Antioxidant and ace-inhibitory peptides from tuna and preparation method and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to an antioxidant peptide and ACE inhibitory peptide from tuna, and a preparation method and application thereof. The amino acid sequence of the polypeptide provided by the present application is shown as SEQ ID NO. 1-6. The present application takes tuna skin waste as raw material, obtains tuna skin collagen through enzymolysis, then performs liquid chromatography-tandem mass spectrometry identification and molecular docking screening on the tuna skin collagen enzymolysis product, and obtains polypeptides with antioxidant activity and polypeptides effectively inhibiting ACE activity. The method simplifies the protein extraction step. Meanwhile, the method can improve the deep processing level of the tuna industry, achieve high-value utilization, and reduce the environmental pressure on processing waste.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a tuna-derived antioxidant peptide and an ACE inhibitory peptide, as well as a preparation method and application thereof. Background Art

[0002] Free radicals are byproducts of cellular metabolism. Furthermore, certain adverse external factors and lifestyle influences can stimulate the body to produce large amounts of free radicals. Free radicals have a dual nature. Under normal circumstances, free radicals generated in the body are maintained in a harmless dynamic equilibrium by the body's own antioxidant enzyme system. However, when the body develops pathological changes or metabolic disorders, overoxidation occurs, and excessive free radicals can damage DNA, proteins, and cellular structures. Reactive oxygen species and free radicals are also associated with aging. Furthermore, the occurrence of some diseases, such as cardiovascular disease, cancer, inflammation, and neurodegenerative diseases like Parkinson's disease, is associated with excessive free radicals or a decreased ability to scavenge them. Therefore, people are increasingly realizing that to reduce the damage caused by free radicals, in addition to relying on the body's own scavenging system, it is also necessary to identify and explore exogenous free radical scavengers. Commonly used chemically synthesized antioxidants are potentially harmful and toxic to humans and animals. Furthermore, antioxidant active substances such as vitamin C, polyphenols, and flavonoids are unstable and susceptible to air oxidation and thermal decomposition. Antioxidant peptides have attracted attention due to their safety, effectiveness, stability, and ease of absorption. Currently, antioxidant peptides are mainly obtained from soybeans, corn, milk, aquatic animals, etc.

[0003] Angiotensin I-converting enzyme (ACE) is a key enzyme in regulating blood pressure, contributing to elevated blood pressure. The development of hypertension is primarily associated with overactivation of the renin-angiotensin-aldosterone system (RAAS) and disruption of the kallikrein-kinin system (KKS). ACE is generally believed to play a key role in regulating the RAAS and KKS systems, influencing blood pressure levels.

[0004] In the RAAS system, the juxtaglomerular apparatus secretes renin hydrolase into the bloodstream, breaking down angiotensinogen secreted by the liver into type I angiotensin Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO. 7). ACE, by releasing His-Leu from the inactive type I angiotensin, further degrades it into type II angiotensin Asn-Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO. 8), a vasoconstrictor. Type II angiotensin is one of the main contributors to elevated blood pressure. In the KKS, bradykininogen is hydrolyzed by kallikrein to form bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (SEQ ID NO. 9). ACE catalyzes the degradation of bradykinin, removing two amino acid residues at its carboxyl terminus, rendering it inactive and leading to elevated blood pressure. ACE inhibitory peptides can effectively prevent the increase in blood pressure by inhibiting ACE activity and interfering with the regulatory function of ACE in RAAS and KKS.

[0005] In recent years, chemically prepared antioxidant and ACE inhibitors have developed rapidly, but their toxic side effects have significantly restricted their application. Studies have shown that a large number of natural extracts possess antioxidant and ACE inhibitory activity, with fish, shrimp, and shellfish peptides, soybean peptides, and peanut peptides showing particular effectiveness. Food protein-derived antioxidant and ACE inhibitory peptides play an important role in these fields due to their high specificity, low toxicity, relative ease of synthesis, and suitability as carriers for other molecules. Food protein-derived antioxidant and ACE inhibitory peptides are widely available.

