Grape seed protein peptide with ACE inhibitory activity and preparation method thereof

Abstract

The invention discloses a grape seed protein peptide with ACE inhibitory activity and a preparation method thereof, and belongs to the field of comprehensive utilization of wine fermentation byproducts. The invention provides a grape seed protein peptide with ACE (Angiotensin Converting Enzyme) inhibitory activity. The grape seed protein peptide is FFYPL, SGMFPFP or WFFPGPR. The grape seed protein peptide disclosed by the invention can be used for medicines, health-care products or dietary supplements, or can be used as a food base material to be added into common foods such as drinks, dairy products and the like; as used for prevention or auxiliary treatment of hypertensive patients or people suffering from chronic metabolic comprehensive disorder diseases with diseases such as obesity and heart and cerebral vessels, the product has a wide application prospect in the fields of functional foods, medicines, health care products and the like.

Description

Technical Field

[0001] The present invention relates to grape seed protein peptides with ACE inhibitory activity and a preparation method thereof, belonging to the field of comprehensive utilization of wine fermentation by-products. Background Art

[0002] Hypertension is one of the major global public cardiovascular diseases. Patients are usually accompanied by other metabolic disorders such as obesity, pre-diabetes, and atherosclerosis, and there are often no obvious symptoms in the early stage. Relevant research shows that the increase in blood pressure in the human body is related to angiotensin II. The renin-angiotensin system (RAS) is the key hormonal regulatory mode for regulating the synthesis of angiotensin II, which mainly occurs in the blood circulation system and organs such as the aorta, kidneys, lungs, and brain. Angiotensinogen is cleaved into angiotensin I by the renin released from prorenin, and then converted into angiotensin II under the catalysis of angiotensin-converting enzyme (ACE), thus leading to an increase in blood pressure. The main mechanism of action of the angiotensin-converting enzyme (ACE) inhibitory peptide in the present invention is to inhibit the conversion of angiotensin I to angiotensin II catalyzed by angiotensin-converting enzyme, thereby preventing the continuous increase in blood pressure in patients.

[0003] Grapes are one of the most valuable fruits recognized worldwide. 75-80% of the grapes planted worldwide are put into the wine fermentation industry, and the large amount of grape by-products generated has attracted wide attention. Therefore, in order to improve the reasonable resource utilization of wine processing waste, the present invention proposes that after the grape seeds generated by wine fermentation are pressed to extract oil, the defatted grape seeds formed are also a high-quality source for exploring ACE inhibitory active functional peptides.

[0004] At present, the development of natural source blood pressure-lowering functional peptides includes: the Chinese patent application text with publication number CN119264219A discloses two ACE inhibitory peptides extracted, separated and purified from rice wine residues, and explored their pathway mechanisms through cell model experiments; the Chinese patent application text with publication number CN117756886A discloses a preparation method of seabuckthorn leaf antihypertensive peptides and explained the action potential of the inhibitory peptides from a molecular perspective; the angiotensin-converting enzyme inhibitors sold on the market were initially isolated from snake venom, and drugs such as captopril, lisinopril, and enalapril were developed. However, patients taking them for a long time will experience some serious side effects, such as dry cough, rash, and taste disorders. Based on this, natural source ACE inhibitory peptides have become one of the main research directions in this field due to their advantages of no side effects, high safety, and easy absorption. Therefore, while promoting the research and development of wine by-products, we are committed to exploring safe and efficient plant-derived functional peptides with ACE inhibitory activity. Summary of the Invention

[0005] To solve the above existing problems and further improve the reuse method of grape seed, a by-product of wine fermentation, the present invention evaluates the potential of grape seed protein as a source of ACE (angiotensin-converting enzyme) inhibitory peptides, verifies that the grape seed protein hydrolysate has good ACE inhibitory effects, and provides a grape seed protein peptide with ACE inhibitory activity and a preparation method thereof.

[0006] The present invention provides a grape seed protein peptide with ACE inhibitory activity, and the amino acid sequence of the grape seed protein peptide is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR), as shown in SEQ ID NO.1-3;

[0007] SEQ ID NO.1: FFYPL;

[0008] SEQ ID NO.2: SGMFPFP;

[0009] SEQ ID NO.3: WFFPGPR.

[0010] In one embodiment of the present invention, the grape seed protein peptide has good ACE inhibitory activity, and the ACE inhibitory activity (Ic50 value) is less than 0.5 μg / mL.

[0011] In one embodiment of the present invention, the grape seed protein peptide is prepared from defatted grape seeds.

[0012] In one embodiment of the present invention, the grape seed protein peptide can be used as a medicine, a health product or a dietary supplement, or added to ordinary foods such as beverages and dairy products as a food base material; it is used to inhibit ACE and lower blood pressure, and is used for the prevention or adjuvant treatment of hypertensive patients or people with chronic metabolic syndrome such as obesity, immune deficiency, cardiovascular and cerebrovascular diseases.

[0013] The present invention provides a preparation method of a grape seed protein peptide with ACE inhibitory activity, and the method includes:

[0014] 1) Select defatted grape seeds from wine fermentation by-products as raw materials, grind the grape seeds into powder to obtain defatted grape seed powder; mix the obtained defatted grape seed powder with 50% ethanol at a solid-liquid ratio of 1:4 - 6, let it stand and then filter to obtain defatted and dephenolized grape seed powder; dissolve the defatted and dephenolized grape seed powder with sodium hydroxide at a solid-liquid ratio of 1:15 - 18, adjust the pH to 10 - 12, perform ultrasonic extraction while stirring, with a frequency of 30 - 50 kHz and a time of 15 - 30 min, filter, add cross-linked polyvinylpyrrolidone at a mass-volume ratio of 4 - 6% (w / v) to the obtained filtrate, mix and react for 20 - 30 min, and remove the lower solid phase to obtain grape seed protein alkaline solution;

[0015] 2) Adjust the pH of the grape seed protein alkaline solution obtained in step 1) to the isoelectric point of 3.5 - 4, centrifuge to obtain acidic precipitated protein; finally, add distilled water to redissolve the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder;

[0016] 3) Dissolve the grape seed protein powder prepared in step 2) above in a phosphate buffer system, at 40 - 45 °C, pH = 9.5 - 10.5, with a stirring speed of 100 - 150 r / min, add alkaline protease at an enzyme / protein concentration ratio of 3800 - 4200 U / g for the next enzymatic hydrolysis; after reacting for 3.5 - 4 h, terminate with a boiling water bath, add trichloroacetic acid solution to precipitate and centrifuge to remove excess protein, and freeze-dry to obtain a grape seed protein peptide mixture.

