Tuna-sourced antioxidant peptide and ACE inhibitory peptide as well as preparation method and application of tuna-sourced antioxidant peptide and ACE inhibitory peptide

Abstract

The invention relates to the technical field of biology, in particular to antioxidative peptide and ACE (angiotensin converting enzyme) inhibitory peptide from tuna as well as a preparation method and application of the antioxidative peptide and the ACE inhibitory peptide. The amino acid sequences of the polypeptide provided by the invention are as shown in SEQ ID NO.1-6. The preparation method comprises the following steps: by taking processing waste tuna skin as a raw material, carrying out enzymolysis to obtain tuna skin collagen, then carrying out liquid chromatography-tandem mass spectrometry identification and molecular docking screening on a tuna skin collagen zymolyte, and extracting to obtain polypeptide with antioxidant activity and polypeptide capable of effectively inhibiting ACE (Angiotensin Converting Enzyme) activity. According to the method, the protein extraction step is simplified; meanwhile, on one hand, the deep processing level of the tuna industry can be improved, high-value utilization is achieved, and on the other hand, the bearing pressure of the environment on the processing waste can be reduced.

Description

Technical Field

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

[0002] Free radicals are byproducts of cell metabolism. In addition, under the influence of certain external adverse factors and living habits, the body will also be stimulated to produce a large number of free radicals. Free radicals have duality. Under normal circumstances, the free radicals produced in the body are maintained in a harmless dynamic equilibrium state under the scavenging of the body's own antioxidant enzyme system. However, when the body is diseased or the metabolism is abnormal, peroxidation will occur. Excessive free radicals will damage DNA, destroy proteins and cell structures; reactive oxygen and free radicals are also related to human aging. In addition, the occurrence of some diseases is related to excessive free radicals in the body or decreased scavenging ability, such as cardiovascular disease, tumors, inflammation and neurodegenerative diseases such as Parkinson's disease. Therefore, people gradually realize that in order to reduce the damage caused by free radicals to the human body, in addition to relying on their own scavenging system, they also need to find and explore some exogenous free radical scavengers. Commonly used chemically synthesized antioxidants have potential hazards and toxicity to humans and animals. At the same time, antioxidant active substances such as vitamin C, polyphenols, and flavonoids have poor stability, are susceptible to air oxidation, and are easy to thermally decompose. Antioxidant peptides have attracted people's attention due to their safety, high efficiency, good stability, and easy absorption. Currently, antioxidant peptides are mainly obtained from soybeans, corn, milk, aquatic animals, etc.

[0003] Angiotensin I-converting enzyme (ACE) can promote blood pressure increase and is a key enzyme in regulating human blood pressure. The development of hypertension is mainly related to the over-activation of the renin-angiotensin-aldosterone system (RAAS) and the destruction of the kallikrein-kinin system (KKS). It is generally believed that ACE plays a key role in regulating the RAAS and KKS systems, affecting the blood pressure level of the human body.

[0004] In the RAAS system, the human body's juxtaglomerular apparatus secretes a renin hydrolase into the blood, breaking down the angiotensinogen secreted by the liver into type I angiotensin Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO.7). ACE releases His-Leu from the extremely low-activity type I angiotensin, and then degrades it into type II angiotensin Asn-Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO.8) with vasoconstriction ability. Type II angiotensin is one of the main substances that cause high blood pressure. In KKS, bradykininogen is hydrolyzed by kallikrein to form bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (SEQ ID NO.9). ACE can catalyze bradykinin, causing it to lose its activity by removing two amino acid residues at the carboxyl end, resulting in high 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, antioxidant inhibitors and ACE inhibitors prepared by chemical methods have developed rapidly, but their toxic side effects have greatly restricted their application. Studies have found that a large number of natural extracts have antioxidant activity and ACE inhibitory activity, among which fish, shrimp and shellfish peptides, soybean peptides, peanut peptides, etc. have better effects. Food protein-derived antioxidant active peptides and ACE inhibitory active peptides play an important role in these fields due to their high specificity, low toxicity, relatively easy synthesis and applicability as other molecular carriers. Food protein-derived antioxidant active peptides and ACE inhibitory active peptides have a wide range of sources.

