Multifunctional bioactive peptide based on highland barley vinasse protein and preparation method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a multifunctional bioactive peptide based on highland barley vinasse protein and a preparation method thereof, and the amino acid sequence of the bioactive peptide is shown as SEQ ID NO: 1. The bioactive peptide disclosed by the invention is small in molecular weight and easy to absorb, can inhibit tyrosinase activity and efficiently scavenge free radicals, and has the advantages of no toxicity, no carcinogenicity, high hydrophilicity, longer half-life period and the like through biosignal analysis and prediction; the compound can be applied to development of drugs for treating diseases related to skin pigmentation and oxidative stress or daily chemical products for whitening, anti-aging, anti-oxidation and the like, and has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a multifunctional bioactive peptide based on hulless barley distillers' grains protein and a preparation method thereof. Background Art

[0002] Hulless barley is an important plateau specialty cereal, and its distillers' grains are by-products generated during the brewing of hulless barley wine, containing rich nutrients such as protein, dietary fiber, vitamins and minerals. As a high-quality plant protein source, hulless barley distillers' grains protein has good nutritional value and development potential. However, at present, the utilization efficiency of hulless barley distillers' grains protein is relatively low, and most of it is treated as feed or fertilizer, failing to fully exert its potential bioactive value.

[0003] By methods such as enzymatic hydrolysis, small molecule polypeptides can be obtained from hulless barley distillers' grains protein. Among them, some polypeptides may have specific biological activities, such as antioxidant and blood pressure-lowering functions. However, at present, the research on the characteristic peptide segments with clear functions in the enzymatic hydrolysate of hulless barley distillers' grains protein is relatively less.

[0004] Therefore, screening out hulless barley distillers' grains protein polypeptides with multiple biological activities and clear efficacy is of great significance for the application of hulless barley distillers' grains protein products in the development of drugs or daily chemical products. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, the present invention provides a multifunctional bioactive peptide based on hulless barley distillers' grains protein and a preparation method thereof. The bioactive peptide of the present invention has a small molecular weight, is easy to absorb, can inhibit the activity of tyrosinase and efficiently scavenge free radicals, and through bioinformatics analysis, it is predicted to have advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity and a relatively long half-life. It can be applied to the development of drugs for the treatment of skin pigmentation and oxidative stress-related diseases or daily chemical products for whitening, anti-aging, antioxidant and other uses, and has a wide application prospect.

[0006] The present invention is based on the inventor's discovery and understanding of the following problems:

[0007] During the research process of hulless barley distillers' grains protein bioactive peptides, the inventor obtained a bioactive peptide DHWDIM with the activity of inhibiting tyrosinase through a large number of virtual screening works and experimental verifications. The amino acid sequence of this bioactive peptide is: Aspartic acid-Histidine-Tryptophan-Asparagine-Isoleucine-Methionine (Asp-His-Trp-Asn-Ile-Met, DHWDIM); the inventor further prepared this bioactive peptide by the Fmoc solid-phase synthesis method and verified its biological activities of simultaneously inhibiting tyrosinase activity and efficiently scavenging free radicals through experiments.

[0008] In the first aspect of the present invention, a bioactive peptide is proposed. According to an embodiment of the present invention, the amino acid sequence of the bioactive peptide is as shown in SEQ ID NO: 1. The bioactive peptide of the present invention has a small molecular weight, is easily absorbed, can inhibit the activity of tyrosinase and efficiently scavenge free radicals, and through bioinformatics analysis, it is predicted to have advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a relatively long half-life. It can be applied to the development of drugs for the treatment of skin pigmentation and oxidative stress-related diseases, or daily chemical products for whitening, anti-aging, antioxidant and other purposes, and has broad application prospects.

[0009] In the second aspect of the present invention, a nucleic acid molecule is proposed. According to an embodiment of the present invention, the nucleic acid molecule encodes the bioactive peptide described in the first aspect. According to an embodiment of the present invention, the bioactive peptide encoded by the nucleic acid molecule has a small molecular weight, is easily absorbed, can inhibit the activity of tyrosinase and efficiently scavenge free radicals, and through bioinformatics analysis, it is predicted to have advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a relatively long half-life. Through the nucleic acid molecule, the efficient expression of the bioactive peptide can be achieved, which helps to realize the large-scale industrial production of the bioactive peptide, and provides convenience for the development and application of subsequent products such as drugs for the treatment of skin pigmentation and oxidative stress-related diseases, or daily chemical products for whitening, anti-aging, antioxidant.

[0010] In the third aspect of the present invention, a construct is proposed. According to an embodiment of the present invention, it includes the nucleic acid molecule described in the second aspect. According to an embodiment of the present invention, the construct enables the nucleic acid molecule to be highly expressed in a host cell, thereby realizing the stable and efficient production of the aforementioned bioactive peptide.

