**1. Introduction**

Hemp (*Cannabis sativa* L.) is a widely cultivated plant, and has an especially important role in industry. It is divided into two types according to the 9-tetrahydrocannabinol (THC) content: industrial hemp and drug hemp [1]. A 0.3% THC standard has been established by the European Union for this classification. If the THC is less than 0.3%, industrial hemp is allowed to be grown in China and Canada. Hemp is grown for industrial use and harvested for its fiber, seeds, and oil. Hemp seeds are rich in phytosterols, omega-3, and omega-6 essential fatty acids and protein (approximately 25% of dry weight). Moreover, there are many kinds of essential amino acids, and all of the essential amino acids needed by the human body are contained, with relatively high glutamic acid and arginine content [2,3]. Histidine is an essential amino acid for infants less than eight months old, and it plays an important role in the prevention of cardiovascular diseases in middle-aged and elderly people and in the growth of children. Hemp seeds are considered to be a good source of high-quality protein suitable for the elderly and infants. Therefore, they have been

**Citation:** Pang, X.-H.; Yang, Y.; Bian, X.; Wang, B.; Ren, L.-K.; Liu, L.-L.; Yu, D.-H.; Yang, J.; Guo, J.-C.; Wang, L.; et al. Hemp (*Cannabis sativa* L.) Seed Protein–EGCG Conjugates: Covalent Bonding and Functional Research. *Foods* **2021**, *10*, 1618. https://doi.org/ 10.3390/foods10071618

Academic Editor: Barry J. Parsons

Received: 4 June 2021 Accepted: 1 July 2021 Published: 13 July 2021

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used in the production of various foods with high nutritional value. Importantly, the emergence of hemp varieties with low THC content has increased the utilization of hemp in food production [4]. Hemp seeds contain more than 30% oil and 25% protein. Currently, hemp is mainly used for the extraction of hemp oil because of the high oil content in the seeds [5]. However, compared with other plant proteins, hemp protein has low solubility, which is attributed to the aggregation of edestin (11S globulin) at pH values lower than 7.0. Therefore, hemp protein needs to be modified to increase its range of applications.

In recent years, various chemical modification methods, such as phosphorylation, glycosylation, deamidation, and succinylation, have been proven to be effective in improving the functional properties of proteins [6]. Phosphorylation, deamidation, and succinylation methods all result in chemical residues, which lead to a decrease in the nutritional value of the protein. The Maillard reaction is the main method of glycosylation, but it is difficult to control. Overreaction affects the flavor and quality of the products. Therefore, some researchers have proposed the use of polyphenols to modify proteins. In recent years, many researchers have begun to study the interaction between polyphenols and proteins. Proteins and polyphenols can interact through both non-covalent and covalent bonds. Non-covalent interactions are reversible, whereas covalent interactions are irreversible [7]. There are two types of methods used to form protein–polyphenol conjugates: non-enzymatic (alkaline and free radical reactions) and enzymatic (polyphenol oxidase, laccase, and tyrosinase) [8]. The mechanism of the alkaline reaction involves the oxidation of phenolic compounds to quinone compounds in an alkaline solution [9]. Quinone compounds usually react further with nucleophilic amino acid residues in the protein chain. Wei conducted research on the covalent complex of sodium caseinate, β-lactoglobulin, lactoferrin, and α-whey protein with EGCG and found that the covalent complex significantly improved the stability of the β-carotene emulsion [10]. Some researchers have also covalently combined phenolic compounds with flaxseed protein isolate (FPI). They found that the covalent complex of polyphenols and has a higher emulsifying ability than FPI [11]. When He studied the covalent binding of ovalbumin (OVA) and EGCG, he also found that the emulsification of OVA was improved [12]. The addition of phenol allows the protein to acquire phenolic hydroxyl groups, thereby changing the functional properties of the protein.

(−)-Epigallocatechin gallate (EGCG) is the main component of polyphenols and catechins in green tea [13]. EGCG has beneficial physiological activities, such as protecting against free radical DNA damage, antioxidative effects [14], inhibiting tumor growth, reducing serum low-density lipoprotein and cholesterol levels, improving vascular proliferation, and protecting against cardiovascular diseases [15].

Research on polyphenols and proteins has mainly focused on non-covalent interactions, but these interactions are reversible and unstable. Therefore, we chose to study covalent interactions between polyphenols and proteins. The covalent complexes formed by hemp protein isolate (HPI) and EGCG were used to investigate these interactions. Zeta potential values, CD spectra, and three-dimensional fluorescence spectra were used to explore the structure of the HPI-EGCG complex. The functional characteristics of the covalent complex were determined by analyzing the reactive groups, polyphenol content, the size of the particles, and other relevant indicators. The results of this research provide some theories and provide a research basis for future researchers for the application of the HPI-EGCG complex in the food industry as an emulsifier.

#### **2. Materials and Methods**

#### *2.1. Experimental Materials*

Hemp was purchased from the Heilongjiang Academy of Sciences (Harbin, China). Hemp protein samples were prepared by alkali-soluble acid precipitation. EGCG, *o*phthaldialdehyde (OPA), and 5,5- -dithio-2-nitrobenzoic acid (DTNB) were purchased from Solarbio Life Science Co., Ltd. (Beijing, China). Other reagents were of analytical grade and were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China).