[0017] In an embodiment of the present invention, the method for preparing grape seed protein peptides includes the steps:

[0018] On the one hand, the present invention uses a method of ultrasonic-assisted alkali solution and acid precipitation to extract proteins, and adsorbs phenols in the protein solution by adding cross-linked polyvinylpyrrolidone to improve the extraction rate and extraction effect; on the other hand, alkaline protease is selected for enzymatic hydrolysis to release grape seed protein peptides with ACE inhibitory activity.

[0019] In an embodiment of the present invention, the method for preparing grape seed protein peptides includes the steps:

[0020] 1) Extract proteins from grape seed powder by ultrasonic-assisted alkali solution and acid precipitation;

[0021] 2) Enzymatically hydrolyze the grape seed protein prepared in step 1) to release protein peptides;

[0022] 3) Purify the hydrolyzate obtained in step 2) by ultrafiltration and RP-HPLC, rotary evaporate and freeze-dry to obtain it.

[0023] In order to obtain 100% grape seed protein peptides, the above grape seed extraction process preferably may include the following steps:

[0024] Using defatted grape seeds in wine fermentation by-products as raw materials, the whole grape seeds are ground into powder under the protection of liquid nitrogen, mixed with 50% absolute ethanol and left standing in the dark for 1 h, the top phase is filtered off, and defatted and dephenolized grape seed powder is obtained. The defatted grape seed powder is dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, its pH is adjusted to 11 with 6 M sodium hydroxide, ultrasonic extraction is carried out while stirring, the lower solid phase is filtered off, then pvpp (cross-linked polyvinylpyrrolidone) is added to the obtained filtrate, and the mixture is reacted for 30 min, and the lower solid phase is removed to obtain grape seed protein alkaline solution; then the pH of the obtained alkaline solution is adjusted to the isoelectric point of 3.5 with hydrochloric acid, refrigerated for 2 h, centrifuged to obtain an acidic precipitate, and freeze-dried to obtain grape seed protein powder.

[0025] In a preferred embodiment of the present invention, the solid-liquid ratio of the mixture of grape seed powder and 50% absolute ethanol is 1:5.

[0026] In a preferred embodiment of the present invention, the ultrasonic wave for assisted extraction is 40 Hz, the power is 200 w, and the reaction time is 40 min. In the embodiment of the present invention, the use of ultrasonic wave technology with a specific frequency for extraction can significantly improve the extraction efficiency and reduce the time cost.

[0027] In a preferred embodiment of the present invention, the amount of cross-linked polyvinylpyrrolidone for adsorbing polyphenols is 4% (w / w), which can significantly reduce the polyphenol content in grape seed protein. Due to the significant reduction of anthocyanins, the color of the obtained protein powder is also purer.

[0028] In a preferred embodiment of the present invention, the specific steps of enzymatic hydrolysis in step 2) are as follows:

[0029] The grape seed protein powder prepared in step 1) is dissolved in a 0.01 M phosphate buffer (PBS) system, adjusted to the optimal reaction conditions of temperature 45 °C and pH = 10.0, and alkaline protease is added at an enzyme / protein concentration ratio of 4000 U / g for the next enzymatic hydrolysis; the pH and temperature of the whole reaction system are maintained stable during the process, the reaction is terminated by boiling water bath after the reaction, a 15% trichloroacetic acid solution is added for precipitation and centrifuged to remove excess protein, a 15% trichloroacetic acid solution is added for precipitation and centrifuged to remove excess protein, and freeze-dried to obtain a concentrated grape seed protein hydrolysate.

[0030] In a preferred embodiment of the present invention, it has been verified that alkaline protease is the enzyme with the most ACE inhibitory potential, and the optimal enzymatic hydrolysis time is 4 h. Under this enzymatic hydrolysis method, the obtained grape seed protein hydrolysate has the highest degree of hydrolysis and the strongest ACE inhibitory potential.

[0031] In order to make the inhibitory effect of the obtained grape seed protein solution the best, the ultrafiltration operation in step 3) is specifically as follows:

[0032] The grape seed protein hydrolysate concentrate obtained in step 2) was purified using a Labscale TFF ultrafiltration system with a molecular weight cut-off of 5 kDa to obtain grape seed protein hydrolysate with a molecular weight of <5 kDa. It was then purified again using an ultrafiltration centrifuge tube with a molecular weight cut-off of 3 kDa to obtain grape seed protein hydrolysates with molecular weights of 3-5 kDa and <3 kDa.

[0033] In a preferred embodiment of the present invention, the reverse-phase HPLC in step 3) can be as follows:

[0034] The grape seed protein hydrolysate with a molecular weight of <3 kDa was separated by RP-HPLC (reverse-phase high-performance liquid chromatography). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, mobile phase B was HPLC-grade acetonitrile, the detection wavelength was 214 nm, the elution peak at 9-12 min was collected, and the solvent was removed by rotary evaporation to obtain the crude grape seed protein peptide.