[0006] Tuna, also known as tuna, belongs to the class of Osteichthyes, order Pereiformes, family Scombridae. It is distributed in the Pacific, Atlantic and Indian Oceans. It is an oceanic highly migratory fish. The tuna species with high commercial value mainly include bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares), Atlantic bluefin tuna (Thunnus thynnus) and skipjack tuna (Katsuwonus pelamis). The annual catch of tuna in the world is about 6 million tons, accounting for one-tenth of the international seafood trade. It is an important commercial economic fish in the ocean. The processing of tuna is mainly sashimi and canned food, and the remaining parts are directly discarded as by-products, resulting in a lot of waste. At the same time, there is no report on the preparation of antioxidant active peptides and ACE inhibitory active peptides from tuna skin. Summary of the invention

[0007] The purpose of the present invention is to provide an antioxidant peptide and ACE inhibitory peptide derived from tuna, and a preparation method and application thereof, in order to solve the problems existing in the above-mentioned prior art. Tuna processing byproducts 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 improve the deep processing level of the tuna industry and achieve high value utilization on the one hand, and can reduce the pressure on the environment to bear processing waste on the other hand.

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

[0009] The present invention provides a polypeptide derived from tuna skin, wherein the amino acid sequence of the polypeptide is shown in any one or several sequences of SEQ ID NO.1-6.

[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 identified by liquid chromatography-tandem mass spectrometry and screened by molecular docking to obtain the polypeptide.

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

[0015] And / or, the acid soaking comprises the step of mixing an alkali 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 is carried out at a temperature of 60°C for 5 hours;

[0017] And / or, the first centrifugal treatment is performed at a rotation speed of 10000 r / min and for 20 min.

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

[0019] And / or, the enzymatic treatment time is 4 hours, the temperature is 50° C., and the pH is pH 8.0;

[0020] And / or, the second centrifugal treatment is performed at a rotation speed of 10000 r / min and for 10 min.

[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 several sequences of SEQ ID NO.1-3.

[0023] More preferably, the antioxidant also includes 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 several sequences of SEQ ID NOs. 4 to 6.

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

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

[0027] The present invention provides a blood pressure lowering 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. The polypeptide provided by the present invention can improve the deep processing level of the tuna industry and achieve high value utilization on the one hand, and can reduce the pressure on the environment to bear processing waste on the other hand. 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, and extracts polypeptides with antioxidant activity and polypeptides that effectively inhibit ACE activity, and this method simplifies the protein extraction step. The present invention extracts tetrapeptides MGPR (SEQ ID NO.1), tetrapeptide PRGP (SEQ ID NO.2) and tetrapeptide PGPM (SEQ ID NO.3) that can effectively inhibit ACE activity, and polypeptide decapeptides GPSGPPGKNG (SEQ ID NO.4), heptapeptide SGPAGPR (SEQ ID NO.5) and heptapeptide SGPAGPK (SEQ ID NO.6), which have effective antioxidant activity, 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0032] Figure 1 It is a schematic diagram of the binding position of 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 position of polypeptide PRGP and Keap1;

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

[0036] Figure 5 It is a schematic diagram of the binding position of peptide PGPM and Keap1;

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

[0038] Figure 7 This is a schematic diagram of the binding position of the peptide GPSGPPGKNG and ACE;

[0039] Figure 8 This is a schematic diagram of molecular docking of peptide GPSGPPGKNG and ACE;

[0040] Fig. 9 Schematic diagram of the binding position of peptide SGPAGPR and ACE;

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

[0042] Fig.11 It is a schematic diagram of the binding position of the peptide SGPAGPK and ACE;

[0043] Fig.12 Schematic diagram of molecular docking of 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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), aliphatic amino acids (such as serine, histidine, threonine, leucine, glycine, lysine) and hydrophobic amino acids (such as leucine) at the C and N termini 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, the mass ratio of tuna skin to NaOH solution is 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 to 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., after 5 hours, centrifuge at 10000 r / min for 20 minutes, then take the supernatant and concentrate it to obtain tuna skin collagen extract.

