Morchella esculenta peptide and use thereof

Abstract

The present invention relates to the field of protein engineering, and specifically relates to a Morchella esculenta peptide and the use thereof. By means of comprehensively applying bioinformatic methods such as virtual enzymolysis, activity prediction, physicochemical property and safety evaluation, and molecular docking, one Morchella esculenta peptide having the potential effects of "inhibiting oxidation + skin whitening" is screened from Morchella esculenta, which peptide has a sequence of WWVCAK. In-vitro chemical experiments verify that WWVCAK is the most effective antioxidant and tyrosinase-inhibitory peptide. Therefore, WWVCAK can exert the effects of inhibting oxidation and skin whitening via multiple targets, multiple functions and multiple pathways. WWVCAK is expected to be developed and used in cosmetics as a functional skin-care ingredient with the twin effects of "inhibiting oxidation + skin whitening".

Description

Morelloide and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of protein engineering, and particularly relates to a "antioxidant + whitening" dual-function morelloide and application thereof. BACKGROUND

[0002] Some intrinsic factors (such as age growth and endocrine disorders, etc.) and many external factors (such as ultraviolet radiation, air pollution, smoking, long time facing computer, etc.) can accelerate skin aging, cause skin to lose elasticity, produce wrinkles, appear loose and pigmentation, etc., and consume people's "youth". Preventing and delaying skin aging has become one of the hot issues in the scientific community, and antioxidant and whitening as the focus of the skin care industry at this stage are more and more concerned by people. Although synthetic compounds such as arbutin, kojic acid, resveratrol, hydroquinone and vitamin E have been applied in cosmetics and can produce certain antioxidant and whitening effects, there are also some limitations (high cost, poor permeability, instability) and side effects (such as skin irritation, inflammatory reaction and allergy, etc.). Therefore, the research and development of safe and healthy natural "antioxidant + whitening" skin care ingredients have become a hot issue for researchers at home and abroad.

[0003] Bioactive peptides are active polypeptides with multiple functions composed of 2-20 natural amino acids. Bioactive peptides have simple structure, good safety and easy absorption, and can exert a wide range of biological functions through enzyme inhibition, antibacterial, antioxidant and other different ways. Although its biological activity may not be comparable to synthetic drugs, it is rarely accumulated in the human body and has no side effects, and is an ideal candidate for the development of functional skin care ingredients. In recent years, the research on the potential application of bioactive peptides in cosmetics has gradually increased, and some bioactive peptides (such as carnosine, silk peptide and palmitoyl peptide) have been commercialized in cosmetics.

[0004] Traditional bioactivity research usually adopts enzymatic method for preparation, and then uses ultrafiltration, chromatography, chromatography and mass spectrometry for separation and identification, and finally conducts functional evaluation through in vivo and in vitro activity detection. The whole process is complicated, high cost, long time, low yield, and sometimes the target peptide segment with low content but high activity is lost. In recent years, the application of bioinformatics in bioactive peptide research has increased year by year. Bioinformatics methods can realize the analysis and optimization of precursor proteins, optimize the controllable hydrolysis of parent proteins, and also can predict the activity and physicochemical properties of target peptides. Compared with traditional bioactive peptide research, the application of bioinformatics tools can greatly improve the screening efficiency of bioactive peptides. Recently, bioinformatics methods have identified a variety of antioxidant peptides and tyrosinase inhibitory peptides from different raw material proteins such as sesame, sea cucumber and pea.

[0005] Morel (Morchella esculenta) is also known as Morel mushroom, Morel vegetable, etc. It is a kind of extremely rare medicinal and edible fungus, and is named because of its shape similar to a sheep's stomach. Morel has extremely high nutritional value and various excellent biological activities. Studies have shown that Morel protease hydrolysate has good antioxidant activity and tyrosinase inhibitory activity, and is expected to be developed and utilized as a functional ingredient in the fields of medicine and cosmetics. However, so far, there is no report on screening of Morel peptides with dual functions of "antioxidation + whitening" by bioinformatics methods.

[0006] SUMMARY

[0007] To solve the above problems, the present application provides a Morel peptide, and the amino acid sequence of the Morel peptide is WWVCAK.