[0011] In the fourth aspect of the present invention, a recombinant cell is proposed. According to an embodiment of the present invention, it includes the nucleic acid molecule described in the second aspect or the construct described in the third aspect. According to an embodiment of the present invention, the recombinant cell can highly express the aforementioned bioactive peptide under suitable conditions. The bioactive peptide has a small molecular weight, is easily absorbed, can inhibit the activity of tyrosinase and efficiently scavenge free radicals, and through bioinformatics analysis, it is predicted to have advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a relatively long half-life. Through the recombinant cell, the efficient expression of the bioactive peptide can be achieved, which helps to realize its large-scale industrial production.

[0012] In the fifth aspect of the present invention, a pharmaceutical composition is proposed. According to an embodiment of the present invention, it includes one or more of the bioactive peptide described in the first aspect, the nucleic acid molecule described in the second aspect, the construct described in the third aspect, and the recombinant cell described in the fourth aspect.

[0013] Those skilled in the art can understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells are equally applicable to the pharmaceutical composition, and will not be elaborated herein.

[0014] According to an embodiment of the present invention, the above pharmaceutical composition may further have the following additional technical features:

[0015] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0016] In the sixth aspect of the present invention, the present invention provides a daily chemical product. According to an embodiment of the present invention, it includes: the bioactive peptide described in the first aspect. The daily chemical product according to the embodiment of the present invention has functions such as whitening and antioxidant.

[0017] According to an embodiment of the present invention, the above daily chemical product may further have the following additional technical features:

[0018] According to an embodiment of the present invention, the daily chemical product further comprises an excipient acceptable in the daily chemical product.

[0019] According to an embodiment of the present invention, the daily chemical product includes one or more of creams, lotions, aqueous solutions, gels, oils, wax-based products and film products.

[0020] In the seventh aspect of the present invention, the present invention provides an antioxidant product. According to an embodiment of the present invention, it includes: the bioactive peptide described in the first aspect. The antioxidant product according to the embodiment of the present invention contains the aforementioned bioactive peptide capable of reducing the free radical level, and this bioactive peptide has antioxidant effects and can be applied to antioxidant.

[0021] In the eighth aspect of the present invention, the present invention provides the use of the bioactive peptide described in the first aspect, the nucleic acid molecule described in the second aspect, the construct described in the third aspect or the recombinant cell described in the fourth aspect in the preparation of pharmaceuticals, daily chemical products or antioxidant products. The pharmaceutical has at least one of the following uses: preventing and / or treating skin pigmentation; preventing and / or treating oxidative stress; the daily chemical product has at least one of the following uses: whitening; anti-aging; antioxidant; the antioxidant product is used for antioxidant.

[0022] Those skilled in the art can understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells are equally applicable to this use, and will not be elaborated herein.

[0023] In the ninth aspect of the present invention, a method for inhibiting the tyrosinase activity of a sample is proposed. According to an embodiment of the present invention, it includes: contacting the sample with the bioactive peptide described in the first aspect. According to the method of the embodiment of the present invention, by co-culturing the sample with the bioactive peptide of the present invention, the tyrosinase activity level in the sample is further inhibited.

[0024] In the tenth aspect of the present invention, a tyrosinase inhibitor is proposed. According to an embodiment of the present invention, it includes: the bioactive peptide described in the first aspect. According to the inhibitor of the embodiment of the present invention, it can specifically inhibit the tyrosinase activity through the action of the bioactive peptide described in the first aspect.

[0025] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The above and / or additional aspects and advantages of the present invention will become apparent and be easily understood from the description of the embodiments in conjunction with the following drawings, where:

[0027] Figure 1 It is the HPLC detection result diagram of the pure sample of the bioactive peptide DHWDIM in Example 1 of the present invention;

[0028] Figure 2 It is the MS detection result diagram of the pure sample of the bioactive peptide DHWDIM in Example 1 of the present invention;

[0029] Figure 3 It is the schematic diagram of the molecular docking simulation of the bioactive polypeptide DHWDIM and tyrosinase in Example 2 of the present invention;

[0030] Figure 4 It is the evaluation result diagram of the whitening and antioxidant functions of the bioactive polypeptide DHWDIM in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0032] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0033] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0034] As used herein, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0035] As used herein, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where such events or conditions occur and the cases where such events or conditions do not occur.