[0052] (2) Add pancreatin to the tuna skin collagen extract (the amount of pancreatin added is 3% of the protein mass in the tuna skin collagen extract), and keep the reaction at pH 8.0 and 50° C. for 4 h with shaking. After the reaction, inactivate the enzyme in a boiling water bath for 10 min, filter, and centrifuge at a speed of 10,000 r / min for 10 min. Take the supernatant and dry it to obtain tuna skin collagen enzymatic hydrolyzate powder.

[0053] (3) The tuna collagen enzymatic hydrolysate powder was analyzed by liquid chromatography-tandem mass spectrometry equipped with an online nanospray ion source. The sample volume of the liquid chromatograph was 2 μL, the sample separation gradient was 60 min, the column flow rate was controlled at 350 nL / min, the mobile phase A phase was 0.1% formic acid aqueous solution, the mobile phase B phase was 0.1% formic acid in acetonitrile solution, the column temperature was 40°C, and the electrospray voltage was 2 kV; the mass spectrometer parameters were set as follows: ①MS: scanning range (m / z): 200-1500; resolution: 60000; Normalized AGCtarget: 300%; maximum injection time: 25 ms. ②HCD-MS / MS: resolution: 15000: Normalized AGCtarget: 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. The "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 be: PeptideRanker activity score> 0.5, non-toxic, 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 by molecular docking software AutodockVina, and 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), and 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), and they were visualized using Pymol and Discovery Studio software.

[0055] Keap1 is a BTB-Kelch substrate adaptor protein that regulates the steady-state level of Nrf2 in response to oxidative stress. Angiotensin-converting enzyme is a key enzyme that causes elevated blood pressure in humans. Inhibiting the activity of angiotensin-converting enzyme can maintain the relative stability of human blood pressure. The Keap1 structure (PDB ID: 2FUL) and the human angiotensin-converting enzyme structure (PDB ID: 1O8A) were obtained from the RCSB protein library. Before docking, Keap1 and ACE enzymes were modified by Pymol. The secondary structures of tetrapeptide MGPR, tetrapeptide PRGP and tetrapeptide PGPM, decapeptide GPSGPPGKNG, heptapeptide SGPAGPR and heptapeptide SGPAGPK were drawn using Chem Draw software and energy minimization was performed using the Minimize Energy module. GridBox was set in AutoDock Vina software, and the X, Y, Z and grid center coordinates were adjusted, and the box size was set to cover the entire protein molecule.

[0056] Molecular docking screening was performed on the peptide sequences identified by liquid chromatography-tandem mass spectrometry, and three peptides with the highest antioxidant activity were obtained, namely, the three polypeptides shown in SEQ ID NO.1 to 3, and their molecular weights and molecular docking results are shown in Table 1; three peptides with ACE inhibitory activity were obtained, namely, the three polypeptides shown in SEQ ID NO.4 to 6. The docking results showed that the minimum binding energy of the six peptides with the enzyme was less than -6 kcal / mol, which indicated that the ligand receptors could bind spontaneously. In addition, it is generally believed that when the binding free energy is ≤-5.0 kcal / mol, the binding of the ligand molecule to the target protein can be considered to have practical significance.

[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 were 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 , Fig. 9 and Fig.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 , Fig.10 and Fig.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, each peptide has certain similarities with the residues binding to the corresponding macromolecular enzyme.

