A preparation method of an active peptide from aquatic products with high blood pressure lowering efficiency

Abstract

The present invention belongs to the technical field of the preparation of blood pressure-lowering products. A preparation method of an active peptide of a high-efficiency blood pressure-lowering aquatic product is characterized by comprising the following steps: Step 1, using pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides, separating, purifying and identifying; Step 2, synthesizing the polypeptide sequence YLRLHF by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform; Step 3, dissolving the nanoscale material in water and preparing a dispersion of 0.1 mg / mL by ultrasonic treatment; Step 4, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide to the dispersion; Step 5, adding the ACE inhibitory peptide YLRLHF, mixing evenly and stirring to obtain a reaction solution; Step 6, centrifuging the reaction solution obtained in Step 5, washing the precipitate, and freeze-drying to obtain the active peptide of the high-efficiency blood pressure-lowering aquatic product. The product obtained by this method has a good inhibitory effect on angiotensin-converting enzyme (ACE).

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of blood pressure-lowering products, and particularly relates to a method for preparing an active peptide from aquatic products with blood pressure-lowering effect. Background Art

[0002] As one of the risk factors for stroke, hypertension affects 20%-45% of the global population, especially 50%-60% of the elderly. Angiotensin-I-converting enzyme (EC 3.4.15.1; ACE) is a metallo-carboxypeptidase that regulates blood pressure in the renin-angiotensin system. It hydrolyzes the C-terminal dipeptide His-Leu of angiotensin I to obtain angiotensin II. Angiotensin II enhances the contractility of the myocardium, leading to vasoconstriction of vascular smooth muscle and an increase in blood pressure. Therefore, inhibiting ACE activity is an important means of lowering blood pressure. Synthetic angiotensin-converting enzyme (ACE) inhibitors can cause side effects such as allergic reactions and renal failure. Therefore, they are not suitable for long-term use. Natural blood pressure-lowering peptides extracted from animal proteins have attracted extensive attention worldwide due to their advantages such as environmental protection, sustainability, low cost, and no toxic side effects. From the perspective of sustainable development and utilization of biological resources, the protein resources in by-products or wastes generated during animal processing have great potential for the production of bioactive peptides.

[0003] However, at present, the effects of angiotensin-converting enzyme (ACE) inhibitory peptides from natural sources still need to be improved. Their stability in vivo is poor and they are easily hydrolyzed by enzymes in the gastrointestinal tract, and thus cannot exert their blood pressure-lowering effect well. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing an active peptide from aquatic products with high blood pressure-lowering effect, and the product obtained by this method has a good inhibitory effect on angiotensin-converting enzyme (ACE).

[0005] To achieve the above purpose, the technical solution adopted by the present invention is: a method for preparing an active peptide from aquatic products with high blood pressure-lowering effect, which is characterized by comprising the following steps:

[0006] Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, select the peptide segment YLRLHF with the greatest potential ACE activity. The specific steps are as follows:

[0007] 1) Preparation of crude polypeptides:

[0008] The bighead carp meat was treated under ultra-high pressure of 300 MPa for 20 min to obtain the bighead carp meat after ultra-high pressure treatment; according to the mass ratio of the bighead carp meat after ultra-high pressure treatment: water = 1:5, water was added to the bighead carp meat after ultra-high pressure treatment (adjusting the pH to 3), 4000 μ / g of pepsin was added, and the reaction was carried out in a water bath at 55 °C for 6 h; the reaction was terminated by heating in a water bath at 95 °C for 10 min, the hydrolysis product was centrifuged, and the supernatant was collected and dialyzed against salt using a dialysis bag (MD77-5M) to obtain the crude polypeptide;

[0009] 2) Separation and purification of the polypeptide:

[0010] The crude polypeptide was ultrafiltered using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa was freeze-dried and the amino acid sequence was determined by mass spectrometry. Peptides with a confidence value >99% were selected and scored for activity using the PeptideRanker software. Peptides with a score result >0.5 were subjected to molecular docking, and the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the greatest potential ACE activity was screened according to the molecular docking results;

[0011] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF;

[0012] Step 3: The nanoscale material was dissolved in water and sonicated for 2 h to prepare a dispersion of 0.1 mg / mL;

[0013] The nanoscale materials are: graphene oxide at the nanoscale (GO), carboxyl mesoporous silica nanoparticles (Carboxyl Mesoporous Silica Nanoparticles, CMSN), polyethylene glycol-coated gold nanoparticles (modified with carboxyl groups at the end) (Gold Nanoparticles-PEG-N3, GN-P), carboxyl-functionalized magnetite (DMSA@Fe 3 O 4 );

[0014] Step 4: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of the nanoscale material and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of the nanoscale material were added to the dispersion and stirred for 5 min;

[0015] Step 5: According to the mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2: the nanoscale material is 1-2:2-1, and then the ACE inhibitory peptide YLRLHF obtained in Step 2 was added. After mixing evenly, the reaction was carried out on a magnetic stirrer for 6 h to obtain the reaction solution;

[0016] Step 6: Centrifuge the reaction solution obtained in Step 5. Wash the precipitate with ultrapure water three times, and then freeze-dry to obtain the active peptide of the high-efficiency blood pressure-lowering aquatic product (or: RVPSL-Nano powder with high ACE inhibitory activity).

[0017] According to the above technical solution, in the preparation of the crude polypeptide in Step 1, the hydrolysis product is centrifuged under the conditions that the hydrolysis product is centrifuged at 10,000 g for 20 min.

[0018] According to the above technical solution, in Step 5, the mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2 to the nanoscale material is preferably 1:2 - 2:1.