[0035] In a preferred embodiment of the present invention, the grape seed protein peptides with specific ACE inhibitory function prepared by the above method include polypeptides with the following amino acid sequences: Phe-Phe-Tyr-Pro-Leu;

[0036] In a preferred embodiment of the present invention, the grape seed protein peptides with ACE inhibitory function prepared by the above method include polypeptides with the following amino acid sequences: Ser-Gly-Met-Phe-Pro-Phe-Pro;

[0037] In a preferred embodiment of the present invention, the grape seed protein peptides with ACE inhibitory function prepared by the above method include polypeptides with the following amino acid sequences: Trp-Phe-Phe-Pro-Gly-Pro-Arg;

[0038] The grape seed protein peptides containing any one of the above three polypeptides have good ACE inhibitory activity, and the ACE inhibitory activity (IC50 value) is less than 0.5 μg / mL.

[0039] The present invention also provides an expression vector or a recombinant microorganism, which contains at least one of the above grape seed protein peptides.

[0040] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0041] In one embodiment of the present invention, the recombinant microorganism is a bacterium or a fungus.

[0042] The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises any one or more of the above grape seed protein peptides: Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR);

[0043] The above grape seed protein peptides can significantly inhibit ACE activity.

[0044] In one embodiment of the present invention, the excipient refers to a conventional pharmaceutical carrier in the pharmaceutical field.

[0045] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose, etc.; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch, etc.; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid esters, and fatty acid glycerides, etc.; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc powder, and polyethylene glycol, etc.; coating materials such as acrylic resin, hydroxypropyl methylcellulose, polyvinylpyrrolidone, cellulose acetate phthalate, etc.; additionally, other adjuvants such as flavoring agents, sweetening agents, etc. can also be added to the composition.

[0046] The present invention also provides a food, medicine, health product, or nutritional product, which contains at least one of the above grape seed protein peptides in an effective dose.

[0047] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0048] In one embodiment of the present invention, the medicine also contains a pharmaceutically acceptable excipient; the excipient refers to a conventional pharmaceutical carrier in the pharmaceutical field.

[0049] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, crospovidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbates, sorbitan fatty acids, and glycerol fatty acid esters; coloring agents such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resins, hypromellose, povidone, and cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition.

[0050] In one embodiment of the present invention, the dosage forms of the drug include, but are not limited to, oral dosage forms, injection dosage forms, and inhalation dosage forms.

[0051] In one embodiment of the present invention, the oral dosage forms include, but are not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0052] In one embodiment of the present invention, the injection dosage forms include, but are not limited to, injection solutions and injection powder for injection.

[0053] In one embodiment of the present invention, the inhalation dosage forms include, but are not limited to, aerosols and powder inhalations.

[0054] In one embodiment of the present invention, the foods include, but are not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, beverages; the foods also include special dietary foods.

[0055] In one embodiment of the present invention, the health products also contain acceptable excipients.

[0056] The present invention also provides the use of at least one or more of the above-mentioned grape seed protein peptides in the preparation of foods, drugs, health products, or nutritional products; it is used to inhibit ACE activity, lower blood pressure, prevent or treat hypertension or diseases benefited from ACE inhibition, or prevent or treat chronic metabolic syndrome with conditions such as obesity, immune deficiency, or cardiovascular and cerebrovascular diseases.

[0057] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0058] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0059] In one embodiment of the present invention, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbates, sorbitan fatty acids, and glycerol fatty esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resins, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition.

[0060] In one embodiment of the present invention, the dosage forms of the drug include, but are not limited to, oral dosage forms, injection dosage forms, and inhalation dosage forms.

[0061] In one embodiment of the present invention, the oral dosage forms include, but are not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

[0062] In one embodiment of the present invention, the injection dosage forms include, but are not limited to, injection solutions and injection powder injections.

[0063] In one embodiment of the present invention, the inhalation dosage forms include, but are not limited to, aerosols and powder aerosols.

[0064] In one embodiment of the present invention, the foods include, but are not limited to, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, beverages; the foods also include special dietary foods.

[0065] In one embodiment of the present invention, the health products also contain acceptable excipients.

[0066] The present invention also provides a method for preparing the above-mentioned foods, drugs, health products, or nutritional products, the method comprising mixing at least one of the above-mentioned grape seed protein peptides with at least one acceptable excipient.

[0067] In one embodiment of the present invention, the amino acid sequence of the grape seed protein peptide is Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0068] Beneficial effects

[0069] (1) The present invention obtains grape seed protein peptides with ACE inhibitory activity. The specific sequences are Phe-Phe-Tyr-Pro-Leu or Ser-Gly-Met-Phe-Pro-Phe-Pro or Trp-Phe-Phe-Pro-Gly-Pro-Arg. They have novel structures and are easy to prepare. They can be obtained from grape seed protein or synthesized artificially.

[0070] (2) The three polypeptides obtained in the present invention have strong ACE inhibitory activity. Among them, the half-inhibitory concentration (IC 50 value) of Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR) is 25.27 ± 0.7393 nmol / L; the half-inhibitory concentration (IC 50 value) of Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) is 66.39 ± 26.13 nmol / L; the half-inhibitory concentration (IC 50 value) of Phe-Phe-Tyr-Pro-Leu (FFYPL) is 81.71 ± 5.847 nmol / L.

[0071] (3) The grape seed protein peptides of the present invention can be used in pharmaceuticals, health products or dietary supplements, or added as food ingredients to common foods such as beverages and dairy products. They have broad application prospects in the fields of functional foods, medicine, health products, etc. for the prevention or adjuvant treatment of hypertension patients or people with chronic metabolic syndrome disorders such as obesity, cardiovascular and cerebrovascular diseases. Description of the drawings

[0072] Figure 1 It is a graph of peptide concentration results of grape seed protein under different hydrolases and different hydrolysis times.

