Egg white-derived active peptide and use thereof

Abstract

The present application relates to the technical field of bioactive peptides, and particularly relates to an egg white-derived active peptide and application thereof. The egg white-derived active peptide comprises amino acids Asp-Glu-Lys and / or Gly-Asp-Val-Ala. The application provides application of the egg white-derived active peptide in preparation of tyrosinase inhibitors and whitening cosmetics. Advantages are that the active peptides Asp-Glu-Lys and Gly-Asp-Val-Ala can stably and effectively bind to tyrosinase, inhibit the activity of tyrosinase, thereby inhibiting the generation of melanin and helping to improve the pigmentation of human faces. The active peptide can be applied to preparation of cosmetics and functional foods with whitening effects, and is safe, non-toxic, easy to digest and absorb.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to an egg white-derived bioactive peptide and its applications. Background Technology

[0002] Tyrosinase (EC 1.14.18.1) is a copper-containing polyphenol oxidase that plays a crucial role in the first two steps of melanin biosynthesis, catalyzing the hydroxylation of L-tyrosine to L-DOPA and the oxidation of DOPA to dopaquinone. While melanin plays a vital role in protecting the skin from UV damage, excessive production and accumulation of melanin can lead to solar melanosis, freckles, age spots, and post-inflammatory hyperpigmentation, negatively impacting appearance and increasing psychological stress.

[0003] Commonly used tyrosinase inhibitors include ascorbic acid, hydroquinone, kojic acid, and arbutin. However, their application is greatly limited due to several adverse side effects. For example, arbutin and hydroquinone have been reported to cause contact dermatitis and exogenous okra, respectively. Kojic acid has been shown to be unstable and long-term use can lead to skin cancer. Therefore, the search for potential natural, non-toxic, stable, and highly effective tyrosinase inhibitors is of great significance in the food and cosmetic industries.

[0004] In recent years, research on peptide-based skin whitening agents has attracted much attention. Studies have found that hydrolysates of some natural proteins have whitening functions, inhibiting tyrosinase activity and melanin production. Eggs are an important source of dietary protein and a rich source of bioactive peptides. The proteins in egg white mainly include 54% ovalbumin, 12% ovotransferrin, and 11% ovomucoid. Chinese patent application CN115353548A discloses a pearl oyster tyrosinase inhibitory peptide with whitening effects and its application. The sequence of this pearl oyster tyrosinase inhibitory peptide is WLL(Trp-Leu-Leu). This pearl oyster tyrosinase inhibitory peptide exhibits good tyrosinase inhibitory activity at both the molecular and biological levels, inhibiting melanin synthesis and demonstrating good whitening activity. However, no egg white-derived tyrosinase inhibitors have been reported in existing literature. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an egg white-derived active peptide and its applications.

[0006] The primary objective of this invention is to provide an egg white-derived bioactive peptide, wherein the egg white-derived bioactive peptide comprises the amino acid Asp. Glu Lys and / or Gly-Asp-Val-Ala.

[0007] Preferably, the egg white-derived bioactive peptides are prepared by solid-phase synthesis or targeted enzymatic hydrolysis and purification of egg white protein.

[0008] The second objective of this invention is to provide an application of an egg white-derived active peptide in the preparation of tyrosinase inhibitors.

[0009] The third objective of this invention is to provide an application of egg white-derived active peptides in the preparation of whitening cosmetics.

[0010] Compared with the prior art, the present invention can achieve the following beneficial effects: This invention contains two active peptides, DEK and GDVA, derived from egg white protein, which have tyrosinase inhibitory activity. These peptides can stably and effectively bind to tyrosinase, inhibiting its activity and thus suppressing melanin production, helping to improve facial pigmentation. They can be used to prepare cosmetics with whitening effects; they are safe, non-toxic, and easily digested and absorbed. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the active peptide screening process provided in an embodiment of the present invention.

[0012] Figure 2 This is a 2D diagram of the interaction between the active peptide DEK and tyrosinase according to an embodiment of the present invention.

[0013] Figure 3 This is a 2D diagram of the interaction between the active peptide GDVA and tyrosinase according to an embodiment of the present invention. Detailed Implementation

[0014] In the following description, embodiments of the invention will be described with reference to the accompanying drawings. In the description below, the same modules are denoted by the same reference numerals. Where the same reference numerals are used, their names and functions are also the same. Therefore, their detailed description will not be repeated.

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof.

[0016] Example 1 I. Computer-aided virtual screening of bioactive peptides The amino acid sequences of ovotransferrin, ovalbumin, and ovomucin from eggs were obtained using the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and virtual enzyme digestion was performed on the obtained sequences using the online enzyme digestion tool ExPASy PeptideCutter. The water solubility and toxicity of the peptides were predicted using the online programs Peptide Property Calculator and ToxinPred. Non-toxic and water-soluble peptides were defined as ligands. The 3D structure of tyrosinase (PDB ID: 2Y9X) was obtained from the Protein Data Bank (PDB) database and used as the receptor protein. Molecular docking was performed using DiscoveryStudio (DS) software to screen for peptides that could bind tightly to the active site of tyrosinase, preliminarily identifying potential tyrosinase inhibitory peptides DDSK, AQSD, GDVA, and DEK.

