A soybean antihypertensive peptide and its screening method and application

Abstract

The present application provides a soybean antihypertensive peptide and its screening method and application, which belongs to the field of polypeptides. The soybean antihypertensive peptide provided in the present application includes one or more of soybean antihypertensive peptide I with a molecular weight of less than 1000Da, soybean antihypertensive peptide II with a molecular weight of less than 1000Da, and soybean antihypertensive peptide III with a molecular weight of less than 1000Da; wherein, the soybean antihypertensive peptide I with a molecular weight of less than 1000Da is prepared by nattokinase enzymatic hydrolysis of soybean protein isolate, the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by Bacillus subtilis liquid fermentation of soybean protein hydrolysate, and the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by Bacillus subtilis solid fermentation of soybeans. The soybean antihypertensive peptide of the present application is a plant-derived antihypertensive peptide with high angiotensin converting enzyme inhibition rate and good antihypertensive effect. And it has been verified by animal experiments that the soybean antihypertensive peptide of the present application can significantly reduce the heart rate, diastolic blood pressure and systolic blood pressure of rats, and has less toxic and side effects.

Description

Technical Field

[0001] The present application relates to the field of polypeptides, and specifically to a soybean antihypertensive peptide and a screening method and application thereof. Background Art

[0002] Hypertension is a disease characterized by increased blood pressure in the systemic arteries. According to data published by the World Health Organization (WHO), more than one billion people suffer from this disease. Every year, 9.4 million people die from complications of hypertension, accounting for 55% of deaths from cardiovascular diseases. At the same time, hypertension causes at least 45% of deaths from heart disease and 51% of deaths from stroke. In addition, hypertension is closely related to diseases such as aneurysms, renal failure, and arteriosclerosis. Since hypertension has become a very difficult problem facing global health, the prevention and treatment of hypertension is particularly important.

[0003] Dietary regulation is an important means of alleviating and assisting the treatment of hypertension. Plant-derived antihypertensive peptides are very popular in the field of developing antihypertensive functional foods because of their obvious efficacy, natural nature, and no toxic side effects. Plant-derived antihypertensive peptides are small molecule peptides obtained by protease hydrolysis of plant proteins under appropriate conditions. They can inhibit the activity of angiotensin-converting enzyme (ACE) by competitively binding to ACE, thereby exerting a blood pressure-lowering effect. Plant-derived antihypertensive peptides developed into health foods and medicines can provide new means for the prevention and control of hypertension, but currently only a few fermented plant-derived antihypertensive peptides have been commercialized. Summary of the invention

[0004] In view of this, the present application provides a soybean antihypertensive peptide and a screening method and application thereof, aiming to provide a plant-derived antihypertensive peptide that is effective in lowering blood pressure and has lower toxic side effects.

[0005] The embodiment of the present application is implemented as follows: a soybean antihypertensive peptide is provided, wherein the soybean antihypertensive peptide includes one or more of a soybean antihypertensive peptide I with a molecular weight of less than 1000Da, a soybean antihypertensive peptide II with a molecular weight of less than 1000Da, and a soybean antihypertensive peptide III with a molecular weight of less than 1000Da; wherein the soybean antihypertensive peptide I with a molecular weight of less than 1000Da is prepared by a nattokinase enzymatic hydrolysis of soy protein isolate, the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by a Bacillus subtilis liquid fermentation soybean protein hydrolysate method, and the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by a Bacillus subtilis solid fermentation soybean method.

[0006] In some embodiments, the soybean antihypertensive peptide includes the soybean antihypertensive peptide II having a molecular weight of less than 1000 Da.

[0007] In some embodiments, the soybean antihypertensive peptide II with a molecular weight of less than 1000 Da includes a polypeptide with an amino acid sequence of VFPPPW.

[0008] In some embodiments, the method for enzymatically hydrolyzing soy protein isolate with nattokinase comprises: enzymatically hydrolyzing the soy protein isolate with nattokinase, then inactivating the enzyme, separating and drying the obtained protein; and / or,

[0009] The method of liquid fermentation of soybean protein hydrolysate by Bacillus subtilis comprises: preparing a fermentation medium using glucose, soybean protein hydrolysate, disodium hydrogen phosphate dodecahydrate, disodium hydrogen phosphate monohydrate, magnesium sulfate heptahydrate and calcium chloride as culture medium raw materials, inoculating Bacillus subtilis on the fermentation medium and fermenting, and centrifuging and filtering after the fermentation is completed to obtain the obtained product; and / or,

[0010] The Bacillus subtilis solid fermentation soybean method comprises: washing, soaking and steaming raw soybeans, inoculating Bacillus subtilis for fermentation, freeze-drying after the fermentation to obtain fermentation powder, mixing the fermentation powder with water and heating to a certain temperature, further enzymolysis by using nattokinase generated during the fermentation process, killing the enzyme after the enzymolysis, and separating to obtain soybean antihypertensive peptide III with a molecular weight of less than 1000Da.