[0073] Figure 2 It is a graph of ACE inhibition rate results of grape seed protein under different hydrolases and different hydrolysis times. Detailed implementation manners

[0074] The following combines examples to further describe in detail the specific implementation manners of the present invention. The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0075] Unless otherwise specified, the technical means used in the examples are conventional technical means well-known to those skilled in the art. Unless otherwise specified, the reagents used in the examples are commercially available.

[0076] The defatted grape seed powder involved in the following examples can be prepared with reference to the following method: Mix grape seed powder with n-hexane at a solid-liquid ratio of 1:5, let it stand and then filter to obtain defatted grape seed powder;

[0077] Alternatively, defatted grape seeds in wine fermentation by-products can be directly selected as raw materials.

[0078] The sources of raw materials involved in the following examples are as follows:

[0079] PPVP (crosslinked polyvinylpyrrolidone) was purchased from Shanghai Merck Biochemical Technology Co., Ltd., with the product number M86828-100G; Folin-Ciocalteu reagent was purchased from Shanghai Titan Technology Co., Ltd., with the product number 4140369A;

[0080] Alkaline protease was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 200 U / mg and the product number S10154-100g;

[0081] Papain was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 800 U / mg and the product number S10011-100g;

[0082] Pepsin was purchased from Merck Life Science China, with an enzyme activity of ≥500 U / mg and the product number 77160-25g;

[0083] Bromelain was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 300 U / mg and the product number S10009-100g;

[0084] Neutral protease was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with an enzyme activity of 100 U / mg and the product number S10013-250g;

[0085] BCA Protein Assay Kit (Enhanced) was purchased from Beyotime Biotechnology Co., Ltd., with the product number: P0010S;

[0086] Angiotensin-converting enzyme (ACE) inhibitor activity detection kit was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., with the product number: A502.

[0087] The measurement methods involved in the following examples are as follows:

[0088] ACE (angiotensin-converting enzyme) inhibition activity measurement method:

[0089] The method is based on the Angiotensin Converting Enzyme Inhibitor Activity Detection Kit (ACE Kit-WST, product number: A502) of Dongren Chemical Technology (Shanghai) Co., Ltd. Using 3-hydroxybutyryl-glycyl-glycyl-glycine (3HB-GGG) as a substrate to screen for ACE inhibitors. Under the action of ACE and aminacylase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The ACE inhibition rate is calculated by detecting the absorbance of 3HB. The kit includes ACE enzyme working solution, substrate diluent, and chromogenic solution. The activity is determined using a 96-well plate.

[0090] Samples: Add 20 μl of sample, 20 μl of substrate, and 20 μl of enzyme working solution to the wells in sequence;

[0091] Negative control: Add 20 μl of pure water, 20 μl of substrate, and 20 μl of enzyme working solution to the wells in sequence;

[0092] Background blank: Add 40 μl of pure water and 20 μl of substrate to the wells in sequence;

[0093] Sample blank: Add 20 μl of sample and 240 μl of pure water to the wells in sequence;

[0094] Place the above wells in a constant temperature incubator at 37 °C and let them stand for 60 min. Add 200 μl of chromogenic solution to each well of the sample, negative control, and background blank respectively, and incubate at room temperature for 10 min. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay reader.

[0095] Ic 50 Value, the half-inhibitory concentration, that is, the sample concentration when the ACE inhibition rate is 50%. Dilute the sample to be tested into 5 concentration gradients spanning 50% according to the inhibition rate data of the preliminary experiment, measure the ACE inhibition rate of each gradient concentration, and import the data into GraghPad for non-linear fitting curve to obtain the Ic 50 Value.

[0096] Calculation formula:

[0097] ACE inhibition rate (%) = A 阴性对照 -(A 样品 -A 样品空白 ) / A 阴性对照 -A 背景空白

[0098] BCA protein concentration determination method:

[0099] The method is based on the instruction manual of Beyotime Biotechnology BCA Protein Concentration Assay Kit (Enhanced) (Catalog No.: P0010S). The kit contains BCA working solution, protein standard (20 mg BSA), and protein standard preparation solution. The activity assay is carried out using a 96-well plate.

[0100] Dissolve the protein standard with the protein standard preparation solution. After complete dissolution, prepare a protein standard solution with a concentration of 0.5 mg / ml, and then dilute it with distilled water to 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 mg / ml. In a 96-well plate, add 20 μl of the protein standard and the sample solution, and then add 200 μl of BCA working solution to each well. Incubate at 37 °C for 25 min, measure the absorbance at A562 using a microplate reader, and prepare a protein standard curve. Calculate the protein concentration of the sample according to the standard curve.

[0101] In the following examples, (w / v) refers to g / ml.

[0102] Example 1: Extraction process of grape seed protein

[0103] 1. Preparation of grape seed protein

[0104] 1) Select defatted grape seeds of Cabernet Sauvignon from wine fermentation by-products as raw materials, and grind the whole grape seeds into powder under liquid nitrogen protection; obtain defatted grape seed powder.

[0105] Mix the prepared defatted grape seed powder with 50% (v / v) ethanol aqueous solution at a solid-liquid ratio of 1:5 (w / v) and let it stand in the dark for 1 h. Filter to remove the top phase, and repeat the above operation twice for the residue to obtain defatted and dephenolized grape seed powder.

[0106] 2) Dissolve the defatted and dephenolized grape seed powder obtained in step 1) with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16 (w / v), adjust its pH to 11 with 6 M sodium hydroxide, extract with stirring assisted by ultrasound (frequency 40 kHz) for 40 min, and filter to remove the lower solid phase. Then add pvpp (crosslinked polyvinylpyrrolidone) to the obtained filtrate at a solid-liquid ratio of 4% (w / v), mix and react for 30 min, and remove the lower solid phase to obtain grape seed protein alkaline solution.