[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 hydrolyzate 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 polypeptide solution sample (the amino acid sequence of the polypeptide is shown in SEQ ID NO.1-3) for the sample group. Take 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] Determination of ACE inhibition rate: Prepare 5mmol / L hippuryl-histidyl-leucine (N-hippuryl-His-Leu tetrahydrate, HHL) solution, 0.1U / mL ACE enzyme solution and 1mg / mL polypeptide solution sample (the amino acid sequence of the polypeptide is shown in SEQ ID NO.4-6) with borate buffer (0.1mol / L, pH 8.3, containing 0.3mol / L NaCl); prepare 1mol / L HCL solution separately;

[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 it was placed in an oven at 120 ° C for 30 min. It was cooled to room temperature, dissolved in 4 mL of deionized water, shaken thoroughly, and its absorbance at a wavelength of 228 nm was measured using an ultraviolet spectrophotometer. Three groups of parallel measurements were performed 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] In the formula, 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 results of the antioxidant activity test 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 increased after trypsin hydrolysis, and the half inhibition rate IC 50 The peptide sequences identified by liquid chromatography-tandem mass spectrometry were screened by molecular docking to obtain three peptides with the highest predicted activity. The DPPH free radical scavenging activity of the three peptides was ranked as PRGP>MGPR>PGPM. The ACE inhibitory activity of tuna skin collagen was low, and its half inhibition rate IC 50 The inhibitory activity was significantly improved after trypsin hydrolysis, and the half inhibition rate IC 50 It was 0.937 mg / mL; molecular docking screening of the peptide sequences identified by liquid chromatography-tandem mass spectrometry technology obtained three peptides with the highest predicted activity, and the ACE inhibition rate of the three peptides was ranked as SGPAGPR>GPSGPPGKNG>SGPAGPK.

[0072] Table 3 DPPH free radical scavenging rate of tuna skin collagen, tuna skin enzymatic hydrolyzed powder, three polypeptides and ACE inhibition rate of three polypeptides

[0073]

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polypeptide derived from tuna skin, characterized in that: The amino acid sequence of the polypeptide is shown in any one or several sequences of SEQ ID NO.1-6.

2. The method for preparing the polypeptide according to claim 1, 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 is identified by liquid chromatography-tandem mass spectrometry and screened by molecular docking to obtain the polypeptide.

3. The preparation method according to claim 2, characterized in that: 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 to obtain an alkaline soaking treatment solution; the alkaline soaking treatment time is 1.5 hours; And / or, the acid soaking treatment comprises the step of mixing an alkali 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; And / or, the water bath treatment is carried out at a temperature of 60°C for 5 hours; And / or, the first centrifugal treatment is performed at a rotation speed of 10000 r / min and for 20 min.

4. The preparation method according to claim 2, characterized in that: The protease includes pancreatin; the amount of the pancreatin is 3% of the protein mass in the tuna skin collagen extract; And / or, the enzymatic treatment time is 4 hours, the temperature is 50° C., and the pH is pH 8.0; And / or, the second centrifugal treatment is performed at a rotation speed of 10000 r / min and for 10 min.

5. Use of the polypeptide according to claim 1 in the preparation of antioxidants and / or ACE inhibitors.

6. An antioxidant, characterized in that The antioxidant comprises a polypeptide having an amino acid sequence as shown in any one or several sequences in SEQ ID NO. 1-3.

7. An ACE inhibitor, characterized in that The ACE inhibitor includes a polypeptide having an amino acid sequence as shown in any one or more sequences of SEQ ID NOs. 4 to 6.

8. Use of the ACE inhibitor according to claim 7 in the preparation of antihypertensive drugs.

9. A blood pressure lowering drug, characterized in that: The antihypertensive drug comprises the ACE inhibitor according to claim 7.

10. The antihypertensive drug according to claim 9, characterized in that: The antihypertensive drug also includes pharmaceutically acceptable auxiliary materials.

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