[0006] Tuna, also known as bluefin tuna, belongs to the class Osteichthyes, order Pereiformes, and family Scombridae. It is found in the Pacific, Atlantic, and Indian Oceans and is a highly migratory oceanic fish. Commercially valuable tuna species include bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus salbacares), Atlantic bluefin tuna (Thunnus thynnus), and skipjack tuna (Katsuwonus pelamis). The annual global catch of bluefin tuna is approximately 6 million tons, accounting for one-tenth of the international seafood trade, making it a key commercial fish species in the ocean. Tuna processing primarily focuses on sashimi and canning, with the remaining parts discarded as a by-product, resulting in significant waste. Furthermore, there are currently no reports on the preparation of antioxidant and ACE inhibitory peptides from tuna skin. Summary of the Invention

[0007] The purpose of the present invention is to provide an antioxidant peptide and an ACE inhibitory peptide derived from tuna, as well as a preparation method and application thereof, in order to solve the problems existing in the above-mentioned prior art. Tuna processing by-products contain about 15% crude protein, and the fish skin is rich in collagen, which is a high-quality protein resource. The present invention selects bigeye tuna skin as raw material to prepare antioxidant active peptides and ACE inhibitory active peptides. The polypeptide provided by the present invention can, on the one hand, improve the deep processing level of the tuna industry and achieve high value utilization, and on the other hand, it can reduce the pressure on the environment to withstand processing waste.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a polypeptide derived from tuna skin. The amino acid sequence of the polypeptide is shown in any one or several sequences among SEQ ID NOs. 1 to 6.

[0010] The present invention provides a method for preparing the above-mentioned polypeptide, comprising the following steps:

[0011] The tuna skin is sequentially subjected to an alkali soaking treatment, an acid soaking treatment, a pulping treatment, a water bath treatment and a first centrifugation treatment to obtain a tuna skin collagen extract;

[0012] The tuna skin collagen extract and protease are mixed, followed by enzymatic hydrolysis, a second centrifugal treatment and freeze-drying to obtain tuna skin enzymatic hydrolysis powder;

[0013] The tuna skin enzymatic hydrolyzed powder is subjected to liquid chromatography-tandem mass spectrometry identification and molecular docking screening to obtain the polypeptide.

[0014] Preferably, the alkali soaking treatment comprises the steps of mixing tuna skin and NaOH solution in a mass ratio of 1:10, and then soaking the mixture in alkali to obtain an alkali soaking treatment solution; the alkali soaking treatment time is 1.5 hours;

[0015] And / or, the acid soaking comprises the step of mixing an alkaline soaking treatment solution and hydrochloric acid in a mass ratio of 1:10, and then performing an acid soaking treatment to obtain an acid soaking treatment solution; the acid soaking treatment time is 2 hours;

[0016] And / or, the water bath treatment temperature is 60° C. and the time is 5 h;

[0017] And / or, the first centrifugal treatment is performed at a rotation speed of 10,000 r / min and for 20 min.

[0018] Preferably, the protease comprises pancreatin; the amount of pancreatin used is 3% of the protein mass in the tuna skin collagen extract;

[0019] and / or, the enzymatic hydrolysis treatment is carried out for 4 hours, at a temperature of 50° C. and at a pH of 8.0;

[0020] And / or, the second centrifugal treatment is performed at a rotation speed of 10,000 r / min and for 10 minutes.

[0021] The present invention provides the use of the above polypeptide in the preparation of antioxidants and / or ACE inhibitors.

[0022] The present invention provides an antioxidant, which comprises a polypeptide having an amino acid sequence as shown in any one or more sequences of SEQ ID NOs. 1 to 3.

[0023] More preferably, the antioxidant further comprises auxiliary materials.

[0024] The present invention provides an ACE inhibitor, which comprises a polypeptide having an amino acid sequence as shown in any one or more of SEQ ID NOs. 4 to 6.

[0025] Further preferably, the ACE inhibitor further comprises an excipient.

[0026] The present invention provides the use of the above-mentioned ACE inhibitor in the preparation of antihypertensive drugs.

[0027] The present invention provides an antihypertensive drug, which comprises the above-mentioned ACE inhibitor.

[0028] Preferably, the antihypertensive drug further comprises pharmaceutically acceptable excipients.