[0019] The present invention adopts the "preparation of crude polypeptide" process conditions (ultra-high pressure treatment, pepsin). The functions are as follows: Ultra-high pressure can make the protein structure looser, and expose more cleavage sites by changing non-covalent bonds such as hydrogen bonds and ionic bonds. At the same time, ultra-high pressure can easily induce protein aggregation through disulfide bonds. Therefore, the sensitivity of the protein to the enzyme has changed. Pepsin is an endopeptidase that preferentially cleaves C-terminal aromatic amino acid (phenylalanine, tyrosine, and tryptophan) residues. Therefore, the hydrolysate obtained by using gastrointestinal proteolytic enzymes has higher angiotensin-converting enzyme inhibitory activity because more peptides with C-terminal aromatic amino acid residues are released by pepsin hydrolysis.

[0020] The function of the "separation and purification of polypeptide" adopted in the present invention is as follows: The components in the crude polypeptide are complex. In order to understand the specific polypeptide that plays an ACE inhibitory role and its mechanism of action.

[0021] The function of the present invention of "adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of nanoscale graphene oxide (GO) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of nanoscale graphene oxide (GO) to the dispersion liquid" is that the combination of graphene oxide and polypeptide requires EDCl and NHS as initiators to activate the carboxyl groups of the nanoparticles.

[0022] To solve the effect of the angiotensin-converting enzyme (ACE) inhibitory peptide from natural sources, the present invention loads the ACE inhibitory peptide YLRLHF (active peptide YLRLHF) on nanoscale graphene oxide (GO). Nanomaterials have unique physical and chemical properties such as small size, large specific surface area, and strong permeability, which can effectively enhance the blood pressure-lowering effect of the ACE inhibitory peptide in vivo and its stability in vivo, and improve the bioavailability of the ACE inhibitory peptide. Graphene oxide (GO), silicon dioxide (SiO 2) Gold Nanoparticles, carboxyl-functionalized magnetite (Fe 3 O 4 ), zinc oxide (ZnO), titanium dioxide (TiO 2 ), chitosan and other nanoparticles are potential materials for loading ACE inhibitory peptides.

[0023] When the polypeptide not combined with GO was at 0.1 mg / mL, the ACE inhibition rate was 43.36%. After binding with GO, the inhibition rates were 46.75% (peptide:GO = 1:2), 51.72% (peptide:GO = 1:1), and 52.44% (peptide:GO = 2:1), respectively.

[0024] The beneficial effects of the present invention are as follows: The method of the present invention uses nanomaterials to load the bioactive peptide YLRLHF and compares it with the bioactive peptide without loaded nanomaterials. When the sample concentration is 0.1 mg / mL, the angiotensin-converting enzyme (ACE) inhibition rate is increased by 19.23%. At the same time, the loaded nanoparticles can enhance the stability of the bioactive peptide YLRLHF in the gastrointestinal tract. It shows that the blood pressure-lowering effect and stability of the bioactive peptide after being loaded with nanomaterials are better than those of the bioactive peptide without loaded nanoparticles. It provides certain technical support for the related research of angiotensin-converting enzyme (ACE) inhibitory peptides. Detailed implementation manners

[0025] In order to make the technical solutions and advantages of the present invention clearer, the following further details the present invention in conjunction with embodiments. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] Method for measuring ACE inhibition rate:

[0027] The reaction solution contains 30 μL of ACE (0.1 U / mL), 40 μL of the peptide, and 80 μL of N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycine (FAPGG) (1 mmol / L). The blank control sample includes 30 μL of ACE (0.1 U / mL), 80 μL of FAPGG, and 40 mL of PBS (80 mmol / L, pH = 8.2). The absorbance of the pre-reaction mixture at 340 nm is measured by a multi-functional microplate reader. After the mixture is shaken at 37 °C for 30 min, the absorbance of the reaction mixture is measured again. The ACE inhibitory activity is calculated according to the following formula.

[0028] ACE inhibitory activity (%) = (1 - ΔA2 / ΔA1) × 100%,

[0029] ΔA1: The change in absorbance of the blank control within 30 min; ΔA2: The change in absorbance of the reaction sample within 30 min.

[0030] Example 1:

[0031] A preparation method of an active peptide from aquatic products with high efficiency in reducing blood pressure, comprising the following steps:

[0032] Step 1: Using pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides, separating, purifying, and identifying the crude polypeptides, and then screening out the peptide segment YLRLHF with the greatest potential ACE activity; the specific steps are as follows:

[0033] 1) Preparation of crude polypeptides:

[0034] Subject bighead carp meat to ultra-high pressure treatment at 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment; according to the mass ratio of bighead carp meat after ultra-high pressure treatment: water = 1:5, add water (adjust the pH to 3) to the bighead carp meat after ultra-high pressure treatment, add 4000 μ / g of pepsin, and react in a water bath at 55 °C for 6 h; terminate the reaction by heating in a water bath at 95 °C for 10 min, centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min, and collect the supernatant and dialyze it using a dialysis bag (MD77 - 5M) to remove salts to obtain crude polypeptides;

[0035] 2) Separation and purification of polypeptides:

[0036] Ultrafilter the crude polypeptides using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane, with a membrane pressure of 0.05 MPa, to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is freeze-dried and the amino acid sequence is determined by mass spectrometry. Select the peptide segments with a confidence value >99% and perform activity scoring through the PeptideRanker software. For the peptide segments with a scoring result >0.5, perform molecular docking, and screen out the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the greatest potential ACE activity according to the molecular docking results;

[0037] Step 2: Synthesize the polypeptide sequence YLRLHF by solid-phase synthesis method using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF;

[0038] Step 3: Dissolve nanoscale graphene oxide (GO) in water and ultrasonicate for 2 h to prepare a dispersion of 0.1 mg / mL;

[0039] Step 4: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of nanoscale graphene oxide (GO) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of nanoscale graphene oxide (GO) to the dispersion, and stir for 5 min;

[0040] Step 5: The mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2 to nano-sized graphene oxide (GO) is 1:2. Then add the ACE inhibitory peptide YLRLHF obtained in Step 2, mix evenly, and react on a magnetic stirrer for 6 h to obtain a reaction solution.