[0008] The present application also provides application of the above-mentioned Morel peptide in preparation of food, medicine, skin care product or health care product with whitening effect.

[0009] The present application also provides application of the above-mentioned Morel peptide in preparation of food, medicine, skin care product or health care product with antioxidant effect.

[0010] The present application also provides application of the above-mentioned Morel peptide in preparation of food, medicine, skin care product or health care product with antioxidant and whitening effect.

[0011] On the other hand, the present application provides a screening method of Morel peptide, which is screened by using bioinformatics methods of virtual enzymolysis, activity prediction, physicochemical property and safety evaluation and molecular docking.

[0012] The present application also provides a skin care product, and the main active ingredient is the above-mentioned Morel peptide.

[0013] The present application has the following beneficial effects:

[0014] The application adopts a bioinformatics method to efficiently screen a "antioxidant + whitening" function Morchella esculenta peptide WWVCAK from Morchella esculenta. The double-function Morchella esculenta peptide has a molecular weight less than 1000 and strong hydrophobicity. Molecular docking results show that WWVCAK can be combined with key amino acid residues of myeloperoxidase (MPO), Kelch-like epoxide chloropropane-related protein 1 (keap1) and tyrosinase active site to play the "antioxidant + whitening" function. The IC50 of WWVCAK for scavenging DPPH and ABTS, chelating ability and tyrosinase inhibition activity is 70.24±0.79 μg / mL, 51.28±0.79 μg / mL, 1944±41 μg / mL and 1128±18 μg / mL respectively, the Trolox equivalent of reducing power is 127.29±3.77 mg / g, the Trolox equivalent of DPPH scavenging is 75.17±2.55 mg / g, the Trolox equivalent of ABTS scavenging is 969.53±16.88 mg / g, the EDTA-2Na equivalent of chelating ability is 7.69±0.22 mg / g, and the kojic acid equivalent of tyrosinase inhibition activity is 119.80±3.75 mg / g. At the same time, WWVCAK has no significant effect on cell viability, can significantly improve the total antioxidant capacity of cells, significantly slow down the generation of intracellular free radicals (ROS), malondialdehyde (MDA) and melanin, and has a strong inhibitory effect on intracellular tyrosinase activity, so WWVCAK has good antioxidant and whitening effects. In addition, based on the research results of bioinformatics, the double-function Morchella esculenta peptide of the application has no hemolytic activity, immunogenicity, allergenicity and toxicity. The application provides a new idea for the development of peptide cosmetic ingredients, and has certain theoretical guiding significance and expanding scientific significance. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0016] Figure 1 is a mass spectrum of WWVCAK

[0017] Figure 2 3D and 2D diagram of WWVCAK docking with myeloperoxidase (MPO), Kelch-like epoxide hydroprene associated protein 1 (keap1) and tyrosinase molecules; A is the 3D diagram of WWVCAK interacting with MPO, keap1 and tyrosinase, in which the receptors are represented in cartoon form and WWVCAK is represented in yellow surface form; B is the 2D diagram of WWVCAK interacting with receptors, in which the green dotted line represents the hydrogen bond, the purple solid line represents the ligand bond, and the eyelash-like structure represents the hydrophobic interaction. Different lower case letters in Figures 3-8 represent significant differences (P < 0.05) between different treatments

[0018] Figure 3 Effect of WWVCAK on cell viability;

[0019] Figure 4 Effect of WWVCAK on cell MDA content;

[0020] Figure 5 Effect of WWVCAK on cell ROS content;

[0021] Figure 6 Effect of WWVCAK on cell total antioxidant capacity;

[0022] Figure 7 Effect of WWVCAK on cell melanin content;

[0023] Figure 8 Effect of WWVCAK on cell tyrosinase activity. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present application are now described in detail. The methods of the examples are carried out in accordance with conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description is not to be taken in a limiting sense and is understood to be merely a description of certain aspects, features, and embodiments of the present application.

[0025] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise stated, each intervening value of the parameter is also specifically included within the scope of the present application. The intervening values are based on the application's teachings. In addition, where a range of values is provided, it is understood that each intervening value, to the extent that it does not expressly exclude any other intervening value, along with each individual value selected from the provided ranges is also individually and specifically included within the scope of the present application. The same applies to ranges of values included in statements of scope of the disclosed or claimed subject matter.