[0107] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point of 3.5 with 6 M hydrochloric acid, refrigerate at 4 °C for 2 h, centrifuge at 12000 rpm to obtain acidic precipitated protein; finally, add distilled water to redissolve the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge at 12000 rpm to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0108] 2. Optimization of grape seed protein extraction process

[0109] To verify the effects of ethanol extraction pretreatment and polyvinylpyrrolidone (PVPP) impurity removal on the preparation efficiency of grape seed protein, and select Cabernet Sauvignon defatted grape seeds from the same four groups of wine fermentation by-products. Under liquid nitrogen protection, the whole grape seeds were ground into powder to obtain defatted grape seed powder (the preparation method is the same as 1) in step 1). Four control experiments were designed in this invention (Table 1):

[0110] Table 1: Four control experiments

[0111]

[0112] The specific method is as follows:

[0113] 1) Preparation of #1 grape seed protein:

[0114] The obtained defatted grape seed powder was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH was adjusted to 11 with 6 M sodium hydroxide. It was extracted for 40 min with stirring assisted by ultrasound (frequency 40 kHz), and the lower solid phase was removed by filtration. Then, 4% (w / w) PVPP (crosslinked polyvinylpyrrolidone) was added to the obtained filtrate, and the mixture was reacted for 30 min. The lower solid phase was removed to obtain grape seed protein alkaline solution, which was freeze-dried to obtain #1 grape seed protein (GSP#1).

[0115] 2) Preparation of #2 grape seed protein:

[0116] The obtained defatted grape seed powder was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH was adjusted to 11 with 6 M sodium hydroxide. It was extracted for 40 min with stirring assisted by ultrasound (frequency 40 kHz), and the lower solid phase was removed by filtration to obtain grape seed protein alkaline solution, which was freeze-dried to obtain #2 grape seed protein (GSP#2).

[0117] 3) Preparation of #3 grape seed protein:

[0118] The obtained defatted grape seed powder was mixed with 50% anhydrous ethanol at a solid-liquid ratio of 1:5 and allowed to stand in the dark for 1 h. The top phase was removed by filtration, and the residue was repeated the above operation twice to obtain defatted and dephenolized grape seed powder. Then, it was dissolved in 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH was adjusted to 11 with 6 M sodium hydroxide. It was extracted for 40 min with stirring assisted by ultrasound (frequency 40 kHz), and the lower solid phase was removed by filtration. Then, 4% (w / w) PVPP (crosslinked polyvinylpyrrolidone) was added to the obtained filtrate, and the mixture was reacted for 30 min. The lower solid phase was removed to obtain grape seed protein alkaline solution, which was freeze-dried to obtain #3 grape seed protein (GSP#3).

[0119] 4) Preparation of #4 grape seed protein:

[0120] The obtained defatted grape seed powder was mixed with 50% absolute ethanol at a solid-liquid ratio of 1:5 and left to stand in the dark for 1 h. The top phase was removed by filtration, and the above operation was repeated twice for the residue to obtain defatted and dephenolized grape seed powder. Then it was dissolved with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, and its pH was adjusted to 11 with 6 M sodium hydroxide. It was extracted with ultrasonic assistance (frequency 40 kHz) while stirring for 40 min, and the lower solid phase was removed by filtration to obtain grape seed protein alkaline solution, which was freeze-dried to obtain #4 grape seed protein (GSP#4).

[0121] The protein content was determined using the Kjeldahl method in GB 5009.5-2016 Determination of Protein in Foods:

[0122] Weighed a well-mixed solid grape seed protein powder sample into a digestion tube, and then added 0.4 g of copper sulfate, 6 g of potassium sulfate and 20 ml of sulfuric acid to the digestion furnace for digestion. When the temperature of the digestion furnace reached 420 °C, digestion continued for 1 h. At this time, the liquid in the digestion tube was green and transparent. After taking it out and cooling, 50 ml of water was added, and automatic liquid addition, distillation, titration and recording of titration data were realized on an automatic Kjeldahl apparatus. According to the consumption of acid multiplied by the conversion factor 6.25, it was the protein content.

[0123] The polyphenol content was determined using the Folin-phenol method:

[0124] First, a standard curve was established with the standard phenolic substance catechin. Take 0.1 mL of a sample with a concentration of 1 mg / ml and add it to a 10 mL volumetric flask, add 7 mL of distilled water and shake well, then add 0.5 mL of 1 M Folin-phenol. After 1 min, add 1.5 mL of 20% (v / v) sodium carbonate solution, mix well, and make up the volume to 10 mL with distilled water. React in the dark for 120 min, measure the absorbance at 765 nm, and the result was expressed as the equivalent value of the catechin standard curve.

[0125] 5) Detection of experimental results

[0126] The protein content and polyphenol content of the defatted grape seed powder and the obtained #1 grape seed protein, #2 grape seed protein, #3 grape seed protein, and #4 grape seed protein were determined, and the determination results are shown in Table 2.

[0127] Table 2: Grape seed protein content under different extraction methods

[0128]

[0129] It can be seen from Table 2 that the protein content of #3 grape seed protein (GSP#3) is the highest and the total phenol content is the lowest. Basically, it can be seen that the effect of removing impurities is good, the operation is simple, and the cost is low. It is an effective method for extracting grape seed protein.

[0130] Example 2: Preparation of grape seed protein hydrolysate concentrate

[0131] The preparation method of grape seed protein peptides with ACE inhibition is as follows:

[0132] 1. Preparation of grape seed protein

[0133] 1) Select Cabernet Sauvignon defatted grape seeds in the by-products of wine fermentation as raw materials, and grind the whole grape seeds into powder under liquid nitrogen protection to obtain defatted grape seed powder.