[0036] Terms and Definitions

[0037] As used herein, the term "radical scavenging rate" is one of the important indicators for measuring antioxidant capacity. Specifically, in the radical scavenging experiment, by comparing the absorbance changes of the radical solution before and after adding the scavenger, in the radical scavenging rate determination experiment of the embodiments of the present invention, the scavenger is the bioactive polypeptide DHWDIM of the present invention.

[0038] As used herein, the term "tyrosinase" refers to a copper-containing oxidoreductase, which is the rate-limiting enzyme for melanin synthesis and is widely present in the human body. It participates in the conversion process of tyrosine to melanin. By inhibiting the activity of tyrosinase, the production of melanin can be reduced, thereby improving skin color, whitening the skin, and being used for the treatment of diseases related to pigmentation and delaying skin aging.

[0039] Bioactive Peptide

[0040] The present invention provides a bioactive peptide. According to the embodiments of the present invention, the amino acid sequence of the bioactive peptide is as shown in SEQ ID NO: 1. The bioactive peptide of the present invention has a small molecular weight, is easy to absorb, can inhibit the activity of tyrosinase and efficiently scavenge free radicals, and through bioinformatics analysis, it is predicted to have the advantages of non-toxicity, non-carcinogenicity, high hydrophilicity, long half-life, etc. It can be applied to the development of drugs for the treatment of skin pigmentation and oxidative stress-related diseases or daily chemical products for whitening, anti-aging, antioxidant and other uses, and has broad application prospects.

[0041] According to the embodiments of the present invention, the amino acid sequence of the bioactive peptide is as shown in SEQ ID NO: 1.

[0042] DHWDIM (SEQ ID NO: 1).

[0043] It should be noted that the amino acid sequences described in the present invention are shown in the N-terminal to C-terminal manner.

[0044] Exemplarily, the bioactive peptide of the present invention has the ability to inhibit tyrosinase activity, and thus can be further used to develop drugs for treating diseases such as skin pigmentation or daily chemical products for whitening and anti-aging; the bioactive peptide of the present invention also has the ability to efficiently scavenge free radicals, and thus can be further used to develop drugs for treating diseases related to oxidative stress or daily chemical products for antioxidant use.

[0045] Nucleic acid molecule

[0046] The present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned bioactive peptide. According to an embodiment of the present invention, the aforementioned bioactive peptide encoded by the nucleic acid molecule has a small molecular weight, is easily absorbed, can inhibit tyrosinase activity and efficiently scavenge free radicals, and is predicted by bioinformatics analysis to have advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a relatively long half-life. Through the nucleic acid molecule, the efficient expression of the bioactive peptide can be achieved, which helps to realize the large-scale industrial production of the bioactive peptide, and provides convenience for the development and application of subsequent products such as drugs for treating skin pigmentation and oxidative stress-related diseases or daily chemical products for whitening, anti-aging, and antioxidant use.

[0047] It should be noted that for the nucleic acid molecules mentioned in this article, those skilled in the art should understand that it actually includes any one or both of the complementary double strands; although usually only one strand is given, in fact, the other complementary strand is also disclosed; in addition, the nucleic acid molecule sequences in the present invention include DNA form or RNA form, and disclosing one means the other is also disclosed.

[0048] Construct

[0049] The present invention provides a construct. According to an embodiment of the present invention, it includes the aforementioned nucleic acid molecule. According to an embodiment of the present invention, the construct enables the nucleic acid molecule to be efficiently expressed in a host cell, thereby realizing the stable and efficient production of the aforementioned bioactive peptide.

[0050] Exemplarily, the construct may be a vector; it should be noted that the vector mentioned in this article refers to a nucleic acid molecule that can self-replicate after being inserted into a suitable host, and transfers the inserted nucleic acid molecule into host cells and / or between host cells; the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and an expression vector mainly used for transcription and / or translation of DNA or RNA; the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell; by culturing a suitable host cell containing the vector, the vector can produce the aforementioned bioactive peptide, and the vector includes viral vectors, plasmids, phages, etc.

[0051] Recombinant cell

[0052] The present invention provides a recombinant cell. According to an embodiment of the present invention, it includes the aforementioned nucleic acid molecule or the aforementioned construct. According to an embodiment of the present invention, the recombinant cell can efficiently express the aforementioned bioactive peptide under suitable conditions. The bioactive peptide has a small molecular weight, is easily absorbed, can inhibit the activity of tyrosinase and efficiently scavenge free radicals, and is predicted by bioinformatics analysis to have advantages such as non-toxicity, non-carcinogenicity, high hydrophilicity, and a relatively long half-life. The efficient expression of the bioactive peptide can be achieved through the recombinant cell, which helps to realize its large-scale industrial production.

[0053] Pharmaceutical composition

[0054] The present invention provides a pharmaceutical composition. According to an embodiment of the present invention, it includes one or more of the aforementioned bioactive peptide, the aforementioned nucleic acid molecule, the aforementioned construct, and the aforementioned recombinant cell.