[0026] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains unless otherwise specifically defined herein. Although preferred methods and materials are described herein, any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described herein. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0027] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0028] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and methods.

[0029] Example 1 Bioinformatics-based screening of "antioxidant + whitening" dual-function Morchella peptides

[0030] The Morchella main structural protein sequences were downloaded from the UniProt KB protein database (https: / / www.uniprot.org / ). The protein sequences were optimized using the CALCULATIONS module of the BIOPEPUWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ). Twenty Morchella proteins were screened as experimental research materials based on the comprehensive ΣA value (antioxidant peptide frequency + tyrosinase inhibitor peptide frequency).

[0031] The 20 Morchella proteins were virtually digested by the ENZYME(S) ACTION module in the BIOPEPUWM database, and the release frequency (ΣAE) of "antioxidant + whitening" dual-function peptides was comprehensively optimized. 54S ribosomal protein L4, mitochondrial was selected as the raw material protein for preparing "antioxidant + whitening" dual-function Morchella peptides, and ficin was selected as the preparation enzyme (Table 1).

[0032] Table 1 Optimization and virtual digestion results of Morchella proteins

[0033] The potential of the virtual enzymatic peptides to have biological activity was predicted using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), and the cell permeability of the peptides was evaluated by CPPpred (http: / / distilldeep.ucd.ie / CPPpred); the peptides with PeptideRanker scores greater than 0.6 and CPPpred scores greater than 0.5 were used to predict their antioxidant activity and tyrosinase inhibitory activity by AnOxPePred (https: / / services.healthtech.dtu.dk / service.php?AnOxPePred-1.0) and pepsite2 (http: / / pepsite2.russelllab.org / ) respectively, and the peptides with high antioxidant activity and tyrosinase inhibitory activity scores were intersected using the Venn diagram tool (https: / / bioinfogp.cnb.csic.es / tools / venny / ), to preliminarily screen a series of "antioxidant + whitening" dual-function Morchella peptides. These peptides were then respectively docked with myeloperoxidase (MPO), Kelch-like epoxide hydrolase-related protein 1 (keap1) and tyrosinase by HPEPDOCK (http: / / huanglab.phys.hust.edu.cn / hpepdock / ), and the docking scores are shown in Table 2, and a novel Morchella peptide WWVCAK with antioxidant and tyrosinase inhibitory activity was screened by combining novelty detection (Table 3). The mass spectrum of WWVCAK is shown in Figure 1.

[0034] Table 2 Molecular docking scores of WWVCAK with receptor proteins

[0035] Table 3 "Antioxidant + whitening" dual-function Morchella peptides screened by virtual screening

[0036] The molecular weight, isoelectric point and solubility of WWVCAK were predicted by Innovagen (http: / / www.innovagen.com); the net charge and hydrophobicity were predicted by Pepdraw (http: / / www.tulane.edu / ~biochem / WW / PepDraw / ); the allergenicity was predicted by Allergy lab (https: / / sortaller.gzhmu.edu.cn / ); the hemolyticity was predicted by HemoPI (https: / / webs.iiitd.edu.in / raghava / hemopi / design.php); the aggregation was predicted by ChemAGG (https: / / admet.scbdd.com / ChemAGG / index / ); the immunogenicity was predicted by IEDB (http: / / tools.iedb.org / immunogenicity / ); and the toxicity was predicted by ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / design.php). The prediction results of the physicochemical properties of WWVCAK are shown in Table 3.

[0037] Table 4 Physicochemical properties and safety of WWVCAK

[0038] As can be seen from Table 4, the molecular weight of WWVCAK is less than 1000, the solubility is poor, the hydrophobicity is strong, the net charge is +1, and the isoelectric point is alkaline; there is no aggregation, and there is no hemolyticity, immunogenicity, allergenicity and toxicity. The "antioxidant + whitening" dual-function Morel peptide of the present application has good physicochemical properties and good safety.