[0134] 2) Mix the obtained defatted grape seed powder with 50% absolute ethanol at a solid-liquid ratio of 1:5 and let it stand in the dark for 1 h. Filter to remove the top phase, and repeat the above operation twice for the residue to obtain defatted and dephenolized grape seed powder. Then dissolve it with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, adjust its pH to 11 with 6 M sodium hydroxide, extract with ultrasonic (frequency 40 kHz) for 40 min while stirring, and filter to remove the lower solid phase. Then add 4% (w / w) pvpp (cross-linked polyvinylpyrrolidone) to the obtained filtrate, mix and react for 30 min, and remove the lower solid phase to obtain grape seed protein alkaline solution;

[0135] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point of 3.5 with 6 M hydrochloric acid, refrigerate at 4 °C for 2 h, and centrifuge at 12000 rpm to obtain acidic precipitated protein; finally, add distilled water to redissolve the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge at 12000 rpm to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0136] 2. Preparation of grape seed protein hydrolysate concentrate with ACE inhibition

[0137] Dissolve the grape seed protein powder prepared in step 1 above in a 0.01 M phosphate buffer (PBS) system, adjust the magnetic stirrer connected to the pH electrode to the optimal reaction conditions of temperature 45 °C, pH = 10.0, and stirring speed 40 rpm, add alkaline protease at an enzyme / protein concentration ratio = 4000 U / g for the next enzymatic hydrolysis; maintain the pH and temperature of the whole reaction system stable during the process, terminate the reaction by boiling water bath after 4 h, add 15% (v / v) of 1 M trichloroacetic acid solution to precipitate and centrifuge at 12000 rpm to remove excess protein, and freeze-dry to obtain grape seed protein hydrolysate concentrate.

[0138] Example 3: Optimization of the preparation method of grape seed protein hydrolysate concentrate

[0139] The specific implementation method is the same as that of Example 2, except that in step 2, (1), the reaction time of 4 h is replaced with 0.5, 1, 2, 3, 4, 5 or 6 h, and the alkaline protease in step 2, (2) is replaced with papain, pepsin, neutral protease or bromelain (Table 3). The remaining steps are the same as those in Example 2 to obtain the grape seed protein hydrolysate concentrate. For the grape seed protein hydrolysate concentrates prepared by hydrolysis with alkaline protease, papain, pepsin, neutral protease or bromelain for different reaction times, the ACE inhibition rate and polypeptide concentration were measured using the above ACE inhibition activity measurement method and protein concentration measurement method.

[0140] The results are as Figures 1-2 shown in Table 3.

[0141] Table 3: ACE inhibition Ic of different proteases for grape seed protein at different hydrolysis times 50 Value

[0142]

[0143]

[0144] It was found that the hydrolysate by alkaline protease had the strongest biological activity and the highest polypeptide content at 3 h.

[0145] The ACE inhibition Ic values of the grape seed protein hydrolysate concentrates prepared by hydrolysis with alkaline protease, papain, pepsin, neutral protease or bromelain for 3 h were detected respectively, and the results are shown in Table 4: 50 Value

[0146] Table 4: ACE inhibition Ic values of hydrolysates of different proteases for grape seed protein hydrolyzed for 3 h 50 Value

[0147]

[0148] Among them, it was preliminarily verified that the hydrolysate by alkaline protease had the most significant inhibitory effect on ACE enzyme, and the Ic 50 was 19.82 ± 1.256 μg soluble protein hydrolysate / mL (pepsin Ic 50 = 21.98 ± 0.018 μg soluble protein hydrolysate / mL, neutral protease Ic 50 = 35.18 ± 1.461 μg soluble protein hydrolysate / mL, bromelain Ic 50 = 24.58 ± 4.163 μg soluble protein hydrolysate / mL, papain Ic 50 = 104.5 ± 12.47 μg soluble protein hydrolysate / mL).

[0149] Example 4: Preparation of Grape Seed Protein Peptide with ACE Inhibition

[0150] The preparation method of grape seed protein peptide with ACE inhibition is as follows:

[0151] 1. Preparation of grape seed protein

[0152] 1) Select Cabernet Sauvignon defatted grape seeds in wine fermentation by-products as raw materials, and grind the whole grape seeds into powder under liquid nitrogen protection to obtain defatted grape seed powder.

[0153] 2) Mix the obtained defatted grape seed powder with 50% absolute ethanol at a solid-liquid ratio of 1:5 and let it stand in the dark for 1 h, filter to remove the top phase, and repeat the above operation twice for the residue to obtain defatted and dephenolized grape seed powder. Then dissolve it with 0.1 M sodium hydroxide at a solid-liquid ratio of 1:16, adjust its pH to 11 with 6 M sodium hydroxide, extract with ultrasonic (frequency 40 kHz) for 40 min while stirring, and filter to remove the lower solid phase. Then add 4% (w / w) pvpp (cross-linked polyvinylpyrrolidone) to the obtained filtrate, mix and react for 30 min, and remove the lower solid phase to obtain grape seed protein alkaline solution;

[0154] 3) Adjust the pH of the grape seed protein alkaline solution obtained in step 2) to the isoelectric point of 3.5 with 6 M hydrochloric acid, refrigerate at 4 °C for 2 h, centrifuge at 12000 rpm to obtain acidic precipitated protein; finally, add distilled water to redissolve the obtained acidic precipitated protein, adjust its pH to neutral, centrifuge at 12000 rpm to obtain grape seed protein precipitate, and freeze-dry to obtain grape seed protein powder.