[0011] In some embodiments, in the nattokinase enzymatic hydrolysis of soy protein isolate: the amount of nattokinase added is 4000-8000 IU / g; and / or the temperature of the enzymatic hydrolysis is 50-60°C, and the time is 4-6h; and / or the temperature of the enzyme inactivation is above 95°C, and the time is 10-15min; and / or the separation is specifically: solid-liquid separation by a disc centrifuge, first passing through a 5000Da filter membrane, taking the permeate and then passing through a 1000Da filter membrane, and drying the permeate.

[0012] In some embodiments, in the Bacillus subtilis liquid fermentation soybean protein hydrolysate method: the fermentation medium includes the following components in weight percentage: glucose 1%-5%, soybean protein hydrolysate 5%-15%, disodium hydrogen phosphate dodecahydrate 1%-3%, sodium dihydrogen phosphate monohydrate 0.5%-2%, magnesium sulfate heptahydrate 0.1%-1%, CaCl2 0.1%-1%; and / or, the soybean protein hydrolysate is specifically soy peptone; and / or, the Bacillus subtilis is deposited in the China Center for Type Culture Collection with the accession number CCTCCNO: M20221746; and / or, the inoculation amount of the Bacillus subtilis is 1%-5%; and / or, the fermentation temperature is 35°C-40°C, and the time is 24h-48h; and / or, the centrifugation and filtration specifically includes: after centrifugation, taking the supernatant for membrane filtration, after filtration, first passing through a 5000Da filter membrane, taking the permeate and then passing through a 1000Da filter membrane to obtain the permeate.

[0013] In some embodiments, in the solid fermentation soybean method with Bacillus subtilis: the raw soybean is small-grain soybean with a protein content of >35%; and / or, the soaking is specifically: soaking at 5°C to 20°C for 8h-12h; and / or, the steaming is specifically steaming at 105°C to 115°C and 0.02MPa for 20-40min; and / or, after the steaming is completed, Bacillus subtilis is inoculated when the temperature is cooled to 70-90°C; and / or, the inoculation amount of Bacillus subtilis is 3%-8%; and / or, the Bacillus subtilis is deposited in the China Center for Type Culture Collection with the accession number of CCTCC NO:M20221746; and / or, the fermentation conditions are: fermentation at 35-40℃ for 18h-24h; and / or, the baking powder and water are mixed according to the solid-liquid mass ratio of 1:(15-25); and / or, the temperature of the enzymatic hydrolysis is 40-60℃, and the time is 2h-4h; and / or, the temperature of the enzyme inactivation is above 95℃, and the time is 10-15min; and / or, the separation is: using a disc centrifuge for solid-liquid separation to take the supernatant, first pass it through a 5000Da filter membrane, take the permeate and then pass it through a 1000Da filter membrane to obtain the permeate.

[0014] The present application also provides a soybean antihypertensive peptide, which includes a polypeptide with an amino acid sequence of VFPPPW.

[0015] The present application also provides a method for screening soybean antihypertensive peptides, comprising:

[0016] Providing a plurality of peptide segments to be screened, wherein the plurality of peptide segments to be screened are determined by mass spectrometry identification of the soybean antihypertensive peptide II having a molecular weight of less than 1000 Da;

[0017] The probability of biological activity was predicted using the Peptide Ranker database, and peptides with a biological activity prediction score > 0.9 were screened out;

[0018] The peptides with a predicted biological activity score of >0.9 were subjected to molecular docking screening with angiotensin converting enzyme to obtain soybean antihypertensive peptides;

[0019] Wherein, the soybean antihypertensive peptide II with a molecular weight of less than 1000Da is the soybean antihypertensive peptide II with a molecular weight of less than 1000Da among the above-mentioned soybean antihypertensive peptides.