[0017] The specific analysis steps are as follows: The amino acid sequences of ovalbumin (Accession: AAA48998.1), ovotransferrin (Accession: NP_990635.2), and ovomucin (Accession: NP_001295423.1) from eggs were obtained using the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). The obtained sequences were then virtually digested using trypsin (EC 3.4.21.4), pepsin (EC 3.4.23.1), and chymotrypsin (EC 3.4.21.1) using the online enzyme digestion tool ExPASyPeptideCutter (http: / / web.expasy.org / peptide_cutter / ), yielding 213 peptides. The water solubility and toxicity of the peptides were predicted using the online programs Peptide Property Calculator and ToxinPred, and 127 peptides with good water solubility and non-toxicity were selected. The 3D structure of tyrosinase (PDB ID: 2Y9X) was obtained from the Protein Data Bank (PDB) database and used as a receptor protein. The active site was defined in the receptor-ligand interaction module of the DS program. Non-toxic and water-soluble peptides were screened and defined as ligands, and energy minimization was performed. The CDOCKER module in the DS program was used to screen for bioactive peptides that could effectively bind to the active site of tyrosinase, with docking coordinates x: 10.0, y: 28.8, z: The diameter is 43.6, and the radius is 9 Å. A lower CDOCKER-ENERGY value indicates a tighter binding between the active peptide and the tyrosinase. Docking results showed that 70 peptides could bind to the active site of the tyrosinase.

[0018] Using known tyrosinase inhibitory peptides DGL (-112.13 kcal / mol) and FPY (-40.183 kcal / mol) as positive controls, 70 peptides that can bind to the active site of tyrosinase were screened, and the results are shown in Table 1. Four peptides with the most potential tyrosinase inhibitory activity, DDSK, AQSD, GDVA and DEK (-139.6, -126.2, -122.8 and -122.5 kcal / mol), were selected, and their docking energies were all lower than those of DGL and FPY.

[0019] Table 1. Water solubility, toxicity, and molecular docking results of potential egg white-derived tyrosine-inhibiting peptides.

[0020] II. In vitro tyrosinase inhibitory activity assay The inhibitory activities of the active peptides DDSK, AQSD, GDVA, and DEK on tyrosinase were determined by spectrophotometry. Specifically, 80 μL of solutions of different concentrations of DDSK, AQSD, GDVA, and DEK peptides were taken, and 40 μL (500 U / mL) of tyrosinase solution was added. The mixture was incubated at 25°C for 5 min. Then, 80 μL of L-DOPA solution (0.5 mM) was added to initiate the reaction, and the reaction was carried out at 25°C for 5 min. After the reaction, the absorbance of the reaction solution at 475 nm was quickly measured. Each measurement was repeated three times in parallel. The tyrosinase inhibition rate was calculated using the following formula: Tyrosinase inhibition rate (%) = [(AB) - (CD)] × 100 / (AB) Where A is the absorbance value of the reaction solution without inhibitors, B is the absorbance value of the reaction solution without inhibitors and enzymes, C is the absorbance value of the reaction solution containing L-DOPA, and D is the absorbance value of the reaction solution without enzymes. IC 50 Defined as the inhibitor concentration that inhibits 50% of tyrosinase activity under the assay conditions.

[0021] The results showed that both peptide DEK and GDVA could effectively inhibit tyrosinase activity, and their IC50 values ​​were [not specified]. 50 The values ​​were 0.08 mg / mL and 5.80 mg / mL, respectively (see screening procedure). Figure 1 The bioactive peptides DDSK and AQSD did not exhibit tyrosinase inhibitory activity.

[0022] III. Identification of Two Tyrosinase Inhibitory Peptides Due to known limitations of the DS software, DDSK and AQSD did not exhibit tyrosinase inhibitory activity in in vitro validation experiments. Therefore, two bioactive peptides, DEK and GDVA, were screened and obtained.

[0023] The 2D visualization results of the docking of egg white-derived tyrosinase inhibitory peptides DEK and GDVA with tyrosinase (2y9x) are as follows: Figure 2 , Figure 3 As shown, the results indicate that both active peptides bind to tyrosinase residues His244, Phe292, Val283, Gly281, Asn260, Phe264, and Val248. Hydrogen bonds and hydrophobic interactions are the main forces binding the peptides to tyrosinase.

[0024] The amino acid sequence of DEK is Asp-Glu-Lys (SEQ ID NO.1), and the amino acid sequence of GDVA is Gly-Asp-Val-Ala (SEQ ID NO.2).

[0025] Example 2 Two novel tyrosinase inhibitory peptides, DEK and GDVA, were efficiently identified from egg white protein (ovotransferrin) using computer-generated virtual screening. In actual production, DEK and GDVA can be obtained through solid-phase synthesis or targeted enzymatic hydrolysis and purification methods, and prepared into granules, powders, or water-soluble liquids, which can help improve facial pigmentation. The active peptides can also be added to regular facial cleansers, masks, and creams to prepare whitening cosmetics.

[0026] It should be understood that the various forms of processes shown above can be used to reorder, add, or delete steps. For example, the steps described in this invention disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution disclosed in this invention can be achieved, and this is not limited herein.

[0027] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. The application of an egg white-derived bioactive peptide in the preparation of tyrosinase inhibitors, characterized in that: The amino acid sequence of the egg white-derived active peptide is Gly-Asp-Val-Ala.

2. The application of the egg white-derived active peptide according to claim 1 in the preparation of tyrosinase inhibitors, characterized in that: The egg white-derived active peptides are prepared by solid-phase synthesis and purification of egg white protein.

3. The application of the egg white-derived active peptide according to claim 2 in the preparation of tyrosinase inhibitors, characterized in that: The tyrosinase inhibitor is used to make skin-whitening cosmetics.

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