[0155] 2. Preparation of grape seed protein peptide with ACE inhibition

[0156] 1) Dissolve the grape seed protein powder prepared in step 1 above in a 0.01 M phosphate buffer (PBS) system, adjust the magnetic stirrer connected to the pH electrode to the optimal reaction conditions of temperature 45 °C, pH = 10.0, and stirring speed 40 rpm, and add alkaline protease at an enzyme / protein concentration ratio = 4000 U / g for the next enzymatic hydrolysis; maintain the pH and temperature of the whole reaction system stable during the process, terminate the reaction by boiling water bath after 3 h, add 1 M trichloroacetic acid solution with a final concentration of 15% (v / v) to precipitate and centrifuge at 12000 rpm to remove excess protein, and freeze-dry to obtain grape seed protein hydrolysate concentrate.

[0157] 2) Ultrafiltration separation is carried out on the grape seed protein hydrolysate concentrate obtained in step 1), and a Labscale TFF ultrafiltration system with a molecular weight cut-off of 5 kDa is used for purification to obtain grape seed protein hydrolysate with <5 kDa, and then a ultrafiltration centrifugal tube with a molecular weight cut-off of 3 kDa is used again for purification to obtain grape seed protein hydrolysate with 3 - 5 kDa and <3 kDa.

[0158] 3) Perform RP-HPLC reverse-phase high performance liquid chromatography separation on the <3 kDa grape seed protein hydrolysate obtained in step 2). Mobile phase A is 0.1% (v / v) trifluoroacetic acid aqueous solution, mobile phase B is HPLC-grade acetonitrile, the detection wavelength is 214 nm. From 0 to 5 min, 2% B; from 5 to 9 min, 2% - 7% B; from 9 to 28 min, 7% - 30% B; from 28 to 40 min, 30% - 50% B; from 40 - 41 min, 50% - 100% B; from 41 to 45 min, 100% B; from 45 to 46 min, 100% - 2% B; from 46 to 60 min, 2% B. The flow rate is set at 2 mL / min; collect the elution peak from 9 - 12 min, and rotary evaporate to remove the solvent to obtain the crude grape seed protein peptide.

[0159] 4) Perform mass spectrometry identification on the crude grape seed protein peptide obtained in step 3), and determine the components of the grape seed protein peptide by LC-MS / MS.

[0160] The conditions of LC-MS / MS are as follows:

[0161] The model of the high performance liquid chromatograph is Dionex U3000; the chromatographic column is C18, 3μm, 75μm x 15 cm; mobile phase A: 0.1% Formic acid in water; mobile phase B: 0.1% Formic acid in Acetonitrile; the flow rate is 600 nL / min. From 0 to 8 min, 8% B; from 8 to 16 min, 13% B; from 16 to 39 min, 28% B; from 39 to 50 min, 40% B; from 50 to 51 min, 95% B; from 51 to 55 min, 95% B; from 55 to 56 min, 6% B; from 56 to 60 min, 6% B.

[0162] The model of the mass spectrometer is Thermo Scientific Q Exactive; resolution settings: 70,000@m / z 200 for the first stage, 17,500@m / z 200 for the second stage; parent ion scanning range: m / z 300 - 1400; daughter ion scanning range: m / z 100; MS1 AGC: 3e6, ion injection time: 60 ms; MS2 AGC: 5e4, ion injection time: 80 ms; ion screening window: 3.0 m / z; fragmentation mode: HCD, energy NCE 27; Data-dependent MS / MS: Top 20; dynamic exclusion time: 15 s.

[0163] 5) Screening of grape seed protein peptides with ACE inhibition

[0164] Through computer simulation and in vitro determination experiments, a total of 5 grape seed protein peptides with ACE inhibitory effects were screened (Table 5).

[0165] The determination results of the grape seed protein peptide components were as follows: The amino acid sequences of the three peptides with the highest biological activities in the grape seed protein peptide were Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0166] Example 5: In vitro determination experiment on the ACE inhibitory ability of grape seed protein peptides

[0167] The samples were ACE inhibitory peptides obtained through hydrolysis, ultrafiltration, reverse-phase HPLC, and mass spectrometry identification in Example 4, and their amino acid sequences were Phe-Phe-Tyr-Pro-Leu (FFYPL) or Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP) or Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR).

[0168] Phe-Phe-Tyr-Pro-Leu (FFYPL), Ser-Gly-Met-Phe-Pro-Phe-Pro (SGMFPFP), Trp-Phe-Phe-Pro-Gly-Pro-Arg (WFFPGPR), and the other two screened polypeptides (Table 5) were synthesized by Shanghai Gil Biochemical Co., Ltd., with a purity ≥ 98%.

[0169] Detection of the Ic 50 value of the three peptide segments of FFYPL and SGMFPFP or WFFPGPR.

[0170] Other screened grape seed protein peptides with ACE inhibitory effects (Table 5) were synthesized according to the above method.

[0171] Ic 50 The value determination method is as follows:

[0172] Sample concentration preparation: Dissolve 2 mg of the polypeptide sample (FFYPL) in 100 μl of distilled water to obtain a peptide (FFYPL) of 20 mg / ml, and then dilute it to obtain peptides (FFYPL) of 1 mg / ml, 0.5 mg / ml, 0.1 mg / ml, and 0.01 mg / ml.

[0173] Dissolve 1 mg of the polypeptide sample (SGMFPFP) in 100 μl of distilled water to obtain a peptide (SGMFPFP) at a concentration of 10 mg / ml, and then dilute it to obtain peptides (SGMFPFP) at concentrations of 1 mg / ml, 0.5 mg / ml, 0.1 mg / ml, and 0.01 mg / ml.

[0174] Dissolve 1 mg of the polypeptide sample (WFFPGPR) in 1 ml of distilled water to obtain a peptide (WFFPGPR) at a concentration of 1 mg / ml, and then dilute it to obtain peptides (WFFPGPR) at concentrations of 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml, and 0.01 mg / ml.