[0020] The present application also provides an application of the soybean antihypertensive peptide described above, which is used in food, health food and medicine to treat hypertension or maintain a healthy blood pressure level.

[0021] The soybean antihypertensive peptide of the present application includes one or more of soybean antihypertensive peptide I with a molecular weight of less than 1000Da, soybean antihypertensive peptide II with a molecular weight of less than 1000Da, and soybean antihypertensive peptide III with a molecular weight of less than 1000Da; wherein, the soybean antihypertensive peptide I with a molecular weight of less than 1000Da is prepared by the nattokinase enzymatic hydrolysis of soybean protein isolate, the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by the Bacillus subtilis liquid fermentation soybean protein hydrolysate method, and the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by the Bacillus subtilis solid fermentation soybean method. The soybean antihypertensive peptide of the present application is a plant-derived antihypertensive peptide with high angiotensin converting enzyme inhibition rate and good antihypertensive effect. And it has been verified by animal experiments that the soybean antihypertensive peptide of the present application can significantly reduce the heart rate, diastolic blood pressure and systolic blood pressure of rats, and has less toxic and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 This is a schematic diagram of the process of an embodiment of the screening method of soybean antihypertensive peptides provided in the present application;

[0024] Figure 2 This is the ACE inhibition rate diagram of soybean peptide in Experimental Example 1;

[0025] Figure 3 This is the molecular docking diagram of peptide VFPPPW and human angiotensin converting enzyme. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. In addition, it should be understood that the specific implementation methods described herein are only used to illustrate and explain the present application and are not used to limit the present application. In the present application, unless otherwise stated, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present application, the term "including" means "including but not limited to".

[0027] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0028] In this application, "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0029] In the present application, "at least one" means one or more, and "plurality" means two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0030] The present application provides a soybean antihypertensive peptide, including one or more of a soybean antihypertensive peptide I with a molecular weight of less than 1000Da, a soybean antihypertensive peptide II with a molecular weight of less than 1000Da, and a soybean antihypertensive peptide III with a molecular weight of less than 1000Da; wherein the soybean antihypertensive peptide I with a molecular weight of less than 1000Da is prepared by a nattokinase enzymatic hydrolysis of soybean protein isolate, the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by a Bacillus subtilis liquid fermentation soybean protein hydrolysate method, and the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by a Bacillus subtilis solid fermentation soybean method.

[0031] The soybean antihypertensive peptide of the present application is a plant-derived antihypertensive peptide, which has a high inhibitory activity on angiotensin converting enzyme and has a good antihypertensive effect. Animal experiments have shown that the soybean antihypertensive peptide of the present application can significantly reduce the heart rate, diastolic blood pressure and systolic blood pressure of rats, and has less toxic side effects.

[0032] In some embodiments, the method of enzymatically hydrolyzing soy protein isolate with nattokinase can specifically be: enzymatically hydrolyzing the soy protein isolate with nattokinase, then inactivating the enzyme and separating the soy protein isolate.

[0033] The CAS number of the soy protein isolate (SPI) is 9010-10-0. It is a high molecular organic polymer formed by a series of amino acids bonded by peptide bonds. It is mainly composed of albumin and globulin, of which albumin accounts for about 5% and globulin accounts for about 90%.

[0034] Among them, nattokinase is homemade and obtained through fermentation and cultivation of Bacillus subtilis, with a purity of ≥95%.

[0035] In a specific embodiment, the preparation method of nattokinase is as follows: anhydrous glucose (20-50g / L), soy peptone (10-40g / L), disodium hydrogen phosphate dodecahydrate (1-10g / L), sodium dihydrogen phosphate monohydrate (0.1-2g / L), magnesium sulfate heptahydrate (0.1-1g / L), calcium chloride (0.1-1g / L), pH 7.0-7.6; sterilized at 105-110°C for 10-40 minutes in a fermenter; after the fermentation liquid in the fermenter is cooled to 40-50°C, the mixture is heated to 0.5-3% (V / V) of inoculation amount is inoculated with Bacillus subtilis seed liquid, and fermented in a fermentation equipment for 15-36h, wherein the temperature of the fermentation equipment is controlled at 37°C-42°C, the pressure of the fermentation tank is 0.05-0.08Mpa, and the dissolved oxygen level is set to 10-50%; the fermented fermentation liquid is centrifuged by a tubular centrifuge; the centrifuged fermentation liquid is filtered through a filter membrane; the filtrate after the membrane filtration is separated and purified by a chromatography column, and the purified liquid is spray-dried to obtain a solid powder, which is nattokinase, and the purity of nattokinase is ≥95%.