[0029] The present invention discloses the following technical effects:

[0030] The present invention selects bigeye tuna skin as raw material to prepare antioxidant active peptides and ACE inhibitory active peptides. On the one hand, the polypeptide provided by the present invention can improve the deep processing level of the tuna industry and achieve high value utilization rate. On the other hand, it can reduce the pressure on the environment to bear processing waste. In a specific embodiment of the present invention, the present invention uses processing waste tuna skin as raw material, obtains tuna skin collagenase hydrolysate by enzymatic hydrolysis of tuna skin, and then performs liquid chromatography-tandem mass spectrometry identification and molecular docking screening on the tuna skin collagenase hydrolysate to extract polypeptides with antioxidant activity and polypeptides that effectively inhibit ACE activity. This method simplifies the protein extraction step. The present invention has extracted and obtained the tetrapeptide MGPR (SEQ ID NO.1), tetrapeptide PRGP (SEQ ID NO.2) and tetrapeptide PGPM (SEQ ID NO.3) that can effectively inhibit the activity of ACE, the polypeptide decapeptide GPSGPPGKNG (SEQ ID NO.4), heptapeptide SGPAGPR (SEQ ID NO.5) and heptapeptide SGPAGPK (SEQ ID NO.6), and the antioxidant activity and ACE inhibitory activity of the polypeptide are consistent with the peptide activity obtained by traditional means. The present invention finds that the extracted polypeptide is mainly combined with oxidase and ACE enzyme through hydrogen bond interaction, which can inhibit oxidation reaction and prevent blood pressure from rising. In summary, the present invention provides a new way for the preparation of antioxidant and ACE inhibitory active peptides, and at the same time promotes the high value utilization of low-value aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Schematic diagram of the binding site between the polypeptide MGPR and Keap1;

[0033] Figure 2 Schematic diagram of molecular docking between peptide MGPR and Keap1;

[0034] Figure 3 Schematic diagram of the binding site between the peptide PRGP and Keap1;

[0035] Figure 4 Schematic diagram of molecular docking between peptide PRGP and Keap1;

[0036] Figure 5 Schematic diagram of the binding site between peptide PGPM and Keap1;

[0037] Figure 6 Schematic diagram of molecular docking between peptide PGPM and Keap1;

[0038] Figure 7 Schematic diagram of the binding position of the peptide GPSGPPGKNG and ACE;

[0039] Figure 8 Schematic diagram of molecular docking between peptide GPSGPPGKNG and ACE;

[0040] Figure 9 Schematic diagram of the binding site between the peptide SGPAGPR and ACE;

[0041] Figure 10 Schematic diagram of molecular docking between peptide SGPAGPR and ACE;

[0042] Figure 11 Schematic diagram of the binding site between the peptide SGPAGPK and ACE;

[0043] Figure 12 Schematic diagram of molecular docking between peptide SGPAGPK and ACE. DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] A large number of experimental studies have shown that hydrophobic amino acids (such as methionine, isoleucine, leucine, proline, valine and other amino acid residues) are considered to be the reason why active peptides have antioxidant effects. It is generally believed that active peptides with aromatic amino acids (such as tyrosine and phenylalanine) at the C and N termini, aliphatic amino acids (such as serine, histidine, threonine, leucine, glycine, lysine), and hydrophobic amino acids (such as leucine) are considered to be the reason why active peptides have ACE inhibitory effects.

[0050] Example 1

[0051] (1) Take tuna skin, add 0.05 mol / L NaOH solution, add tuna skin and NaOH solution in a mass ratio of 1:10, soak for 1.5 hours, and then wash with running water until neutral; then add 0.05 mol / L hydrochloric acid (the mass ratio of tuna skin and hydrochloric acid is 1:10), soak for 2 hours, and then wash with running water until neutral, drain and beat, place the slurry in a constant temperature water bath at 60°C, centrifuge at 10000 r / min for 20 minutes after 5 hours, and then take the supernatant and concentrate to obtain tuna skin collagen extract.

