**1. Introduction**

In recent years, the use of plant proteins as an alternative to animal proteins has attracted considerable consumer interest due to their wide availability, affordability, hypoallergenicity, cholesterol-lowering effects, and ease of digestion [1]. Pea protein (PP) is a valuable plant protein with the advantages of being hypoallergenic, non-GMO, aminoacid-balanced and rich in lysine [2,3]. The amino acid ratio of PP is balanced, the content of the other seven kinds of human essential amino acids except methionine is close to the recommended model value of FAO and WHO, and it is easy to digest and absorb. Meanwhile, the lysine content is higher than other plant proteins.

Although PP possesses high nutritional value, its application in formula food is limited due to its poor water solubility and limited functional properties [3,4]. In order to overcome these drawbacks, physical modification, chemical modification, and enzymatic modification have been applied to improve the properties of PP. Physical modification, including heating, magnetic field, freezing, ultrasound, and microfluidization, has been reported to improve the structural, functional, and nutritional value of proteins [5,6]. Although physical modification does not involve the addition of exogenous substances, it is not widely used because the modification effect is limited [7]. Chemical modification was considered as an effective method to improve the functional properties of protein by introducing new organic groups into the protein molecule to break or polymerize the amino, carboxyl, sulfhydryl, or carbonyl groups on the main or side chains of the protein, resulting in changes in the structural and physicochemical properties of the protein [8]. However, chemical modification was also not widely used in the food industry because of the addition of chemicals that may be harmful to human health, such as toxicity.

Enzymatic hydrolysis is a promising technology in the protein field. Compared to physical and chemical modification, enzymatic modification is widely used because of the advantages of low by-products, high specificity, and easy control. It can convert native protein into peptides of various sizes and free amino acids [9]. Enzymatic hydrolysis was considered as one of the most promising methods for the modification of tailor-made protein preparations [10,11]. Arteaga et al. [12] used 11 enzymes to hydrolyze PP under different enzymolysis time, altering the molecular weight distribution and, thus, improving the technical functionality and sensory properties of PP. Klost et al. [13] investigated the ability of trypsin treatment to improve the poor solubility and interfacial properties of PP, and the potential to improve the overall stability of PP emulsions. Tamm et al. [14] found that trypsin treatment improved various properties of PP emulsions through characterization on molecular weight distribution, interfacial activity, expansive characterization, and emulsion properties. Klost et al. [15] discovered that the treatment of PP with trypsin improved the gel properties of fermented PP, which helped to develop PP yogurt alternatives for tailoring such gels. However, most studies mainly focused on single enzyme treatment and comparison of different enzyme treatments at the same enzymatic hydrolysis time. The effects of different enzyme treatments on PP at the same degree of enzymolysis (*DH*) were not clear. In addition, mild enzymatic hydrolysis modification does not improve the protein properties significantly, and excessive enzymatic hydrolysis modification would produce strong bitterness, thus affecting the organoleptic properties of PP [10,12]. Therefore, moderate enzymatic hydrolysis is required to improve the protein properties and produce less bitterness at the same time. Furthermore, systematic studies and comparative information on the effects of different *DH* on PP are limited.

Based on the above analysis, the present study focused on investigating the effects of different enzymes (flavourzyme, neutrase, alcalase, and trypsin) on the structural and functional properties of PP at the same *DH* under moderate enzymatic hydrolysis conditions.

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

#### *2.1. Material*

Pea protein (PP, purity ≥ 80%) was provided by the Shuangta Food Co., Ltd. (Qingdao, China). Enzymes including Alcalase 2.4 L FG (2.4 AU/Ml, endo protease), Neutrase 0.8 L (0.8 AU/g, endo protease), Trypsin (4000 U/g, acts on carboxyl side cleavage of lysine and arginine residues in polypeptide chains), and Flavourzyme 1000 L (1000 LAPU/g, mixture of endopeptidase and exopeptidase) were purchased from Novozymes (Bagsvaerd, Denmark). The SDS-PAGE kit and Micro Total Mercapto Assay Kit were purchased from Solarbio Science and Technology (Beijing, China). 1-anilino-8-naphthalene-sulfonate (ANS), sodium dodecyl sulfate, and ammonium peroxodisulfate were purchased from Sigma company (St. Louis, MO, USA). All other chemicals used were of analytical grade. Doubledistilled water was used throughout the research.