[0036] The amount of nattokinase added can be 4000-8000IU / g, such as 4000-5000IU / g, 5000-6000IU / g, 6000-8000IU / g, etc. The temperature of the enzymolysis can be 50-60°C, and the time of the enzymolysis can be 4-6h. The temperature of the enzyme inactivation is above 95°C, and the enzyme inactivation time can be 10-15min. The separation is specifically: solid-liquid separation by a disc centrifuge, first passing through a 5000Da filter membrane, taking the permeate and then passing through a 1000Da filter membrane, at which time the permeate is <1000Da soybean peptide I. In a specific embodiment, the filtrate is dried after the separation.

[0037] In some embodiments, the Bacillus subtilis liquid fermentation soybean protein hydrolysate method is: using glucose, soybean protein hydrolysate, disodium hydrogen phosphate dodecahydrate, disodium hydrogen phosphate monohydrate, magnesium sulfate heptahydrate, and calcium chloride as culture medium raw materials to prepare a fermentation medium, inoculating Bacillus subtilis on the fermentation medium and fermenting, and after the fermentation is completed, centrifuging and filtering to obtain the obtained product.

[0038] Specifically, the fermentation medium includes the following components in weight percentage: glucose 1%-5%, soybean protein hydrolysate 5%-15%, disodium hydrogen phosphate dodecahydrate 1%-3%, sodium dihydrogen phosphate monohydrate 0.5%-2%, magnesium sulfate heptahydrate 0.1%-1%, CaCl2 0.1%-1%. Further, the soybean protein hydrolysate is specifically soybean peptone. Among them, Bacillus subtilis is Bacillus subtilis CX01, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20221746. Bacillus subtilis with a deposit number of CCTCC NO: M20221746 is deposited in the National Center for Type Culture Collection, named Bacillus subtilis CX01, classified as Bacillus subtilis, with a deposit address of Wuhan, China, and a deposit date of November 9, 2022.

[0039] In a specific embodiment, the inoculation amount of the Bacillus subtilis is 1%-5%, specifically 1%-2%, 2%-3%, 3%-5%, etc. The fermentation temperature is 35° C.-40° C., and the fermentation time is 24h-48h.

[0040] The centrifugal separation and filtration specifically includes: centrifugal separation, taking the supernatant for membrane filtration, and drying after filtration. In a specific embodiment, a disc centrifuge can be used for solid-liquid separation, first passing through a 5000Da filter membrane, taking the permeate and then passing through a 1000Da filter membrane, and the permeate is <1000Da soybean peptide II.

[0041] In some embodiments, the Bacillus subtilis solid fermentation soybean method is as follows: the raw soybeans are washed, soaked and steamed, inoculated with the strain Bacillus subtilis for fermentation, freeze-dried after the fermentation to obtain fermentation powder, the fermentation powder is mixed with water and heated to a certain temperature, further enzymatically hydrolyzed using the nattokinase produced during the fermentation process, and the enzyme is inactivated after the enzymatic hydrolysis to separate the soybean antihypertensive peptide III with a molecular weight of less than 1000Da.

[0042] The raw soybean is small-grain soybean with a protein content of more than 35%. The soaking is specifically soaking at 5°C to 20°C for 8h-12h. The steaming is specifically steaming at 105°C to 115°C and 0.02MPa for 20-40min. After the steaming is completed, Bacillus subtilis is inoculated when the temperature is cooled to 70-90°C, and the inoculation amount is 3%-8%; the fermentation conditions can specifically be: fermentation at 35-40°C for 18h-24h.

[0043] The baking powder and water are mixed in a specific solid-liquid ratio of 1: (15-25), stirred evenly, and further enzymolyzed by using the nattokinase produced during the fermentation process, the enzymolysis temperature is 40-60°C, and the time is 2h-4h. Further, after the enzymolysis is completed, the enzyme is inactivated by heating to above 95°C, and the enzyme inactivation time is 10-15min;

[0044] In the above 3, the separation is specifically as follows: using a disc centrifuge for solid-liquid separation to take the supernatant, first pass it through a 5000Da filter membrane, take the permeate and then pass it through a 1000Da filter membrane, at which point the permeate is <1000Da soybean peptide III. In a specific embodiment, the permeate can be dried to obtain soybean peptide III with a molecular weight of <1000Da.