[0041] Step 6: Centrifuge the reaction solution obtained in Step 5, wash the precipitate with ultrapure water three times, and freeze-dry to obtain a high-efficiency blood pressure-lowering aquatic product active peptide (or: RVPSL-GO powder with high ACE inhibitory activity). When the sample concentration is 0.1 mg / mL, the inhibition rate is measured to be 46.75% (peptide:GO = 1:2).

[0042] Example 2:

[0043] A preparation method of a high-efficiency blood pressure-lowering aquatic product active peptide, comprising the following steps:

[0044] Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, select the peptide segment YLRLHF with the highest potential ACE activity. The specific steps are as follows:

[0045] 1) Preparation of crude polypeptides:

[0046] Treat bighead carp meat at 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment; according to the mass ratio of bighead carp meat after ultra-high pressure treatment to water = 1:5, add water (adjust the pH to 3) to the bighead carp meat after ultra-high pressure treatment, add 4000 μ / g of pepsin, and react in a water bath at 55 °C for 6 h; heat in a water bath at 95 °C for 10 min to terminate the reaction, centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min, and collect the supernatant and dialyze it with a dialysis bag (MD77-5M) to remove salts to obtain crude polypeptides.

[0047] 2) Separation and purification of polypeptides:

[0048] Ultrafilter the crude polypeptides using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is freeze-dried and the amino acid sequence is determined by mass spectrometry. Select the peptide segments with a confidence value >99% and perform activity scoring through the PeptideRanker software. Perform molecular docking on the peptide segments with a scoring result >0.5, and select the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity according to the molecular docking results.

[0049] Step 2: Synthesize the polypeptide sequence YLRLHF by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF.

[0050] Step 3: Dissolve nanoscale graphene oxide (GO) in water and ultrasonicate for 2 h to prepare a dispersion with a concentration of 0.1 mg / mL.

[0051] Step 4: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of nanoscale graphene oxide (GO) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of nanoscale graphene oxide (GO) to the dispersion, and stir for 5 min.

[0052] Step 5: According to the mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2: nanoscale graphene oxide (GO) = 1:1, then add the ACE inhibitory peptide YLRLHF obtained in Step 2, mix evenly and react on a magnetic stirrer for 6 h to obtain a reaction solution.

[0053] Step 6: Centrifuge the reaction solution obtained in Step 5, wash the precipitate 3 times with ultrapure water, and freeze-dry to obtain a high-efficiency blood pressure-lowering aquatic product active peptide (or: RVPSL-GO powder with high ACE inhibitory activity). When the sample concentration is 0.1 mg / mL, the inhibition rate is measured to be 51.72% (peptide:GO = 1:1).

[0054] Example 3:

[0055] A preparation method of a high-efficiency blood pressure-lowering aquatic product active peptide, comprising the following steps:

[0056] Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, select the peptide segment YLRLHF with the highest potential ACE activity. The specific steps are as follows:

[0057] 1) Preparation of crude polypeptides:

[0058] Treat bighead carp meat with ultra-high pressure of 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment; according to the mass ratio of bighead carp meat after ultra-high pressure treatment: water = 1:5, add water (adjust the pH to 3) to the bighead carp meat after ultra-high pressure treatment, add 4000 μ / g of pepsin, and react in a water bath at 55 °C for 6 h; heat in a water bath at 95 °C for 10 min to terminate the reaction, centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min, and collect the supernatant and dialyze it with a dialysis bag (MD77-5M) to remove salts to obtain crude polypeptides.

[0059] 2) Separation and purification of polypeptides:

[0060] The crude polypeptide was ultrafiltered using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa was freeze-dried and the amino acid sequence was determined by mass spectrometry. Peptides with a confidence value >99% were selected and scored for activity using the PeptideRanker software. Peptides with a score result >0.5 were subjected to molecular docking, and based on the molecular docking results, the peptide YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity was screened out;

[0061] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF;

[0062] Step 3: Nanoscale graphene oxide (GO) was dissolved in water and ultrasonicated for 2 h to prepare a dispersion of 0.1 mg / mL;

[0063] Step 4: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of nanoscale graphene oxide (GO) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of nanoscale graphene oxide (GO) were added to the dispersion and stirred for 5 min;

[0064] Step 5: The mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2 to nanoscale graphene oxide (GO) was 2:1. Then, the ACE inhibitory peptide YLRLHF obtained in Step 2 was added, and after mixing evenly, the reaction was carried out on a magnetic stirrer for 6 h to obtain a reaction solution;

[0065] Step 6: The reaction solution obtained in Step 5 was centrifuged, and the precipitate was washed 3 times with ultrapure water and freeze-dried to obtain a high-efficiency blood pressure-lowering aquatic product active peptide (or: RVPSL-GO powder with high ACE inhibitory activity). When the sample concentration was 0.1 mg / mL, the inhibition rate was measured to be 52.44% (peptide:GO = 2:1).