[0039] The molecular docking results of WWVCAK with three receptors are shown in Figure 2, wherein Figure 2A is a 3D interaction diagram of WWVCAK docking with MPO, keap1 and tyrosinase, Figure 2B is a 2D interaction diagram of WWVCAK docking with MPO, keap1 and tyrosinase, and Figure 2 shows that WWVCAK is bonded with His336 of MPO by ligand bond, and is bonded with Ile(339, 402), Leu(338, 406, 417), Glu242, Gln(90, 91), Gly(90, 335), Tyr296, Asp94, Phe332, His95, Arg(239, 333), Phe407 and Met411 by hydrophobic interaction; WWVCAK is bonded with Gly367, Thr560 and Val(604, 606) of keap1 by hydrogen bond, and is bonded with Leu557, Gly(419, 558, 605), Ile(416, 559), Ala(466, 607), Val(369, 418, 420, 463, 467, 514, 561, 608), Arg326, Cys(368, 513) by hydrophobic interaction; WWVCAK is bonded with Arg268, His244, Glu256 of tyrosinase by hydrogen bond, is bonded with Phe292 by ligand bond, and is bonded with Gly(86, 245, 281), His(61, 85, 94, 259, 263, 296), Ser282, Asn260 Phe264, Val(283, 248), Ala286 and Glu322 by hydrophobic interaction.

[0040] In the present application, the Arg239, His336 and Asp94 of the WWVCAK dual-function Morchella peptide of "antioxidation + whitening" combined with MPO are all key amino acid residues of the enzyme active center; the Cys(368, 513), Ile 559, Val(369, 463, 467, 514, 604, 606) amino acid residues combined with keap1 are also important active sites of the protein; in addition, the His(61, 85, 94, 244, 259, 263, 296), Glu256, Asn260 Phe264, Val283, Ala286 and Phe292 combined with tyrosinase are also all important amino acid residues of the enzyme active center.

[0041] Example 2: In vitro chemical experiment for detecting the antioxidant and hypoglycemic activities of WWVCAK

[0042] The in vitro chemical experiment model method was used to evaluate the reducing power, free radical (ABTS and DPPH) scavenging ability, chelating activity and tyrosinase inhibitory activity of WWVCAK (Table 5). It can be seen that WWVCAK not only has strong reducing power, chelating ability, can effectively scavenge DPPH and ABTS free radicals, and also has a strong inhibitory effect on tyrosinase.

[0043] Table 5 Antioxidant and tyrosinase inhibitory activity of WWVCAK Note: The IC50 unit in the table is μg / mL, the equivalent unit is mg / g, and - represents not determined.

[0044] Example 3 Cell experiment to evaluate the antioxidant and tyrosinase inhibitory activity of WWVCAK

[0045] The B16F10 cells in the logarithmic phase were digested, counted, and prepared into a cell suspension with a concentration of 3×10 4 The B16F10 cells in the logarithmic phase were digested, counted, and prepared into a cell suspension with a concentration of 3×10

[0046] The results are shown in Figures 3-8. As can be seen from the results of Figures 3-8, the "antioxidant + whitening" dual-function Morel peptide WWVCAK screened by the application has no significant effect on cell viability, can significantly improve the total antioxidant capacity of cells, significantly slow down the generation of free radicals, malondialdehyde and melanin in cells, and has a strong inhibitory effect on tyrosinase in cells, indicating that WWVCAK has good antioxidant and whitening effects at the cellular level.

[0047] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A morel peptide, characterized in that, The amino acid sequence of the morel peptide is WWVCAK.

2. The application of the morel peptide as described in claim 1 in the preparation of food, medicine, skin care products or health products with whitening effects.

3. The application of the morel peptide as described in claim 1 in the preparation of foods, pharmaceuticals, skin care products or health products with antioxidant effects.

4. The application of the morel peptide as described in claim 1 in the preparation of food, pharmaceuticals, skin care products or health products with antioxidant and whitening effects.

5. A method for screening morel peptides as described in claim 1, characterized in that, It was obtained using bioinformatics methods such as virtual enzymatic digestion, activity prediction, physicochemical property and safety assessment, and molecular docking.

6. A skincare product, characterized in that, The main active ingredient is the morel peptide as described in claim 1.

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