[0045] In a specific embodiment, at least one of the soybean antihypertensive peptide I with a molecular weight of less than 1000Da, the soybean antihypertensive peptide II with a molecular weight of less than 1000Da, and the soybean antihypertensive peptide III with a molecular weight of less than 1000Da comprises a polypeptide with an amino acid sequence of VFPPPW.

[0046] In the above embodiments, the soybean antihypertensive peptides provided use soybean-derived protein as a substrate, and adopt nattokinase hydrolysis process, Bacillus subtilis liquid fermentation process, and Bacillus subtilis solid fermentation process to prepare high-activity soybean antihypertensive peptides through different processes, explore new plant-based antihypertensive peptides, provide new health foods or raw materials, and lay the foundation for the commercial application of antihypertensive peptides.

[0047] In addition, soybean antihypertensive peptide I with a molecular weight of less than 1000Da, soybean antihypertensive peptide II with a molecular weight of less than 1000Da, and soybean antihypertensive peptide III with a molecular weight of less than 1000Da, prepared by the nattokinase enzymatic hydrolysis of soybean protein isolate method, the Bacillus subtilis liquid fermentation soybean protein hydrolysate method, and the Bacillus subtilis solid fermentation soybean method, respectively, all have high activity in inhibiting angiotensin converting enzyme.

[0048] In one embodiment, the soybean antihypertensive peptide includes soybean antihypertensive peptide II with a molecular weight of less than 1000 Da, and the soybean antihypertensive peptide has a relatively better inhibitory activity on ACE enzyme.

[0049] Furthermore, the soybean antihypertensive peptide II with a molecular weight of less than 1000 Da includes multiple peptide segments, specifically including a polypeptide with an amino acid sequence of VFPPPW.

[0050] The present application provides a soybean antihypertensive peptide, including a polypeptide with an amino acid sequence of VFPPPW.

[0051] In the above embodiments, the soybean antihypertensive peptide including a polypeptide having an amino acid sequence of VFPPPW can significantly reduce the heart rate, diastolic blood pressure and systolic blood pressure of rats in animal experiments, and the antihypertensive effect is equivalent to that of the positive control group (conventional antihypertensive drug captopril). Moreover, it is a plant-derived antihypertensive peptide with relatively smaller toxic and side effects.

[0052] The present application also provides a preparation method of the above-mentioned soybean antihypertensive peptide, which can be a nattokinase enzymatic hydrolysis of soybean protein isolate method, a Bacillus subtilis liquid fermentation soybean protein hydrolysate method, or a Bacillus subtilis solid fermentation soybean method. The above three preparation methods use soybean-derived protein as a substrate, adopt a nattokinase hydrolysis process, a Bacillus subtilis liquid fermentation process, and a Bacillus subtilis solid fermentation process, and prepare highly active soybean antihypertensive peptides by different processes, explore new plant-derived antihypertensive peptides, provide new health foods or raw materials, and lay the foundation for the commercial application of antihypertensive peptides.

[0053] The present application also provides a screening method for soybean antihypertensive peptides, see Figure 1 , Figure 1 This is a schematic flow chart of an embodiment of a screening method for soybean hypotensive peptides provided in the present application, comprising:

[0054] Step S11: providing a plurality of peptide segments to be screened, wherein the plurality of peptide segments to be screened are determined by mass spectrometry identification of the soybean antihypertensive peptide II having a molecular weight of less than 1000 Da;

[0055] Step S12: predicting the probability of biological activity through the Peptide Ranker database, and screening out peptides with a biological activity prediction score > 0.9;

[0056] Step S13: performing molecular docking screening on the peptide segments with a biological activity prediction score of >0.9 and angiotensin converting enzyme to obtain soybean antihypertensive peptides.

[0057] Specifically, the molecular docking screening can be performed by AutoDock 4.2.6 software, the angiotensin converting enzyme is specifically human angiotensin converting enzyme, the crystal structure information of the angiotensin converting enzyme is obtained from the RCSB database, and the PDB number of the angiotensin converting enzyme is 1O8A; before the molecular docking, the PDB file of the angiotensin converting enzyme is pre-processed, and the pre-processing includes: deleting water molecules, deleting ligands, and adding hydrogen atoms. One or more.

[0058] In this embodiment, by performing biological activity screening and molecular docking screening, soybean antihypertensive peptides with good lipid-lowering activity can be quickly screened out.