[0055] In this article, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well-known in the pharmaceutical field. All methods include the step of combining the active ingredient with a carrier constituting one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0056] According to an embodiment of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients. Thus, through the addition of excipients, the pharmaceutical composition can better exert the efficacy of the bioactive peptide.

[0057] In this article, the term "pharmaceutically acceptable" is applicable to humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), and "pharmaceutically acceptable excipients" are substances with a reasonable benefit / risk ratio.

[0058] In this text, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for the range where any conventional excipient is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their uses are also within the scope contemplated by the present invention.

[0059] Those skilled in the art can understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells also apply to the pharmaceutical composition and will not be elaborated herein.

[0060] Cosmetics

[0061] The present invention provides a cosmetic. According to an embodiment of the present invention, it includes: the aforementioned bioactive peptide. The cosmetic according to the embodiment of the present invention has effects such as whitening and antioxidant.

[0062] According to an embodiment of the present invention, the cosmetic further includes excipients acceptable in the cosmetic. Thus, through the addition of excipients, the cosmetic can better exert the efficacy of the bioactive peptide and be acceptable to consumers.

[0063] According to an embodiment of the present invention, the cosmetic includes one or more of creams, lotions, aqueous solutions, gels, oils, wax-based products and film products. The cosmetic according to the embodiment of the present invention can meet the usage habits of different consumers and provide more choices while fully exerting its efficacy.

[0064] Exemplarily, the cosmetic may be, including but not limited to: gels, serums, moisturizing creams, facial creams, toners, body washes, shampoos, facial masks, etc.

[0065] Antioxidant products

[0066] The present invention provides an antioxidant product. According to an embodiment of the present invention, it includes: the aforementioned bioactive peptide. The antioxidant product according to the embodiment of the present invention contains the aforementioned bioactive peptide that can reduce the level of free radicals, and this bioactive peptide has antioxidant efficacy and can be applied to antioxidant.

[0067] Use in the preparation of pharmaceuticals, cosmetics or antioxidant products

[0068] The present invention provides the use of the aforementioned bioactive peptide, the aforementioned nucleic acid molecule, the aforementioned construct or the aforementioned recombinant cell in the preparation of pharmaceuticals, cosmetics or antioxidant products, wherein the pharmaceutical has at least one of the following uses: preventing and / or treating skin pigmentation; the cosmetic has at least one of the following uses: whitening; anti-aging; antioxidant; the antioxidant product is used for antioxidant.

[0069] Those skilled in the art can understand that the features and advantages described above for bioactive peptides, nucleic acid molecules, constructs or recombinant cells are equally applicable to this application and will not be elaborated herein.

[0070] Method for inhibiting tyrosinase activity of a sample

[0071] The present invention provides a method for inhibiting the tyrosinase activity of a sample. According to an embodiment of the present invention, it includes: contacting the sample with the aforementioned bioactive peptide. According to the method of the embodiment of the present invention, by co-culturing the sample with the bioactive peptide of the present invention, the tyrosinase activity level in the sample is further inhibited.

[0072] Tyrosinase inhibitor

[0073] The present invention provides a tyrosinase inhibitor. According to an embodiment of the present invention, it includes: the aforementioned bioactive peptide. The inhibitor according to the embodiment of the present invention can specifically inhibit tyrosinase activity through the action of the aforementioned bioactive peptide.

[0074] The amino acid sequences involved in the present invention are shown in detail in Table 1.

[0075] Table 1 Amino acid sequences involved in the present invention

[0076] SEQ ID NO: Sequence 1 DHWDIM 2 MDEWQW 3 EPFPWQPQ 4 SPVPMPQ 5 SDGTWWE 6 ILPPG

[0077] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0078] Example 1: Isolation and preparation of bioactive polypeptide DHWDIM

[0079] 1. Extraction of hulless barley distillers grains protein by the alcohol-alkali method

[0080] Highland barley lees powder (purchased from Tibet Qizheng Tibetan Medicine Co., Ltd.) and alcohol-alkali mixed solution [V (95% ethanol): V (0.5mol / L NaOH) = 1:2] were fully mixed according to the solid-liquid ratio of 1:40, and stirred for 4h; the supernatant was taken after centrifugation, and then the pH of the supernatant was adjusted to 4.7 with 1mol / L HCl; after adjustment, the solution was allowed to stand for 30min, and then centrifuged at 8000×g for 20min, and the precipitate was collected, which was the crude protein precipitate. After washing the crude highland barley lees protein precipitate with distilled water for 3 times, the crude highland barley lees protein precipitate was dissolved with distilled water to obtain a highland barley lees protein solution; the highland barley lees protein solution was adjusted to pH = 7.0 with 0.5mol / L NaOH solution, and then freeze-dried to obtain highland barley lees protein, which was stored at -20℃ for standby use.