[0066] Example 4:

[0067] A method for preparing a high-efficiency blood pressure-lowering aquatic product active peptide, comprising the following steps:

[0068] Step 1: The bighead carp meat after ultra-high pressure treatment was enzymolyzed with pepsin to obtain a crude polypeptide. After the crude polypeptide was separated, purified, and identified, the peptide segment YLRLHF with the highest potential ACE activity was screened out; the specific steps are as follows:

[0069] 1) Preparation of the crude polypeptide:

[0070] The bighead carp meat was treated under ultra-high pressure of 300 MPa for 20 min to obtain the bighead carp meat after ultra-high pressure treatment; according to the mass ratio of the bighead carp meat after ultra-high pressure treatment: water = 1:5, water was added to the bighead carp meat after ultra-high pressure treatment (adjusting the pH to 3), 4000 μ / g of pepsin was added, and the reaction was carried out in a water bath at 55 °C for 6 h; the reaction was terminated by heating in a water bath at 95 °C for 10 min, and the hydrolysis product was centrifuged at a centrifugal force of 10000 g for 20 min, and the supernatant was collected and desalted by dialysis using a dialysis bag (MD77-5M) to obtain the crude polypeptide;

[0071] 2) Separation and purification of polypeptides:

[0072] The crude polypeptide was ultrafiltered using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane, and the membrane pressure was 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa was freeze-dried and the amino acid sequence was determined by mass spectrometry. Peptides with a confidence value >99% were selected and scored for activity using the PeptideRanker software. Peptides with a score result >0.5 were subjected to molecular docking, and the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity was screened according to the molecular docking results;

[0073] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF;

[0074] Step 3: Carboxyl Mesoporous Silica Nanoparticles (CMSN) were dissolved in water and ultrasonicated for 2 h to prepare a dispersion of 0.1 mg / mL;

[0075] Step 4: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of Carboxyl Mesoporous Silica Nanoparticles (CMSN) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of Carboxyl Mesoporous Silica Nanoparticles (CMSN) were added to the dispersion and stirred for 5 min;

[0076] Step 5: The mass ratio of the ACE inhibitory peptide YLRLHF obtained in Step 2 to carboxyl mesoporous silica nanoparticles (CMSN) is 1:2. Then, add the ACE inhibitory peptide YLRLHF obtained in Step 2, mix evenly, and react on a magnetic stirrer for 10 h to obtain a reaction solution.

[0077] Step 6: Centrifuge the reaction solution obtained in Step 5. Wash the precipitate with ultrapure water three times, and then freeze-dry to obtain an active peptide of aquatic products with high blood pressure-lowering effect (or: RVPSL-Si powder with high ACE inhibitory activity). When the sample concentration is 0.1 mg / mL, the inhibition rate is measured to be 50.24% (peptide: CMSN = 1:2).

[0078] Example 5:

[0079] A preparation method of an active peptide of aquatic products with high blood pressure-lowering effect, comprising the following steps:

[0080] Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, screen out the peptide segment YLRLHF with the highest potential ACE activity. The specific steps are as follows:

[0081] 1) Preparation of crude polypeptides:

[0082] Treat bighead carp meat with 300 MPa ultra-high pressure for 20 min to obtain bighead carp meat after ultra-high pressure treatment. According to the mass ratio of bighead carp meat after ultra-high pressure treatment to water = 1:5, add water (adjust the pH to 3) to the bighead carp meat after ultra-high pressure treatment, add 4000 μ / g of pepsin, and react in a water bath at 55 °C for 6 h. Heat in a water bath at 95 °C for 10 min to terminate the reaction. Centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min, and collect the supernatant and dialyze it with a dialysis bag (MD77-5M) to remove salts to obtain crude polypeptides.

[0083] 2) Separation and purification of polypeptides:

[0084] Ultrafilter the crude polypeptides using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is freeze-dried and the amino acid sequence is determined by mass spectrometry. Select the peptide segments with a confidence value >99% and perform activity scoring through the PeptideRanker software. Perform molecular docking on the peptide segments with a scoring result >0.5, and screen out the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity according to the molecular docking results.

[0085] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF;

[0086] Step 3: Carboxyl Mesoporous Silica Nanoparticles (CMSN) were dissolved in water and ultrasonicated for 2 h to prepare a dispersion of 0.1 mg / mL;

[0087] Step 4: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of Carboxyl Mesoporous Silica Nanoparticles (CMSN) and N-Hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of Carboxyl Mesoporous Silica Nanoparticles (CMSN) were added to the dispersion and stirred for 5 min;

[0088] Step 5: The mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2 to Carboxyl Mesoporous Silica Nanoparticles (CMSN) was 1:1. Then, the ACE inhibitory peptide YLRLHF obtained in Step 2 was added, and after mixing evenly, the reaction was carried out on a magnetic stirrer for 10 h to obtain a reaction solution;

[0089] Step 6: The reaction solution obtained in Step 5 was centrifuged, and the precipitate was washed 3 times with ultrapure water and freeze-dried to obtain the active peptide of high-efficiency blood pressure-lowering aquatic products (or: RVPSL-Si powder with high ACE inhibitory activity). When the sample concentration was 0.1 mg / mL, the inhibition rate was measured to be 52.29% (peptide: CMSN = 1:1).

[0090] Example 6:

[0091] A preparation method of an active peptide of high-efficiency blood pressure-lowering aquatic products, comprising the following steps:

[0092] Step 1: The bighead carp meat after ultra-high pressure treatment was enzymolyzed with pepsin to obtain crude polypeptides. After the crude polypeptides were separated, purified, and identified, the peptide segment YLRLHF with the greatest potential ACE activity was screened out. The specific steps are as follows:

[0093] 1) Preparation of crude polypeptides:

[0094] The bighead carp meat was treated at 300 MPa for 20 min to obtain the bighead carp meat after ultra-high pressure treatment. According to the mass ratio of the bighead carp meat after ultra-high pressure treatment to water = 1:5, water (adjusting the pH to 3) was added to the bighead carp meat after ultra-high pressure treatment, 4000 μ / g of pepsin was added, and the reaction was carried out in a water bath at 55 °C for 6 h. The reaction was terminated by heating in a water bath at 95 °C for 10 min, and the hydrolysis product was centrifuged at a centrifugal force of 10000 g for 20 min. The supernatant was collected and dialyzed against salt using a dialysis bag (MD77-5M) to obtain the crude polypeptide.