[0059] The present application also provides the application of the above soybean antihypertensive peptide in food, health food and medicine for treating hypertension or maintaining healthy blood pressure level.

[0060] The technical scheme and technical effects of the present application are described in detail below through specific embodiments, comparative examples and experimental examples. The following embodiments are only some embodiments of the present application and do not specifically limit the present application. It can be understood that the reagents used in each embodiment, comparative example and experimental example are all commercially available reagents unless otherwise specified.

[0061] Example 1

[0062] Soy protein isolate is purchased from the market, with a protein content of ≥90%; nattokinase is homemade, with a purity of ≥95%.

[0063] Weigh soy protein isolate and pure water according to the proportion, put them into a hydrolysis tank, heat them to 80℃, stir and dissolve them, and prepare a soy protein isolate solution with a concentration of 5%; cool them to 50℃, add nattokinase according to the addition amount of 6000IU / g, stir and dissolve; after enzymolysis at 50℃ for 4h, heat them to 95℃ and keep them for 15min to kill the enzyme. After cooling, centrifuge them in a disc centrifuge and take the supernatant. The supernatant passes through a 5000Da filter membrane, and the permeate passes through a 1000Da filter membrane again. The permeate and the concentrated liquid are collected and dried separately to prepare soybean peptide I with a molecular weight of <1000Da and soybean peptide I with a molecular weight of 1000Da-5000Da, which are respectively recorded as <1kD soybean peptide I and 1kD-5kD soybean peptide I.

[0064] Example 2

[0065] The Bacillus subtilis seed liquid culture medium is provided with 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, and the culture time is 18 h at a temperature of 37°C. The fermentation medium is weighed in proportion, including 5% glucose, 15% soy peptone, 3% disodium hydrogen phosphate dodecahydrate, 1.5% sodium dihydrogen phosphate monohydrate, 0.5% magnesium sulfate heptahydrate, and CaCl 2 1%, add pure water and dissolve to obtain. Inoculate the seed liquid at a ratio of 3%, and culture at 37℃ for 24h. Centrifuge with a disc centrifuge and take the supernatant. Pass the supernatant through a 5000Da filter membrane, and pass the permeate through a 1000Da filter membrane again. Collect the permeate and the concentrate, and dry them separately to prepare soybean peptide II with a molecular weight of <1000Da and soybean peptide II with a molecular weight of 1000Da-5000Da, which are respectively recorded as <1kD soybean peptide II and 1kD-5kD soybean peptide II.

[0066] Example 3

[0067] The culture medium for Bacillus subtilis seed liquid is 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, the temperature is 37°C, and the culture time is 18 h.

[0068] The protein content of small-grain soybeans is ≥40%. After removing bad beans and miscellaneous beans and cleaning them, soak the soybeans in pure water at 5°C for 12 hours. After soaking, place them in a cooking tank and cook for 30 minutes. After cooking, when the temperature of the soybeans drops to 80°C, inoculate Bacillus subtilis seed liquid at a ratio of 5% (V / M) and place them in a fermentation cabinet for fermentation. The fermentation conditions are 37°C and fermentation for 24 hours. After fermentation, dry them by vacuum freeze drying, then crush and grind them into powder. After grinding into powder, add pure water at a solid-liquid ratio of 1:20, stir evenly, heat to 50°C, keep for 3 hours, and use the nattokinase produced during the fermentation process for further enzymolysis. Subsequently, heat to 95°C and keep for 10 minutes to inactivate the enzyme. After the enzyme is inactivated, centrifuge with a disc centrifuge and take the supernatant. The clear liquid was filtered through a 5000Da filter membrane, and the permeate was filtered through a 1000Da filter membrane again. The permeate and the concentrated liquid were collected and dried separately to prepare soybean peptide III with a molecular weight of <1000Da and soybean peptide III with a molecular weight of 1000Da-5000Da, which were recorded as <1kD soybean III and 1kD-5kD soybean peptide III, respectively.

[0069] Experimental Example 1 Detection of angiotensin converting enzyme inhibition rate activity of different soybean peptides in the embodiment

[0070] Ultraviolet spectrophotometry was used for detection, and the specific detection steps were carried out with reference to the kit (Solebao angiotensin converting enzyme (ACE) activity detection kit).

