3.5.1. Solubility

The solubility of HPI refers to the percentage of soluble protein in HPI under a specific environment. The solubility of a protein affects its ability to emulsify. HPI had good solubility at 1 mM in (Figure 4). Compared with native HPI, the solubility of HPI-EGCG increased from 39.4% to 50.6%. Thus, the conjugation of HPI with EGCG markedly altered its solubility. The solubility of the conjugate increased at the optimal polyphenol loading (1 mM). The conjugation of EGCG enhanced the solubility of HPI, probably due to EGCG and HPI molecules combining to react, which changed the net charge of the protein molecules. Negatively charged EGCG bound to the hydrophobic area on the HPI surface. On the one hand, it increased the surface electronegativity of the HPI particles due to electrostatic repulsion between the particles, which promoted the dispersion of the protein particles in the aqueous solution to avoid coagulation. On the other hand, the hydrophilic tail of the phenolic hydroxyl group enhanced the interaction between HPI and water molecules [38]. Solubility showed a downward trend at other concentrations (2 mM, 3 mM, 4 mM, and 5 mM) due to excessive polyphenols, which caused protein molecules and polyphenols to form aggregates. The low solubility of such aggregates increased the turbidity of a solution [39]. The result we got is consistent with the result of Quan's study [40], who found that when the concentration of polyphenols exceeds the concentration of protein binding sites, proteins and polyphenols form aggregates, thereby greatly reducing their solubility. Therefore, the covalent bonding of EGCG and HPI resulted in a substantial change in the solubility of HPI.

**Figure 3.** *Cont.*

**Figure 3.** Three-dimensional fluorescence spectra of HPI and covalent HPI-EGCG complexes. (**A**) HPI, (**B**) HPI-1 Mm EGCG, (**C**) HPI-2 mM EGCG, (**D**) HPI-3 mM EGCG, (**E**) HPI-4 mM EGCG, and (**F**) HPI-5 mM EGCG, all suspended in H2O (Ex is the excitation wavelength and Em is the emission wavelength).

**Figure 4.** (a-f) Solubility of HPI and covalent HPI-EGCG complexes at EGCG concentrations of 1, 2, 3, 4, and 5 mM.

#### 3.5.2. Emulsifying Properties of HPI-EGCG Conjugates

The EAI reflects the interfacial tension of protein droplets at the oil–water interface and the ability to stabilize the emulsion. It is determined by protein–protein and protein–lipid interactions. The ESI refers to the stable strength of the emulsion in the dispersion [41]. It can be seen that, except for the 1 mM EGCG complex, the EAI of the HPI-EGCG complexes was lower than that of the control in (Figure 5). Specifically, the ESI value first increased and then decreased with increasing EGCG concentration, reaching a maximum when EGCG was added at 1 mM. Protein–protein and protein-lipid interaction affects the emulsifying properties of proteins and plays an important role in the middle [42]. The combination of protein and the appropriate concentration of EGCG may change the protein–protein interactions between molecules, thereby reducing the interfacial tension of the oil–water interface [43]. The improvement in emulsification performance may be due to the change in the flexibility of HPI after combining with EGCG. The solubility of the protein is improved and the surface hydrophobicity is increased, making protein particles more stable at the oil–water interface [44]. The emulsification of lactoferrin has also been shown to improve when covalently linked with EGCG.

**Figure 5.** (a–f) Emulsifying properties of HPI and covalent HPI-EGCG complexes at EGCG concentrations of 1, 2, 3, 4, and 5 mM (EAI is the Emulsifying Activity Index and ESI is the Emulsifying Stability Index).

#### *3.6. Cryo-Scanning Electron Microscopy Microstructure*

Cryo-scanning electron microscopy images can be seen for HPI and covalent HPI-EGCG complex emulsions in Figure 6. The oil droplets were uniformly attached to the surface of the HPI-EGCG molecules, which had a micro-spherical structure [45]. This result is consistent with the results of a previous study showing that when anthocyanins are covalently bound to HPI, they can disrupt the protein peptide chain and enhance the interactions between droplets to form an emulsion [46]. It can be seen that the native state of the HPI emulsion is flocculated, in which fat globules flocculate without breaking the membrane. The droplets of the emulsion continued to aggregate, and as aggregation increased, flocculation also increased, and emulsification occurred more rapidly [47]. After HPI and EGCG were covalently bound, the flocculation phenomenon in the emulsion was suppressed, with the greatest uniformity seen when EGCG was at 1 mM. This shows that the negative charge of EGCG helped stabilize the emulsion and make it more uniform [48].

**Figure 6.** Cryo-scanning electron microscope images of HPI and covalent HPI-EGCG complexes at EGCG concentrations of 1, 2, 3, 4, and 5 mM. (**A**) HPI, (**B**) HPI-1 Mm EGCG, (**C**) HPI-2 mM EGCG, (**D**) HPI-3 mM EGCG, (**E**) HPI-4 mM EGCG, and (**F**) HPI-5 mM EGCG.

#### **4. Conclusions**

In this study, a protein–polyphenol covalent complex was formed by combining HPI with different concentrations of a polyphenol. The addition of EGCG caused changes in the structure of HPI and improved functional properties. After HPI is combined with 1 mM of EGCG, the emulsification and solubility of the covalent complex are improved. These data provide the theoretical basis for the application of polyphenols and protein covalent complex food processing emulsifier.

**Author Contributions:** X.B. and L.-L.L.: methodology, validation, and writing—original draft. Y.Y. and D.-H.Y.: formal analysis. X.-H.P. and L.-K.R.: data curation and writing—review. J.-C.G., L.W. and X.-M.Z.: methodology and validation. B.W. and J.Y.: editing. N.Z. and H.-S.Y.: supervision, project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** We appreciate the financial support from the National Natural Science Foundation of China (32072258), the Major Science and Technology Program of Heilongjiang (2019ZX08B02, 2020ZX08B02), Harbin University of Commerce "young innovative talents" support program (2019CX06, 2020CX26, 2019CX34), Heilongjiang Academy of Sciences (High Technology Research Institute) (Horizontal Project) Functional evaluation of hemp oil and protein and development of high-value food and central financial support for the development of local colleges and universities.

**Institutional Review Board Statement:** This article does not involve any animal experiments.

**Informed Consent Statement:** This article does not involve any experiments with humans as recipients.

**Data Availability Statement:** Research data are not shared.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **References**