[0052] (2) Add pancreatic enzyme to the tuna skin collagen extract (the amount of pancreatic enzyme added is 3% of the protein mass in the tuna skin collagen extract), keep warm and shake at pH 8.0 and 50°C for 4 hours, and inactivate the enzyme in a boiling water bath for 10 minutes after the reaction. Filter the extract and centrifuge it at a speed of 10,000 r / min for 10 minutes. The supernatant is collected and dried to obtain tuna skin collagen enzymatic hydrolyzed powder.

[0053] (3) Tuna collagen hydrolysate was analyzed by liquid chromatography-tandem mass spectrometry equipped with an online nanospray ion source. The liquid chromatograph loading volume was 2 μL, the sample separation gradient was 60 min, the column flow rate was controlled at 350 nL / min, the mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, the column temperature was 40°C, and the electrospray voltage was 2 kV. The mass spectrometer parameters were set as follows: ① MS: scan range (m / z): 200-1500; resolution: 60,000; normalized AGC target: 300%; maximum injection time: 25 ms. ② HCD-MS / MS: resolution: 15,000; normalized AGC target: 50%; maximum injection time: 22 ms; collision energy: 30%; dynamic exclusion time: 30 s.

[0054] (5) Molecular docking screening: The online tool Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the potential biological activity of the peptides, and the online tool ToxinPred (http: / / crdd.osdd.net / raghava / toxinpred / ) was used to predict the toxicity of the peptides. "Non-Toxin" peptides were selected, and the "Peptide property calculator" (http: / / www.innovagen.com / proteomics-tools) tool of the Innovagen program was used to predict the water solubility of the peptides. The human intestinal adsorption (HIA) properties of the peptides were predicted using admetSAR (http: / / lmmd.ecust.edu.cn / admetsar1 / predict / ). HELPDOCK 2.0 was used to predict the binding energy of the peptides with macromolecular enzymes. The peptides that met the conditions should have: PeptideRanker activity score > 0.5, non-toxicity, good water solubility, and good human intestinal absorption. The smaller the "-Docker Energy" value, the higher the antioxidant activity and ACE inhibitory activity, and the peptides with higher activity scores were selected. The obtained peptides were simulated and docked using the molecular docking software AutodockVina, and the three peptides that bound most tightly to Keap1 were screened out. The sequences of these three peptides were MGPR (SEQ ID NO.1), PRGP (SEQ ID NO.2), and PGPM (SEQ ID NO.3). The three peptides that bound most tightly to ACE were screened out. The sequences of these three peptides were GPSGPPGKNG (SEQ ID NO.4), SGPAGPR (SEQ ID NO.5), and SGPAGPK (SEQ ID NO.6). They were visualized using Pymol and Discovery Studio software.

[0055] Keap1, a BTB-Kelch substrate adaptor protein, regulates Nrf2 homeostasis in response to oxidative stress. Angiotensin-converting enzyme (ACE) is a key enzyme that contributes to elevated blood pressure in humans. Inhibiting ACE activity can maintain relative blood pressure stability. The Keap1 structure (PDB ID: 2FUL) and the human ACE structure (PDB ID: 1O8A) were obtained from the RCSB protein library. Before docking, Keap1 and ACE were modified using Pymol. The secondary structures of the tetrapeptides MGPR, PRGP, and PGPM, and the decapeptides GPSGPPGKNG, SGPAGPR, and SGPAGPK were drawn using ChemDraw software and energy minimized using the Minimize Energy module. A GridBox was set in AutoDock Vina software, with the X, Y, and Z coordinates and the grid center adjusted to accommodate the entire protein molecule.

[0056] Molecular docking screening of the peptide sequences identified by liquid chromatography-tandem mass spectrometry revealed three peptides with the highest antioxidant activity, represented by SEQ ID NOs. 1 to 3. Their molecular weights and docking results are shown in Table 1. Three peptides with ACE inhibitory activity, represented by SEQ ID NOs. 4 to 6, were also identified. Docking results revealed that the minimum binding energies of all six peptides with the enzyme were less than -6 kcal / mol, indicating spontaneous binding of the ligand to the receptor. Furthermore, it is generally accepted that a binding free energy of ≤ -5.0 kcal / mol indicates that the binding of a ligand to the target protein is meaningful.

