsification Activity Index (EAI) and Emulsification Stability Index (ESI):

$$\text{EAI (m}^2\text{/g)} = 2 \times 2.303 \times \text{A}\_0 \times \text{dilution factor} / 100,000 \times \lambda \times \text{C} \tag{3}$$

$$\text{ESI} \left( \% \right) = \text{A}\_{30} / \text{A}\_{0} \tag{4}$$

where the dilution factor is 100, C is the protein concentration (g/mL), λ is the volumetric oil fraction, A0 is the absorbance measured at 0 min, and A30 is the absorbance measured at 30 min.

#### *2.11. Cryo-Scanning Electron Microscopy*

A cryogenic scanning electron microscope (JSM-7100; Jeol Europe, Zaventem, Belgium) was used to observe the microstructure of the emulsion. A field emission scanning electron microscope was used to capture still images of the microstructure of the emulsions. The emulsion was added dropwise into the short tube of a low-temperature scanning electron microscope located on the holder and frozen in nitrogen. Subsequently, the short tube was transferred to an ultra-vacuum low-temperature chamber, and the emulsion was broken and etched at −95 ◦C for 60 s. The broken surface was coated in platinum and then transferred to the freezing stage (−190 ◦C) of the scanning electron microscope. Images were captured using the microscope control software [27].

#### *2.12. Statistical Analysis*

Each group of experiments was repeated in triplicate, and the data were analyzed using SPSS 23.0 software (IBM, Armonk, NY, USA). The variance of each treatment group and an independent-sample Student's *t*-test were used to determine whether there were significant differences between the samples (*p* < 0.05). The results are expressed as mean ± standard deviation (SD).

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

#### *3.1. Determination of HPI and EGCG Covalent Reactive Groups and EGCG Content*

In an alkaline environment, the phenolic hydroxyl group of EGCG is oxidized to produce quinones, which changes the sulfhydryl or amino groups of the protein. C-S or C-N bonds are formed during the covalent bonding of polyphenols and proteins. In this study, an increase or decrease in the content of sulfhydryl and free amino groups concentrations of HPI during the reaction with EGCG and the amount of polyphenol binding in the reaction was measured (Table 1). In the process of covalent bonding of HPI and EGCG, the content of side-chain amino groups in the protein also changed with changes in the amount of EGCG added.

**Table 1.** The sulfhydryl group, free amino group, and EGCG concentrations in HPI-EGCG conjugates.


Values are expressed as the mean ± SD. Different letters in the same column indicate significant differences (*p* < 0.05).

The sulfhydryl contents of HPI and the HPI-EGCG covalent complex were measured within the allowable error range of the method. EGCG was reacted with HPI to form covalent bonds. When the concentration of EGCG continued to rise, the sulfhydryl content of HPI decreased from 28.41 μmol/g HPI to 9.14 μmol/g HPI (Table 1). The process of converting sulfhydryl groups into disulfide bonds can be prevented by urea, and therefore, EGCG can be derived from the decrease in free sulfhydryl content. Covalent bonding occurred with the free sulfhydryl group of HPI; that is, the phenol rings of polyphenols, such as EGCG, can covalently react with the nucleophilic groups of HPI to form C-N and C-S bonds, which illustrates the occurrence of covalent reactions. This is the main reason for the reduction in the number of sulfhydryl groups [28].

The OPA method can sensitively reflect relevant information about the side chain of lysine, the N-terminal residues, and α- or ε-amino groups of proteins, which are the main reactive groups measured by the OPA method. As shown in Table 1, compared with control HPI, there was a downward trend in the content of free amino groups in the covalent complex of HPI and EGCG. When the concentration of EGCG continued to increase, the number of free amino groups in the conjugate greatly decreased. Because free amino groups were quantified in the presence of a 1% concentration sodium dodecyl sulfate, a denaturant that destroys non-covalent protein interactions, we deduced that the decrease in the number of free amino groups may have indicated that the free amino groups of HPI were covalently linked with EGCG. Free amino content groups in HPI were expected to decrease after covalent bonding with EGCG, because the amino acid residues bind to quinone after EGCG oxidation. The ε-amino group contained in lysine was oxidized to form a carbonyl group, and a condensation reaction occurred with the free amino groups, thus reducing the number of amino groups [29].

To further confirm the covalent linkage between HPI and EGCG, the Folin–Ciocalteu method was used to determine the content of EGCG in the solution of the HPI-EGCG conjugate. When determining polyphenol content, HPI samples were pre-measured to have the same protein concentration as the HPI-EGCG samples under the same experimental conditions. Additionally, HPI was used as a blank control to reduce the interference caused by polyphenols. It can be seen from Table 1 that the concentration of EGCG increased and the number of polyphenols increased. However, when the EGCG concentration was 4 mM and 5 mM, the polyphenol content did not significantly change. This implies that, at the concentration of 4 mM, EGCG had reacted with all of the active groups exposed by specific amino acids. It can also be seen from Table 1 that, as more polyphenols were bound, the content of sulfhydryl groups was reduced, as well as the content of free amino groups.