[0081] 2. Enzymatic hydrolysis of highland barley lees protein with alkaline protease

[0082] The highland barley vinasse protein obtained in step 1 is mixed evenly with 35mM sodium phosphate buffer having a pH value of 6.2 at a mass volume ratio of 1:20, and then 1mol / L NaOH is used to adjust the solution pH value to 7.0; 5% alkaline protease (w / w, purchased from Sigma) is added, and mixed to obtain highland barley vinasse protein hydrolysate, which is then placed in a water bath shaker for oscillation enzymolysis, the enzymolysis time is 2h, and the enzymolysis temperature is 50°C; after the enzymolysis is completed, the highland barley vinasse protein hydrolysate is placed in a boiling water bath for 10min to inactivate the residual alkaline protease; after cooling to room temperature, centrifuge at 4°C, 10000×g for 15min, and collect the supernatant to obtain the highland barley vinasse protein hydrolysate solution for standby use.

[0083] 3. Isolation, purification and sequence identification of highland barley lees protein hydrolysate

[0084] The 12 mL highland barley vinasse protein hydrolysate solution obtained in step 2 was transferred to a 3 kDa centrifugal ultrafiltration tube, and centrifuged at 4°C, 5000×g for 30 min to obtain a highland barley vinasse protein hydrolysate sample with a molecular weight of less than 3 kDa, and the obtained highland barley vinasse protein hydrolysate sample with a molecular weight of less than 3 kDa was desalted using a C18 desalting column to obtain a desalted highland barley vinasse protein hydrolysate sample with a molecular weight of less than 3 kDa; and the sample was subjected to LC-MS / MS analysis equipped with an online nanospray ion source, the system used for the analysis was a Q-Exactive Plus mass spectrometer (ThermoFisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200, and the specific LC-MS / MS analysis steps were as follows:

[0085] Load 1 μL of the desalted Qingke distillers' grains protein hydrolysate sample with a molecular weight < 3 kDa fraction (analysis column: Acclaim PepMap C18, 75 μm × 25 cm), and separate the loaded sample with a 60-minute gradient. Column flow rate: 300 nL / min, column temperature: 40 °C, electrospray voltage: 2 kV; mobile phase A: 0.1% formic acid aqueous solution, B: 80% ACN solution containing 0.1% formic acid, the gradient starts from 2% of phase B and increases to 35% in a non-linear gradient at 47 min, increases to 100% within 1 min, and is maintained for 12 min.

[0086] Among them, the Q-Exactive Plus mass spectrometer operates in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisitions. The mass spectrometry parameters are set as follows:

[0087] (1) MS

[0088] Scan range (m / z): 200 - 2000; resolution: 70000; AGC target: 3e6; maximum injection time: 50 ms;

[0089] (2) HCD-MS / MS

[0090] Resolution: 17500; AGC target: 1e5; maximum injection time: 45 ms; collision energy: 28; dynamic exclusion time: 30 s.

[0091] Then, analyze the obtained tandem mass spectra using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database is uniprot-Hordeum_vulgare_subsp_vulgare (version 2023, 34528 entries), and set no enzyme digestion; the search parameters for fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm; variable modification: Oxidation(M) 15.99, peptide score threshold is -10lgP ≥ 20; for peptides not retrieved in the database, set ALC(%) ≥ 80 to obtain the target peptide sequences.

[0092] Partial target peptide sequences are shown in Table 2.

[0093] Table 2 Partial target peptide sequences

[0094] Target peptide sequence Sequence number DHWDIM SEQ ID NO: 1 MDEWQW SEQ ID NO: 2 EPFPWQPQ SEQ ID NO: 3 SPVPMPQ SEQ ID NO: 4 SDGTWWE SEQ ID NO: 5 ILPPG SEQ ID NO: 6

[0095] 4. Virtual screening of bioactive peptides based on bioinformatics

[0096] Non-toxic, non-allergenic, non-carcinogenic and high potential activity are the basic requirements for the screening of bioactive peptides. Small peptides with 2-10 amino acid residues and a molecular weight below 1000 Da can avoid gastrointestinal digestion and overcome the drawback that proteins are destroyed by digestive enzymes and thus cannot be taken orally. Therefore, based on bioinformatics technology, the inventors preliminarily screened out novel bioactive peptides that meet the development requirements from the target peptide sequences obtained in step 3 according to the criteria of non-toxicity, non-allergenicity, non-carcinogenicity, high potential biological activity, easy absorption in the human intestine and stable existence in vitro. The specific steps are as follows:

[0097] First, use the PeptideRanker online system (http: / / bioware.ucd.ie / ~compass / biowareweb / Server_pa ges / peptideranker.Php) to predict the biological activity of the target peptide sequences obtained in step 3; then use ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) to predict the water solubility and toxicity of the target peptide sequences obtained in step 3; then use Expasy (https: / / web.expasy.org / protparam / ) to analyze the isoelectric point and half-life of the target peptide sequences obtained in step 3; finally, use admetSAR (http: / / lmmd.ecust.edu.cn / adm etsar1 / predict / ) to predict the human intestinal absorption and carcinogenicity of the target peptide sequences obtained in step 3.

[0098] The better results of in vitro function prediction of virtual screening of polypeptides are shown in Table 3.

[0099] Table 3 Better results of in vitro function prediction of virtual screening of polypeptides

[0100]

[0101]

[0102] The results show that 6 polypeptide sequences were screened out. Among them, DHWDIM has a high PeptideRanker score (0.819857), a low hydrophilicity (-0.17), and a short half-life (1.1 hours), indicating that it may have good biological activity and stability. Considering comprehensively the activity scores, toxicity, safety and physicochemical properties of the screened polypeptide sequences, and there is no relevant research on DHWDIM in the public database, so DHWDIM was selected for subsequent experiments.

[0103] 5. Artificial Synthesis of Bioactive Peptide DHWDIM

[0104] The inventors prepared the bioactive peptide DHWDIM by Fmoc solid-phase synthesis method, and the specific steps are as follows:

[0105] (1) Solvent Pretreatment

[0106] Before use, N,N-dimethylformamide (DMF) and methanol were respectively soaked overnight with G3 pore molecular sieve to remove impurities and moisture in the solvent.

[0107] (2) Resin Swelling

[0108] Weighed 2.0 g of blank Wang resin into a clean and dry reaction tube, added 15 mL of DMF, and activated it at room temperature for 30 min.

[0109] (3) Incorporation of the First Amino Acid

[0110] At room temperature, the DMF in step 2 was removed by sintered glass filtration, and 1 mmol of 5-fold molar excess of the first amino acid at the C-terminus of the target sequence, 5-fold molar excess of 4-dimethylaminopyridine (DMAP), 5-fold molar excess of N,N-diisopropylcarbodiimide (DIC), and 60 mL of DMF as solvent were added, and the reaction was carried out at room temperature for 3 h; after the reaction was completed, it was washed 5 times with DMF, 6 mL each time; then 6 mL of a 1:1 volume ratio of pyridine and acetic anhydride was added, and the reaction was carried out for 30 min; after the reaction was completed, it was washed 5 times with DMF, 6 mL each time.

[0111] (4) Removal of Fmoc Protecting Group

[0112] The solvent in step 3 was removed by sintered glass filtration, 10 mL of 20% piperidine DMF solution was added to the above resin, stirred for 10 min under N2 protection and then the solution was filtered out; 10 mL of 20% piperidine DMF solution was added again, stirred under N2 protection for 5 min and the solution was filtered out again. After repeating the above operations twice, it was washed 4 times with DMF and 2 times with methanol, 6 mL each time.

[0113] (5) Ninhydrin Detection of the Removal Effect of Fmoc Protecting Group

[0114] A small amount of resin treated in step 4 was taken out, washed three times with methanol, one drop of ninhydrin, KCN, and phenol solution was added, heated at 110 °C for 5 min, and a dark blue color change was a positive reaction, indicating that the Fmoc protecting group was completely removed and the next reaction operation could be carried out; if it was colorless, it indicated that the protecting group was not completely removed, and the above step (4) Removal of Fmoc Protecting Group operation needed to be repeated.

[0115] (6) Incorporation of the Second Amino Acid and Removal of Fmoc Protecting Group

[0116] Weigh 3-fold molar excess of the second amino acid at the C-terminus of the target sequence, 3-fold molar excess of benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 3-fold molar excess of 1-hydroxybenzotriazole (HOBT) into the above reaction tube, add an appropriate amount of DMF to completely dissolve them; then add 10-fold molar excess of N,N-diisopropylethylamine (DIPEA), react at room temperature for 40 min. After the reaction is completed, wash with DMF 5 times, 6 mL each time. Take a small amount of the resin treated as above and detect it with ninhydrin detection reagent, showing colorless. Then add 10 mL of 20% piperidine DMF solution to deprotect the Fmoc group, do this twice, for 10 min and 5 min respectively. After that, wash with DMF 4 times and methanol 2 times, 6 mL each time. Take out a small amount of the resin treated as above again and detect it with ninhydrin detection reagent, detecting blue, then the next step of the reaction can be carried out.