[0057] Table 1 Antioxidant activity of collagen-derived peptides and molecular weight and molecular docking results of ACE inhibitory peptides

[0058] Peptide sequence SEQ ID NO. Molecular weight Docking energy (kcal / mol) MGPR 1 459.2264 -8 PRGP 2 425.2386 -8.9 PGPM 3 400.178 -6.8 GPSGPPGKNG 4 866.4246 -8.6 SGPAGPR 5 640.3293 -8.7 SGPAGPK 6 612.3231 -7.4

[0059] Figure 1 、 Figure 3 and Figure 5 The optimal binding positions of peptides MGPR, PRGP and PGPM after docking with Keap1 are shown respectively, and it was found that all three peptides were docked into the hydrophobic active cavity of Keap1. Figure 2 、 Figure 4 and Figure 6 It can be seen that all three peptides have hydrogen bond interactions with Keap1, and MGPR binds to Keap1 amino acid residues VAL-418, VAL-465, VAL-561, VAL-608, GLY-367, and VAL-608 at a distance of PRGP binds to Keap1 amino acid residues VAL-418, VAL-512, GLY-367, ILE-559, and THR-560 at a distance of PGPM binds to Keap1 amino acid residues ARG-415, LEU-557, and SER-602 at a distance of Figure 7 、 Figure 9 and Figure 11 The optimal binding positions of the peptides GPSGPPGKNG, SGPAGPR and SGPAGPK after docking with ACE were shown respectively, and it was found that all three peptides were docked into the active cavity of ACE. Figure 8 、 Figure 10 and Figure 12 It can be seen that all three peptides have hydrogen bond interactions with Keap1, and GPSGPPGKNG binds to ACE amino acid residues ASN-66, ASN-85, and THR-92 at a distance of SGPAGPR binds to ACE amino acid residues ARG-522, GLY-404, ARG-402, TYR-394, ASP-358, TYR-360, and ASN-66 at a distance of SGPAGPK binds to ACE amino acid residues THR-92, ASN-85, and ASN-66 at a distance of In summary, the residues binding to each peptide and the corresponding macromolecular enzyme have certain similarities.

[0060] (6) Detection of DPPH free radical scavenging rate and ACE inhibition rate of peptides

[0061] The DPPH free radical scavenging rate or ACE inhibition rate of the above-mentioned tuna skin collagen, tuna skin collagen enzymatic hydrolysate powder, MGPR, PRGP, PGPM, GPSGPPGKNG, SGPAGPR and SGPAGPK was detected as follows:

[0062] DPPH free radical scavenging rate determination: Use 95% ethanol to accurately prepare 0.3mmol / L DPPH solution, and use 5mg / mL peptide solution sample (the amino acid sequence of the peptide is shown in SEQ ID NO.1-3). Pipette 100μL sample and measure the absorbance (A) at 517nm. b ), then add 100 μL DPPH solution, mix well and react in the dark for 30 min, and measure the absorbance (A s ). Water was used as the control group instead of the sample, and its blank absorbance and absorbance after reaction were recorded as A cb and A c ; Calculate IC based on clearance 50The calculation formula of DPPH free radical scavenging rate is as follows:

[0063] DPPH free radical scavenging rate = [1-(A s -A b )] / A c -A cb ×100%;

[0064] ACE inhibition rate determination: Prepare 5 mmol / L hippuryl-histidyl-leucine (N-hippuryl-His-Leu tetrahydrate, HHL) solution, 0.1 U / mL ACE enzyme solution, and 1 mg / mL polypeptide solution sample (peptide amino acid sequences are shown in SEQ ID NOs. 4-6) in borate buffer (0.1 mol / L, pH 8.3, containing 0.3 mol / L NaCl). Also prepare 1 mol / L HCl solution.