[0095] 2) Separation and purification of the polypeptide:

[0096] The crude polypeptide was ultrafiltered using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa was freeze-dried and the amino acid sequence was determined by mass spectrometry. Peptides with a confidence value >99% were selected and subjected to activity scoring using the PeptideRanker software. Peptides with a scoring result >0.5 were subjected to molecular docking, and the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity was screened out according to the molecular docking results.

[0097] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM Polypeptide Synthesis Platform (Beijing) to obtain the ACE inhibitory peptide YLRLHF.

[0098] Step 3: Carboxyl Mesoporous Silica Nanoparticles (CMSN) were dissolved in water and ultrasonicated for 2 h to prepare a dispersion of 0.1 mg / mL.

[0099] Step 4: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of Carboxyl Mesoporous Silica Nanoparticles (CMSN) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of Carboxyl Mesoporous Silica Nanoparticles (CMSN) were added to the dispersion and stirred for 5 min.

[0100] Step 5: Take the ACE inhibitory peptide YLRLHF obtained in Step 2 and carboxyl mesoporous silica nanoparticles (CMSN) at a mass ratio of 2:1. Then add the ACE inhibitory peptide YLRLHF obtained in Step 2, mix evenly, and react on a magnetic stirrer for 10 h to obtain a reaction solution.

[0101] Step 6: Centrifuge the reaction solution obtained in Step 5. Wash the precipitate with ultrapure water three times and freeze-dry it to obtain an active peptide of high-efficiency blood-pressure-lowering aquatic products (or: RVPSL-Si powder with high ACE inhibitory activity). When the sample concentration is 0.1 mg / mL, the inhibition rate is measured to be 62.55% (peptide: CMSN = 2:1).

[0102] Example 7:

[0103] A preparation method of an active peptide of high-efficiency blood-pressure-lowering aquatic products, comprising the following steps:

[0104] Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, select the peptide segment YLRLHF with the highest potential ACE activity. The specific steps are as follows:

[0105] 1) Preparation of crude polypeptides:

[0106] Treat bighead carp meat with ultra-high pressure of 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment. According to the mass ratio of bighead carp meat after ultra-high pressure treatment to water = 1:5, add water (adjust the pH to 3) to the bighead carp meat after ultra-high pressure treatment, add pepsin at 4000 μ / g, and react in a water bath at 55 °C for 6 h. Terminate the reaction by heating in a water bath at 95 °C for 10 min. Centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min, and collect the supernatant to remove salts by dialysis using a dialysis bag (MD77-5M) to obtain crude polypeptides.

[0107] 2) Separation and purification of polypeptides:

[0108] Ultrafilter the crude polypeptides using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is freeze-dried and the amino acid sequence is determined by mass spectrometry. Select the peptide segments with a confidence value >99% for activity scoring using PeptideRanker software, perform molecular docking on the peptide segments with a scoring result >0.5, and select the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity according to the molecular docking results.

[0109] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM Polypeptide Synthesis Platform (Beijing), and the ACE inhibitory peptide YLRLHF was obtained;

[0110] Step 3: Polyethylene glycol-coated gold nanoparticles (modified with carboxyl groups at the end) (Gold Nanoparticles-PEG-N3, GN-P) were dissolved in water and ultrasonicated for 2 h to prepare a dispersion of 0.1 mg / mL;

[0111] Step 4: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of polyethylene glycol-coated gold nanoparticles (modified with carboxyl groups at the end) (Gold Nanoparticles-PEG-N3, GN-P) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of polyethylene glycol-coated gold nanoparticles (modified with carboxyl groups at the end) (Gold Nanoparticles-PEG-N3, GN-P) were added to the dispersion and stirred for 5 min;

[0112] Step 5: The mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2 to polyethylene glycol-coated gold nanoparticles (modified with carboxyl groups at the end) (Gold Nanoparticles-PEG-N3, GN-P) was 1:2. Then, the ACE inhibitory peptide YLRLHF obtained in Step 2 was added. After mixing evenly, the reaction was carried out on a magnetic stirrer for 8 h to obtain a reaction solution;

[0113] Step 6: The reaction solution obtained in Step 5 was centrifuged, and the precipitate was washed 3 times with ultrapure water and freeze-dried to obtain an active peptide of aquatic products with high blood pressure-lowering effect (or: RVPSL-Au powder with high ACE inhibitory activity). When the sample concentration was 0.1 mg / mL, the inhibition rate was measured to be 51.37% (peptide:GN-P = 1:2).

[0114] Example 8:

[0115] A preparation method of an active peptide of aquatic products with high blood pressure-lowering effect, comprising the following steps:

[0116] Step 1: Crude polypeptides were obtained by enzymatically hydrolyzing bighead carp meat treated with ultra-high pressure using pepsin. After separating, purifying, and identifying the crude polypeptides, the peptide segment YLRLHF with the greatest potential ACE activity was screened out. The specific steps are as follows:

[0117] 1) Preparation of crude polypeptides:

[0118] The bighead carp meat was treated under ultra-high pressure of 300 MPa for 20 min to obtain the bighead carp meat after ultra-high pressure treatment. According to the mass ratio of the bighead carp meat after ultra-high pressure treatment: water = 1:5, water (adjusting pH = 3) was added to the bighead carp meat after ultra-high pressure treatment, 4000 μ / g of pepsin was added, and the reaction was carried out in a water bath at 55 °C for 6 h. The reaction was terminated by heating in a water bath at 95 °C for 10 min. The hydrolysis product was centrifuged at a centrifugal force of 10000 g for 20 min, and the supernatant was collected and dialyzed against salt using a dialysis bag (MD77-5M) to obtain crude polypeptide.