[0117] (7) Reintroduce the amino acid again

[0118] Repeat the operation described in step (6) until the last amino acid at the N-terminus of the target sequence is synthesized. After removing the Fmoc protecting group, drain the solution to obtain the resin containing the target bioactive peptide DHWDIM.

[0119] (8) Deprotection of the resin and separation and detection of the pure product of the bioactive peptide DHWDIM

[0120] Cleavage: Add a cleavage solution (95% trifluoroacetic acid: 2% triisopropylsilane: 2% ethanedithiol: 1% H2O) with a volume 6 times that of the resin, shake on a shaker for 2 h, filter the reaction solution by suction to obtain a trifluoroacetic acid solution containing the target polypeptide. Dry the cleavage solution with nitrogen, then precipitate with diethyl ether and centrifuge. Then wash the precipitate with anhydrous diethyl ether 3 times to obtain a white solid. Dissolve it with pure water, and then desalt and purify the crude polypeptide by HPLC. After freeze-drying, crystals are precipitated, which is the pure product of the bioactive peptide DHWDIM, and store it at -20 °C for later use.

[0121] (9) Quality detection of the pure product of the bioactive peptide DHWDIM

[0122] Take a small amount of the pure product sample of the bioactive peptide DHWDIM obtained in step (8), dissolve it in trifluoroacetic acid solution by ultrasonic to obtain the test solution; place the test solution in a high performance liquid chromatograph for detection to conduct the quality detection of the pure product of the bioactive peptide DHWDIM.

[0123] Among them, the HPLC parameters are set as follows:

[0124] Chromatographic column: ZORBAX SB-C18, 4.6×250mm, 5μm; Aqueous phase: 100% water plus 0.1% trifluoroacetic acid; Organic phase: 100% acetonitrile plus 0.1% trifluoroacetic acid; Flow rate: 1 mL / min; Injection volume: 10 μL; Detection wavelength: 220 nm;

[0125] Among them, the MS parameter settings are as follows:

[0126] Ion source: electrospray ionization source (ESI source); Nebulizer gas flow rate: 1.5 L / min; CDL: -20.0 V; CDL temperature: 250 °C; Heating block temperature: 200 °C; Ion source voltage: +4.5 kV; Detector voltage: 1.5 kV; Mobile phase flow rate: 0.2 mL / min; Mobile phase ratio: 50% H2O / 50% ACN.

[0127] The HPLC detection results of the pure sample of bioactive peptide DHWDIM are shown in Figure 1 and the MS detection results of the pure sample of bioactive peptide DHWDIM are shown in Figure 2 .

[0128] The results show that: determined by the HPLC detection results, the purity of the pure bioactive peptide DHWDIM obtained in step (8) is greater than 95%; determined by the MS detection results, the polypeptide sequence of the pure bioactive peptide DHWDIM obtained in step (8) is the target sequence DHWDIM.

[0129] Example 2: Molecular docking and interaction force analysis of bioactive polypeptide DHWDIM

[0130] The inventor used the bioactive peptide DHWDIM screened in Example 1 as the ligand and tyrosinase as the receptor, and used molecular docking technology to clarify the binding sites and interaction forces between the peptide DHWDIM and the acting enzyme, so as to realize the theoretical verification of its function in whitening.

[0131] Obtain the crystal structure of tyrosinase (PDB ID: 2Y9X) from the RCSB protein database (http: / / www.rcsb.org / ); then perform molecular docking of the bioactive peptide DHWDIM and tyrosinase using ZDOCK. After screening out the best binding conformation, analyze the binding force between the bioactive peptide DHWDIM and tyrosinase by PDBePISA, and further use LigPlus+ to analyze its interaction forces in detail; then use PyMOL software to visualize the binding of the ligand (bioactive peptide DHWDIM) and the receptor (tyrosinase).

[0132] The schematic diagram of the molecular docking simulation of bioactive polypeptide DHWDIM and tyrosinase is shown in Figure 3 .