[0065] According to the sequence in Table 2, the mixed solution after shaking was centrifuged at 4000 rpm for 15 min, 1 mL of ethyl acetate was taken from the upper part, and dried in an oven at 120°C for 30 min. After cooling to room temperature, it was dissolved with 4 mL of deionized water and shaken thoroughly. The absorbance at a wavelength of 228 nm was measured using a UV spectrophotometer. Three parallel groups were measured for each group, and the average value was taken. The IC was calculated based on the inhibition rate. 50 The calculation formula of ACE inhibition rate is as follows:

[0066] ACE inhibition rate = (A b -A a ) / (A b -A c )×100%;

[0067] Where A a is the absorbance under the condition that both ACE and ACE inhibitory peptide exist (experimental group A); A b A is the absorbance under the condition that ACE inhibitory peptide does not participate in the reaction (control group B); c It is the absorbance under the condition that ACE does not participate in the reaction (blank group and group C).

[0068] Table 2. Sample addition order of ACE inhibition rate determination experiment

[0069]

[0070]

[0071] The antioxidant activity test results of each sample are shown in Table 3. As shown in Table 3, the DPPH free radical scavenging rate of tuna skin collagen is low, and its half inhibition rate IC 50The inhibitory activity was significantly improved after trypsin hydrolysis, and the half inhibition rate IC 50 The ACE inhibitory activity of tuna skin collagen was low, with an IC50% inhibition rate of 1:1. 50 The inhibitory activity was significantly improved after trypsin hydrolysis, and the half inhibition rate IC 50 The concentration of the peptide sequence identified by liquid chromatography-tandem mass spectrometry was 0.937 mg / mL; molecular docking screening was performed on the peptide sequence identified by liquid chromatography-tandem mass spectrometry to obtain three peptides with the highest predicted activity. The ACE inhibition rate of the three peptides was ranked as SGPAGPR>GPSGPPGKNG>SGPAGPK.

[0072] Table 3 DPPH free radical scavenging rates of tuna skin collagen, tuna skin enzymatic hydrolyzed powder, and three peptides, as well as ACE inhibition rates of the three peptides

[0073]

[0074] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a polypeptide, characterized in that: The following steps are involved: The tuna skin is sequentially subjected to an alkali soaking treatment, an acid soaking treatment, a pulping treatment, a water bath treatment and a first centrifugation treatment to obtain a tuna skin collagen extract; The tuna skin collagen extract and protease are mixed, followed by enzymatic hydrolysis, a second centrifugal treatment and freeze-drying to obtain tuna skin enzymatic hydrolysis powder; The tuna skin enzymatic hydrolyzed powder was identified by liquid chromatography-tandem mass spectrometry and molecular docking screening to obtain the polypeptide; the amino acid sequence of the polypeptide is shown in SEQ ID NO.1; The alkaline soaking treatment comprises the steps of mixing tuna skin and NaOH solution in a mass ratio of 1:10, and then soaking in alkaline water to obtain an alkaline soaking treatment solution; the alkaline soaking treatment time is 1.5 h; The acid soaking treatment comprises the steps of mixing an alkaline soaking treatment solution and hydrochloric acid in a mass ratio of 1:10, and then performing an acid soaking treatment to obtain an acid soaking treatment solution; the acid soaking treatment time is 2 hours; The water bath treatment temperature is 60°C and the time is 5 h; The first centrifugal treatment was performed at a speed of 10,000 r / min and for 20 min; The protease is pancreatic enzyme; the amount of pancreatic enzyme used is 3% of the protein mass in the tuna skin collagen extract; The enzymatic hydrolysis treatment lasted for 4 h, the temperature was 50° C., and the pH was 8.0; The second centrifugal treatment was performed at a speed of 10,000 r / min and for 10 min; The molecular docking screening includes using the online tool Peptide Ranker to predict the potential biological activity of peptides, using the online tool ToxinPred to predict the toxicity of peptides, selecting "non-toxin" peptides, using the "Peptide property calculator" tool of the Innovagen program to predict the water solubility of peptides, using admetSAR to predict the human intestinal absorption properties of peptides, and using HELPDOCK 2.0 to predict the binding energy of peptides with macromolecular enzymes. Qualified peptides should have: a Peptide Ranker activity score greater than 0.5, be non-toxic, have good water solubility, and be well absorbed by the human intestinal tract. A smaller "-Docker Energy" value indicates higher antioxidant activity and ACE inhibitory activity. Peptides with higher activity scores are selected, and the obtained peptides are simulated docked using the molecular docking software Autodock Vina to screen out the peptides that bind most tightly to Keap1.

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