#### *3.2. Particle Size Distribution and Zeta Potential Change Analysis*

It can be seen from the particle size distribution and zeta potential changes of the HPI-EGCG complexes and HPI samples that, compared with HPI, the covalent HPI-EGCG complex had larger particles (Figure 1). The particle size of the covalently bound protein was mainly about 200 μm. In addition, with an increase in the amount of EGCG added, the height of the peak representing covalent complexes with a particle size of less than 100 μm decreased. The reason for this phenomenon is related to the covalent bonding of EGCG and the protein, as described in the following.

The zeta potential represents the amount of charge on or near the particle surface. The amount of charge is affected by the liquid environment (pH, ionic strength, or presence of surfactants) [30]. The electric potential is related to the size of the interaction between the charged groups, and plays an important role. It can be seen that, as the EGCG content continued to increase, the absolute zeta potential values of the samples increased (Figure 1). This increase in absolute zeta potential is due to the fact that the EGCG molecule is a negatively charged molecule, and, after binding, the protein is also negatively charged. The dispersion and aggregation of particles are related to their surface charge [31]. The absolute value of the zeta potential becomes larger indicates that the system is relatively stable. In the covalently bound samples, the oxidation and cross-linking of EGCG formed a stable network structure. Therefore, as the content of EGCG continued to increase, so too did both the zeta value and the size of the protein particles.

**Figure 1.** Size distribution (**A**) of HPI and covalent HPI-EGCG complexes at EGCG concentrations of 1, 2, 3, 4, and 5 mM. Zeta potential (**B**) of HPI and covalent HPI-EGCG complexes at EGCG concentrations of 1, 2, 3, 4, and 5 mM.

#### *3.3. CD Spectroscopy*

Far ultraviolet CD spectroscopy can analyze the secondary structure changes of HPI and its conjugates (Figure 2). HPI is a globulin. Its far-UV CD spectrum has a specific negative peak in the range of 210–220 nm. In this study, the maximum value of the negative peak shifted from 213 to 208 nm as the EGCG content continued to rise. Table 2 illustrates the changes in the secondary structure of the HPI-EGCG complex. After the protein was covalently bound to EGCG, the maximum negative value showed an increasing trend. That is, the binding of EGCG and HPI reduced the ellipticity of the protein as measured by CD spectroscopy, which indicated that the secondary structure of HPI had changed after EGCG was added. The secondary structural of natural HPI was composed of 1.79% α-helix, 82.79% β-sheet, and 15.50% unordered. After covalent bonding, the β-sheet content decreased, while the α-helix content and random coil content both increased. The decrease in β-sheet content is the structure that determines the flexibility of protein molecules. The increase in their content indicates that the protein chain becomes loose after covalent interaction and the protein is stretched [32]. Liu also observed this change in a study of the covalent interaction between porcine bone protein hydrolysate and rutin, in which the β-sheet and β-turn content decreased and the α-helix and random helix content increased [33]. In addition, the β-sheet content affects the internal hydrophobic groups of the protein. After covalent bonding, the β-sheet content of the complex was reduced, allowing the internal hydrophobic groups to be exposed, which increased the hydrophobicity of the protein. This change is a sign of a change in protein conformation [34]. Furthermore, Damodaran's research also found that α-helix and random coil structures are more flexible than β-sheet structures, which indicates that covalent bonding increases the flexibility of the secondary conformation of HPI [35]. These changes are brought about by the binding of hydroxyl groups at the protein hydroxyl, amino, or sulfhydryl sites in the reaction system, which significantly changes the secondary structure of the protein.

**Figure 2.** Far-UV CD spectroscopy of HPI and HPI-EGCG conjugates.


**Table 2.** Secondary structure contents of HPI and HPI–EGCG conjugates.

#### *3.4. Three-Dimensional Fluorescence Spectroscopy*

Three-dimensional fluorescence spectroscopy was used to determine the effect of EGCG on the structure of HPI. Peak (a) in the three-dimensional contour map represents Raman scattering. The formation of the complex between EGCG and HPI increased the particle size and enhanced the light scattering effect, thereby increasing the fluorescence intensity of peak (a). Peak 1 shows that the characteristic peaks produced by tryptophan and tyrosine turned blue after the addition of EGCG, which indicated that the fluorescence intensity of the protein decreased after EGCG was combined with HPI [36]. It can be seen from that, as the amount of EGCG increased, the color of the characteristic peak became lighter as the contour line became thinner, and the fluorescence intensity of the protein decreased (Figure 3). The reason for this phenomenon is the covalent interaction between the Trp or Tyr residues in HPI and the quinone formed by the oxidation of polyphenols [37]. Therefore, the fluorescence intensity of HPI decreased after combining with EGCG, which indicated a change in the conformation of HPI. These results confirmed the covalent bonding between EGCG and HPI. Under aerobic conditions, phenol is easily oxidized to quinone, and further oxidation causes a dimerization reaction. Therefore, it is oxidized under aerobic conditions to form *o*-quinone, which can be reduced by the amino groups of the protein side chain, thereby forming a covalent C-N or C-S bond between the HPI and EGCG. These C-N and C-S bonds can form dimers through cross-linking reactions to provide new polymers.