[0133] Results showed that the molecular binding energy between the bioactive polypeptide DHWDIM and the amino acid residues of tyrosinase was -5.1 kcal / mol. The amino acid residues Asp1(C), His2(C), Trp3(C), Asp4(C), Ile5(C), and Met6(C) of the bioactive polypeptide DHWDIM were tightly bound to the key amino acid residues of tyrosinase (TYR) through various interactions. Specifically, Asp1(C) and His2(C) formed stable bindings with Val283(A) and Ser282(A) of tyrosinase through hydrogen bonding, respectively; Trp3(C) and Asp4(C) bound to residues such as Arg268(A), Phe264(A), and Val248(A) of tyrosinase through significant hydrophobic interactions, and these hydrophobic interactions enhanced the stability of the bioactive polypeptide DHWDIM-tyrosinase complex; in addition, the side chains of Met6(C) and Ile5(C) further formed strong hydrophobic interactions with Pro284(A) and His244(A) of tyrosinase, further consolidating the stability of their binding. Thus, it was speculated that the bioactive polypeptide DHWDIM was tightly bound to the active center residues of tyrosinase through the synergistic action of hydrophobic interactions and hydrogen bonds, thereby inhibiting the activity of tyrosinase.

[0134] The above results indicated that the bioactive polypeptide DHWDIM had good affinity with tyrosinase, and it was speculated that it had good whitening effects.

[0135] Example 3: Experimental verification of the whitening and antioxidant functions of the bioactive polypeptide DHWDIM

[0136] 1. Determination of the inhibition rate of tyrosinase activity by the bioactive polypeptide DHWDIM

[0137] After incubating 50 μL of the pure product solution of the bioactive peptide DHWDIM obtained in Example 1 at 8 mg / mL (the blank control group was replaced with 0.2 mol / L phosphate buffer solution with pH = 7.5) and 50 μL of tyrosinase solution at 37 °C for 5 min, 50 μL of the substrate L-tyrosine solution was added; the absorbance value was recorded every 20 s at 475 nm using a microplate reader, and the change in absorbance value within 10 min was continuously recorded. Among them, the inhibition rate of tyrosinase activity was calculated according to Equation 1:

[0138]

[0139] In Equation 1: A: the absorbance of the 0.2 mol / L phosphate buffer solution group with pH = 7.5 (blank control group); B: the absorbance of the sample group of the pure product reaction solution of the bioactive peptide DHWDIM obtained in Example 1 (experimental group).

[0140] 2. Determination of DPPH Free Radical Scavenging Rate of Bioactive Polypeptide DHWDIM

[0141] Mix 50 μL of the pure bioactive peptide DHWDIM solution (replace the blank control group with distilled water) obtained in Example 1 at a concentration of 5 mg / mL and 50 μL of 0.2 mmol / L DPPH (dissolved in methanol) solution, incubate in the dark at 37 °C for 20 min, and record the absorbance at 517 nm using a microplate reader. Among them, the DPPH free radical scavenging rate is calculated according to Equation 2:

[0142]

[0143] In Equation 2: A: Absorbance of the distilled water group (blank control group); B: Absorbance of the sample group of the pure bioactive peptide DHWDIM reaction solution obtained in Example 1 (experimental group).

[0144] The evaluation results of the whitening and antioxidant functions of bioactive polypeptide DHWDIM are shown in Figure 4 .

[0145] The results show that: the inhibition rate of tyrosinase activity of the bioactive peptide DHWDIM obtained in Example 1 is 45.04%, which can exert good whitening and other effects; the DPPH free radical scavenging rate of the bioactive peptide DHWDIM obtained in Example 1 is 46.44%, which can exert good whitening and antioxidant abilities.

[0146] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0147] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A bioactive peptide, characterized in that: The amino acid sequence of the bioactive peptide is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the biologically active peptide according to claim 1.

3. A construct, characterized in that Comprising the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that Comprising the nucleic acid molecule of claim 2 or the construct of claim 3.

5. A pharmaceutical composition, characterized in that include: One or more of the bioactive peptide of claim 1, the nucleic acid molecule of claim 2, the construct of claim 3, and the recombinant cell of claim 4; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

6. A daily chemical product, characterized in that: include: The bioactive peptide according to claim 1; Optionally, the daily chemical product further comprises an auxiliary material acceptable in daily chemical products; Optionally, the daily chemical product includes one or more of creams, lotions, aqueous solutions, gels, oils, wax-based products and film-based products.

7. An antioxidant product, characterized in that: include: The bioactive peptide according to claim 1.

8. Use of the bioactive peptide according to claim 1, the nucleic acid molecule according to claim 2, the construct according to claim 3 or the recombinant cell according to claim 4 in the preparation of a medicine, a daily chemical product or an antioxidant product, wherein the medicine has at least one of the following uses: Prevent and / or treat skin pigmentation; preventing and / or treating oxidative stress; The daily chemical product has at least one of the following uses: Whitening; Anti-aging; Antioxidant; The antioxidant preparation is used for antioxidant function.

9. A method for inhibiting tyrosinase activity of a sample, characterized in that: include: The sample is contacted with the bioactive peptide of claim 1.

10. A tyrosinase inhibitor, characterized in that include: The bioactive peptide according to claim 1.

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