[0119] 2) Separation and purification of polypeptide:

[0120] The crude polypeptide was ultrafiltered using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane, and the membrane pressure was 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa was freeze-dried and the amino acid sequence was determined by mass spectrometry. Peptides with a confidence value >99% were selected and scored for activity using the PeptideRanker software. Peptides with a score result >0.5 were subjected to molecular docking, and the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the greatest potential ACE activity was screened out according to the molecular docking results.

[0121] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF.

[0122] Step 3: Polyethylene glycol-coated gold nanoparticles (modified with terminal carboxyl groups) (Gold Nanoparticles-PEG-N3, GN-P) were dissolved in water and ultrasonicated for 2 h to prepare a dispersion of 0.1 mg / mL.

[0123] Step 4: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of polyethylene glycol-coated gold nanoparticles (modified with terminal carboxyl groups) (Gold Nanoparticles-PEG-N3, GN-P) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of polyethylene glycol-coated gold nanoparticles (modified with terminal carboxyl groups) (Gold Nanoparticles-PEG-N3, GN-P) were added to the dispersion and stirred for 5 min.

[0124] Step 5: According to the mass fraction of the ACE inhibitory peptide YLRLHF obtained in step 2: polyethylene glycol-coated gold nanoparticles (modified with terminal carboxyl groups) (Gold Nanoparticles-PEG-N3, GN-P) being 1:1, the ACE inhibitory peptide YLRLHF obtained in step 2 was added again. After mixing evenly, the reaction was carried out on a magnetic stirrer for 8 h to obtain a reaction solution.

[0125] Step 6: Centrifuge the reaction solution obtained in Step 5. Wash the precipitate three times with ultrapure water, and then freeze-dry to obtain the active peptide of aquatic products with high blood pressure-lowering effect (or: RVPSL-Au powder with high ACE inhibitory activity). When the sample concentration is 0.1 mg / mL, the inhibition rate is measured to be 58.62% (peptide: GN-P = 1:1).

[0126] Example 9:

[0127] A preparation method of an active peptide of aquatic products with high blood pressure-lowering effect, comprising the following steps:

[0128] Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, select the peptide segment YLRLHF with the greatest potential ACE activity. The specific steps are as follows:

[0129] 1) Preparation of crude polypeptides:

[0130] Subject bighead carp meat to ultra-high pressure treatment at 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment. According to the mass ratio of bighead carp meat after ultra-high pressure treatment: water = 1:5, add water to the bighead carp meat after ultra-high pressure treatment (adjust the pH to 3), add 4000 μ / g of pepsin, and react in a water bath at 55°C for 6 h. Terminate the reaction by heating in a water bath at 95°C for 10 min. Centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min, and collect the supernatant and dialyze it using a dialysis bag (MD77-5M) to remove salts to obtain crude polypeptides.

[0131] 2) Separation and purification of polypeptides:

[0132] Ultrafilter the crude polypeptides using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is freeze-dried and the amino acid sequence is determined by mass spectrometry. Select the peptide segments with a confidence value >99% and perform activity scoring using the PeptideRanker software. Perform molecular docking on the peptide segments with a scoring result >0.5, and select the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the greatest potential ACE activity according to the molecular docking results.

[0133] Step 2: Synthesize the polypeptide sequence YLRLHF by solid-phase synthesis method using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF.

[0134] Step 3: Dissolve polyethylene glycol-coated gold nanoparticles (with terminal carboxyl modification) (Gold Nanoparticles-PEG-N3, GN-P) in water and ultrasonicate for 2 h to prepare a 0.1 mg / mL dispersion.

[0135] Step 4: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction of 10 times polyethylene glycol-coated gold nanoparticles (carboxyl-terminally modified) (Gold Nanoparticles-PEG-N3, GN-P) and N-hydroxysulfosuccinimide (NHS) with a mass fraction of 20 times polyethylene glycol-coated gold nanoparticles (carboxyl-terminally modified) (Gold Nanoparticles-PEG-N3, GN-P) are added to the dispersion liquid and stirred for 5 min;

[0136] Step 5: The mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2 to polyethylene glycol-coated gold nanoparticles (carboxyl-terminally modified) (Gold Nanoparticles-PEG-N3, GN-P) is 2:1. Then, the ACE inhibitory peptide YLRLHF obtained in Step 2 is added. After mixing evenly, the reaction is carried out on a magnetic stirrer for 8 h to obtain a reaction solution;

[0137] Step 6: The reaction solution obtained in Step 5 is centrifuged, and the precipitate is washed 3 times with ultrapure water and freeze-dried to obtain an active peptide of aquatic products with high blood pressure-lowering effect (or: RVPSL-Au powder with high ACE inhibitory activity). When the sample concentration is 0.1 mg / mL, the inhibition rate is measured to be 59.71% (peptide: GN-P = 2:1).

[0138] Example 10:

[0139] A preparation method of an active peptide of aquatic products with high blood pressure-lowering effect, comprising the following steps:

[0140] Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, the peptide segment YLRLHF with the highest potential ACE activity is screened out. The specific steps are as follows:

[0141] 1) Preparation of crude polypeptides:

[0142] Bighead carp meat is treated with ultra-high pressure of 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment; according to the mass ratio of bighead carp meat after ultra-high pressure treatment to water = 1:5, water is added to the bighead carp meat after ultra-high pressure treatment (adjusting pH = 3), 4000 μ / g of pepsin is added, and the reaction is carried out in a water bath at 55 °C for 6 h; the reaction is terminated by heating in a water bath at 95 °C for 10 min, and the hydrolysis product is centrifuged at a centrifugal force of 10000 g for 20 min. The supernatant is collected and dialyzed against salt using a dialysis bag (MD77-5M) to obtain crude polypeptides;

[0143] 2) Separation and purification of polypeptides:

[0144] The crude polypeptide was ultrafiltered using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa was freeze-dried and the amino acid sequence was determined by mass spectrometry. Peptides with a confidence value >99% were selected and scored for activity using the PeptideRanker software. Peptides with a score result >0.5 were subjected to molecular docking, and based on the molecular docking results, the peptide YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity was screened out;

[0145] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF;

[0146] Step 3: Carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) was dissolved in water and sonicated for 2 h to prepare a dispersion of 0.1 mg / mL;

[0147] Step 4: 10 times the mass fraction of carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) and 20 times the mass fraction of carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) of N-hydroxysulfosuccinimide (NHS) were added to the dispersion and stirred for 5 min;

[0148] Step 5: According to Step 2, the mass fraction of the ACE inhibitory peptide YLRLHF:carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) was 1:2. Then, the ACE inhibitory peptide YLRLHF obtained in Step 2 was added, and after mixing evenly, the reaction was carried out on a magnetic stirrer for 4 h to obtain a reaction solution;

[0149] Step 6: The reaction solution obtained in Step 5 was centrifuged, and the precipitate was washed 3 times with ultrapure water and freeze-dried to obtain a high-efficiency blood pressure-lowering aquatic product active peptide (or: RVPSL-Fe powder with high ACE inhibitory activity). When the sample concentration was 0.1 mg / mL, the inhibition rate was measured to be 53.41% (peptide:DMSA@Fe 3 O 4 = 1:2).

[0150] Example 11:

[0151] A preparation method of a high-efficiency blood pressure-lowering aquatic product active peptide, comprising the following steps:

[0152] Step 1: Use pepsin to enzymatically hydrolyze the bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, select the peptide segment YLRLHF with the highest potential ACE activity. The specific steps are as follows:

[0153] 1) Preparation of crude polypeptides:

[0154] Treat bighead carp meat with ultra-high pressure of 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment. According to the mass ratio of bighead carp meat after ultra-high pressure treatment: water = 1:5, add water (adjust the pH to 3) to the bighead carp meat after ultra-high pressure treatment, add 4000 μ / g of pepsin, and react in a water bath at 55 °C for 6 h. Terminate the reaction by heating in a water bath at 95 °C for 10 min. Centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min, and collect the supernatant. Use a dialysis bag (MD77 - 5M) to dialyze and desalt to obtain crude polypeptides.

[0155] 2) Separation and purification of polypeptides:

[0156] Ultrafilter the crude polypeptides using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is freeze-dried and the amino acid sequence is determined by mass spectrometry. Select the peptide segments with a confidence value >99% and perform activity scoring using the PeptideRanker software. For the peptide segments with a scoring result >0.5, perform molecular docking, and select the peptide segment YLRLHF (tyrosine - leucine - arginine - leucine - histidine - phenylalanine) with the highest potential ACE activity according to the molecular docking results.

[0157] Step 2: Synthesize the polypeptide sequence YLRLHF by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF.

[0158] Step 3: Dissolve carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) in water and ultrasonicate for 2 h to prepare a dispersion of 0.1 mg / mL.

[0159] Step 4: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) to the dispersion and stir for 5 min.

[0160] Step 5: Take the ACE inhibitory peptide YLRLHF obtained in Step 2 and carboxylated magnetite (DMSA@Fe 3 O 4 ) at a mass ratio of 1:1. Then add the ACE inhibitory peptide YLRLHF obtained in Step 2, mix well, and react on a magnetic stirrer for 4 h to obtain a reaction solution;

[0161] Step 6: Centrifuge the reaction solution obtained in Step 5. Wash the precipitate with ultrapure water three times, and then freeze-dry to obtain an active peptide for high-efficiency blood pressure-lowering aquatic products (or: RVPSL-Fe powder with high ACE inhibitory activity). When the sample concentration is 0.1 mg / mL, the inhibition rate is measured to be 64.91% (peptide:DMSA@Fe 3 O 4 =1:1).

[0162] Example 12:

[0163] A preparation method of an active peptide for high-efficiency blood pressure-lowering aquatic products, comprising the following steps:

[0164] Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, select the peptide segment YLRLHF with the highest potential ACE activity. The specific steps are as follows:

[0165] 1) Preparation of crude polypeptides:

[0166] Treat bighead carp meat at 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment; according to the mass ratio of bighead carp meat after ultra-high pressure treatment: water = 1:5, add water (adjust the pH to 3) to the bighead carp meat after ultra-high pressure treatment, add 4000 μ / g of pepsin, and react in a water bath at 55 °C for 6 h; heat in a water bath at 95 °C for 10 min to terminate the reaction, centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min, and collect the supernatant. Use a dialysis bag (MD77-5M) to dialyze and desalt to obtain crude polypeptides;

[0167] 2) Separation and purification of polypeptides:

[0168] Ultrafilter the crude polypeptides using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is freeze-dried and the amino acid sequence is determined by mass spectrometry. Select the peptide segments with a confidence value >99% and perform activity scoring using the PeptideRanker software. Perform molecular docking on the peptide segments with a scoring result >0.5, and select the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity according to the molecular docking results;

[0169] Step 2: The polypeptide sequence YLRLHF was synthesized by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF;

[0170] Step 3: Carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) was dissolved in water and ultrasonicated for 2 h to prepare a dispersion of 0.1 mg / mL;

[0171] Step 4: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) were added to the dispersion and stirred for 5 min;

[0172] Step 5: According to the mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2: carboxyl-functionalized iron oxide (DMSA@Fe 3 O 4 ) being 2:1, the ACE inhibitory peptide YLRLHF obtained in Step 2 was further added. After mixing evenly, the reaction was carried out on a magnetic stirrer for 4 h to obtain a reaction solution;

[0173] Step 6: The reaction solution obtained in Step 5 was centrifuged, and the precipitate was washed 3 times with ultrapure water and freeze-dried to obtain the active peptide of the high-efficiency blood pressure-lowering aquatic product (or: RVPSL-Fe powder with high ACE inhibitory activity). When the sample concentration was 0.1 mg / mL, the inhibition rate was measured to be 65.87% (peptide: DMSA@Fe 3 O 4 = 2:1).