[0175] The method is based on the angiotensin-converting enzyme inhibitor activity detection kit (ACE Kit-WST, product number: A502) of Dongren Chemical Technology (Shanghai) Co., Ltd. Using 3-hydroxybutyryl-glycyl-glycyl-glycine (3HB-GGG) as a substrate to screen for ACE inhibitors, under the action of ACE and aminopeptidase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The ACE inhibition rate is calculated by detecting the absorbance of 3HB. The kit includes an ACE enzyme working solution, a substrate dilution solution, and a chromogenic solution. A 96-well plate is used for activity determination.

[0176] Samples: Add 20 μl of the sample, 20 μl of the substrate, and 20 μl of the enzyme working solution to the wells in sequence.

[0177] Negative control: Add 20 μl of pure water, 20 μl of the substrate, and 20 μl of the enzyme working solution to the wells in sequence.

[0178] Background blank: Add 40 μl of pure water and 20 μl of the substrate to the wells in sequence.

[0179] Sample blank: Add 20 μl of the sample and 240 μl of pure water to the wells in sequence.

[0180] Place the above wells in a constant temperature incubator at 37 °C and let them stand for 60 min. Add 200 μl of the chromogenic solution to each well of the sample, negative control, and background blank, and incubate at room temperature for 10 min. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay reader.

[0181] Ic 50The value of half inhibitory concentration, that is, the sample concentration when the ACE inhibition rate is 50%. Dilute the sample to be tested into 5 concentration gradients spanning 50% according to the inhibition rate data of the preliminary experiment, measure the ACE inhibition rate of each gradient concentration, and import the data into GraghPad non-linear fitting curve to obtain the Ic 50 value.

[0182] Calculation formula:

[0183] ACE inhibition rate (%) = A 阴性对照 -(A 样品 -A 样品空白 ) / A 阴性对照 -A 背景空白

[0184] The detection method of the Ic 50 value of ACE inhibition of the other two ACE inhibitory peptides is the same as above.

[0185] The results are as follows:

[0186] Table 5: ACE inhibition ability of grape seed protein peptides

[0187]

[0188] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A grape seed protein peptide, characterized in that: The grape seed protein peptide is FFYPL peptide, SGMFPFP peptide or WFFPGPR peptide, and the amino acid sequences thereof are shown in SEQ ID NOs. 1 to 3, respectively.

2. An expression vector or a recombinant microorganism, characterized in that: The grape seed protein peptide according to claim 1 is expressed in the expression vector or recombinant microorganism; Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector; Preferably, the recombinant microorganism is a bacterium or a fungus.

3. A food, medicine, health product or nutritional product, characterized in that: The food, medicine, health product or nutritional product contains an effective dose of the grape seed protein peptide according to claim 1; preferably, the food, medicine, health product or nutritional product may also contain a derivative of the grape seed protein peptide according to claim 1; the derivative of the grape seed protein peptide refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino end or carbonyl end of the grape seed protein peptide; Preferably, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition; Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

4. The food, medicine, health product or nutritional product according to claim 3, characterized in that: The food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods; The health care product also contains acceptable auxiliary materials.

5. A method for preparing a grape seed protein peptide having ACE inhibitory activity, characterized in that: The method comprises: 1) Defatted grape seeds from wine fermentation byproducts are selected as raw materials, and the grape seeds are ground into powder to obtain defatted grape seed powder; the defatted grape seed powder is mixed with a 50% ethanol aqueous solution at a solid-liquid ratio of 1:4-6, and filtered after standing to obtain defatted and dephenolized grape seed powder; the defatted and dephenolized grape seed powder is dissolved in sodium hydroxide at a solid-liquid ratio of 1:15-18, the pH is adjusted to 10-12, and ultrasonic extraction is performed while stirring at a frequency of 30-50kHz for 15-30 minutes, and the extraction is filtered, and cross-linked polyvinyl pyrrolidone is added to the obtained filtrate at a mass volume ratio of 4-6% (w / v), mixed and reacted for 20-30 minutes, and the lower solid phase is removed to obtain grape seed protein alkali solution; 2) adjusting the pH of the grape seed protein alkali solution obtained in step 1) to an isoelectric point of 3.5 to 4, and centrifuging to obtain an acidic precipitated protein; finally, adding distilled water to redissolve the acidic precipitated protein, and adjusting the pH to neutral, centrifuging to obtain a grape seed protein precipitate, and freeze-drying to obtain a grape seed protein powder; 3) The grape seed protein powder prepared in the above step 2) is dissolved in a phosphate buffer system, and alkaline protease is added at 40-45° C., pH 9.5-10.5, stirring speed 100-150 r / min, with an enzyme / protein concentration ratio of 3800-4200 U / g for the next enzymatic hydrolysis; after 3.5-4 hours of reaction, the mixture is terminated in a boiling water bath, trichloroacetic acid solution is added for precipitation, and excess protein is removed by centrifugation, and the mixture is freeze-dried to obtain a grape seed protein peptide mixture.

6. Use of FFYPL peptide, SGMFPFP peptide or WFFPGPR peptide in the preparation of ACE inhibitors, or blood pressure lowering drugs or blood pressure lowering health products, or drugs for treating hypertension.

7. The use according to claim 6, characterized in that: The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

8. The use according to claim 7, characterized in that: The dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays; The health care product also contains acceptable auxiliary materials.

9. An ACE inhibitor, or a blood pressure lowering drug or a health product, or a drug for treating hypertension, characterized in that: The active ingredient is the grape seed protein peptide FFYPL peptide, SGMFPFP peptide or WFFPGPR peptide as claimed in claim 1.

10. A method for preparing the food, medicine, health product or nutritional product according to claim 3 or 4, characterized in that: The method comprises mixing the grape seed protein peptide or the grape seed protein peptide derivative described in claim 1 with at least one acceptable excipient; preferably, the grape seed protein peptide derivative refers to a polypeptide derivative obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification on the amino acid side chain group, amino terminal or carbonyl terminal of the polypeptide.

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