[0174] Control:

[0175] Step 1: Coarse polypeptide was obtained by pepsin hydrolysis of bighead carp meat. After separation, purification, and identification of the coarse polypeptide, the ACE inhibitory peptide YLRLHF was obtained. The specific steps are as follows:

[0176] 1) Preparation of coarse polypeptide:

[0177] Bighead carp meat was treated with 300 MPa ultra-high pressure for 20 min. A certain amount of water (pH = 3) was added to the treated bighead carp meat, and 4000 μ / g of pepsin was added. The reaction was carried out in a water bath at 55 °C for 6 h; the reaction was terminated by heating in a water bath at 95 °C for 10 min. The hydrolysis product was centrifuged at 10000 g for 20 min, and the supernatant was collected and desalted using a dialysis bag (MD77-5M) to obtain the coarse polypeptide;

[0178] 3) Separation and purification of polypeptides:

[0179] Ultrafilter the crude polypeptide solution using an ultrafiltration cup equipped with a 5 kDa cut-off ultrafiltration membrane at a membrane pressure of 0.05 MPa to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is lyophilized and the amino acid sequence is determined by mass spectrometry. Peptides with a confidence value >99% are selected and scored for activity using the PeptideRanker software. Peptides with a score result >0.5 are subjected to molecular docking, and the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity is screened out according to the molecular docking results;

[0180] Step 2: Prepare the ACE inhibitory peptide YLRLHF with a purity of 98% by solid-phase synthesis, and detect the polypeptide purity and molecular weight by liquid phase and mass spectrometry;

[0181] Step 3: Prepare a polypeptide solution (YLRLHF) with a concentration of 0.1 mg / mL and determine the ACE inhibition rate. When the sample concentration is 0.1 mg / mL, the inhibition rate of the control is 43.36%.

Claims

1. A preparation method of an active peptide from aquatic products with high blood pressure lowering efficiency, characterized in that it includes the following steps: Step 1: Use pepsin to enzymatically hydrolyze bighead carp meat after ultra-high pressure treatment to obtain crude polypeptides. After separating, purifying, and identifying the crude polypeptides, select the peptide segment YLRLHF with the highest potential ACE activity. The specific steps are as follows: 1) Preparation of crude polypeptides: Treat bighead carp meat with ultra-high pressure of 300 MPa for 20 min to obtain bighead carp meat after ultra-high pressure treatment; according to the mass ratio of bighead carp meat after ultra-high pressure treatment: water = 1:5, add water to the bighead carp meat after ultra-high pressure treatment, add 4000 μ / g of pepsin, and react in a water bath at 55 °C for 6 h; terminate the reaction by heating in a water bath at 95 °C for 10 min, centrifuge the hydrolysis product, and collect the supernatant. Use a dialysis bag (MD77-5M) to dialyze and desalt to obtain crude polypeptides; 2) Separation and purification of polypeptides: Ultrafilter the crude polypeptides using an ultrafiltration cup equipped with a 5 kDa retention ultrafiltration membrane, with a membrane pressure of 0.05 MPa, to obtain two fractions with molecular weights <5 kDa and >5 kDa. The fraction <5 kDa is freeze-dried and the amino acid sequence is determined by mass spectrometry. Select the peptide segments with a confidence value >99% and perform activity scoring using the PeptideRanker software. Perform molecular docking on the peptide segments with a scoring result >0.5, and select the peptide segment YLRLHF (tyrosine-leucine-arginine-leucine-histidine-phenylalanine) with the highest potential ACE activity according to the molecular docking results; Step 2: Synthesize the polypeptide sequence YLRLHF by solid-phase synthesis using the PepPowerTM polypeptide synthesis platform to obtain the ACE inhibitory peptide YLRLHF; Step 3: Dissolve the nanoscale material in water and ultrasonicate for 2 h to prepare a dispersion of 0.1 mg / mL; The nanoscale materials are: one of nanoscale graphene oxide (GO), carboxyl mesoporous silica nanoparticles (CMSN), polyethylene glycol-coated gold nanoparticles (carboxyl-terminated modification) (Gold Nanoparticles-PEG-N3, GN-P), and carboxyl-functionalized magnetite (DMSA@Fe 3 O 4 ). Step 4: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) with a mass fraction 10 times that of the nanoscale material and N-hydroxysulfosuccinimide (NHS) with a mass fraction 20 times that of the nanoscale material to the dispersion and stir for 5 min; Step 5: According to the mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2: nanoscale material = 1-2:2-1, add the ACE inhibitory peptide YLRLHF obtained in Step 2 again, mix evenly, and react on a magnetic stirrer for 6 h to obtain a reaction solution; Step 6: Centrifuge the reaction solution obtained in Step 5, wash the precipitate 3 times with ultrapure water, and freeze-dry to obtain the active peptide from aquatic products with high blood pressure lowering efficiency.

2. The preparation method of an active peptide from aquatic products with high blood pressure lowering efficiency as described in claim 1, characterized in that, in the preparation of the crude polypeptides in Step 1, the centrifugation of the hydrolysis product is: centrifuge the hydrolysis product at a centrifugal force of 10000 g for 20 min.

3. The preparation method of an active peptide from aquatic products with high blood pressure lowering efficiency as described in claim 1, characterized in that, in Step 5, the optimal mass fraction of the ACE inhibitory peptide YLRLHF obtained in Step 2: nanoscale material is 1:2-2:1.