[0071] According to the instructions of the kit, check the reagents, which are reagent 1, reagent 2, reagent 3, and reagent 4, of which reagent 4 is the positive control. Prepare the working solution of reagent 3 according to the steps in the instructions. Take one bottle and add 50uL of distilled water to dissolve it before use. According to the sample volume, prepare the ratio of reagent 3: reagent 1 = 9uL: 1791uL, and prepare it before use.

[0072] The following 6 groups of samples were tested, specifically: <1kD soybean peptide I and 1kD-5kD soybean peptide I, <1kD soybean peptide II and 1kD-5kD soybean peptide II, <1kD soybean III and 1kD-5kD soybean peptide III. Dissolve the above 6 groups of samples into a sample solution with a concentration of 10mg / mL using reagent 1. Add the sample according to the steps in the table below. Before adding the sample, take an appropriate amount of actual reagent 2 according to the sample volume and preheat it at 37℃ for more than 10 minutes. Add the sample in a micro quartz cuvette according to the steps in the table below:

[0073] Table 1 Angiotensin inhibition rate activity test sample addition table

[0074]

[0075]

[0076] After adding the sample, mix thoroughly and measure the absorbance value A at 340 nm after 10 seconds. 1 , quickly place at 37℃ for accurate reaction for 30min, and measure the absorbance value A at 30min10s 2 , respectively denoted as A1 空白1 、A1 空白2 、A1 测定 and A2 空白1 、A2 空白2 、A2 测定 , calculate ΔA 空白 =(A1 空白1 -A2 空白1 )-(A1 空白2 -A2 空白2 ), ΔA 测定 =A1 测定 -A2 测定 Blank tube 1 and Blank tube 2 only need to be done 1-2 times.

[0077] The inhibition rate was calculated according to the following formula (1):

[0078] ACE inhibition rate (%) = (ΔA 空白 -ΔA 测定 )÷ΔA 空白 ×100% (1)

[0079] During the detection process, the concentration of soybean peptides in different examples was 10 mg / mL. Figure 2 The ACE inhibition rate of soybean peptides in different embodiments is shown in the figure. It can be seen from the figure that the ACE inhibition rates of soybean peptides Ⅰ, Ⅱ, and Ⅲ <1KD are 64.80%, 79.22%, and 75.01%, respectively, which are higher than 35.53%, 42.93%, and 38.11% of 1KD-5KD soybean peptides Ⅰ, Ⅱ, and Ⅲ. The results show that the blood pressure lowering effect of soybean peptides is related to the molecular weight of the peptides. Short-chain peptides can better bind to the catalytic active center of ACE, thereby having a higher ACE inhibition rate. That is, soybean peptides with a molecular weight of less than 1000kDa have better ACE inhibition activity.

[0080] Specifically, the ACE inhibition rate of <1KD soybean peptide II is the highest at 79.22%, followed by <1KD soybean peptide III (inhibition rate 75.01%), and then <1KD soybean peptide I (inhibition rate 64.80%). <1KD soybean peptide II is produced by microbial liquid fermentation. The microbial fermentation method uses various enzyme catalysts produced during the growth of microorganisms to hydrolyze the protein in the raw materials and then produce a hydrolysis polypeptide process. This process produces more proteases and more protein cleavage sites. The types of polypeptides produced by microbial fermentation are richer, which can increase the specific cleavage ratio of hydrophobic amino acid residues and expose them at the C-terminus of the polypeptide sequence, thereby enhancing the binding ability of the polypeptide to the catalytic active center of the ACE molecule.

[0081] Experimental Example 2 LC-MS / MS analysis of peptide sequences

[0082] According to the results of Experimental Example 1, mass spectrometry identification was performed on soybean peptide II with the highest ACE inhibition rate <1KD.

[0083] Dissolve the polypeptide sample in Washing buffer (0.1% FA, 2% ACN), transfer the polypeptide sample solution to a 10KD ultrafiltration centrifuge tube, and centrifuge at 12000g for 10 min; desalt the ultrafiltered solution using a C18 desalting column; elute the sample with Elution buffer (0.1% FA, 60% ACN), and transfer the eluted solution to a new EP tube; centrifuge and concentrate the eluted sample to dryness for mass spectrometry analysis.

[0084] After desalting, the sample was centrifuged and dried, then redissolved in 100uL Nano-LC mobile phase A (0.1% formic acid / water) and loaded into a bottle for online LCMS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3μm, ), and then 20ul volume was used for washing and desalting. The liquid phase was Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA). The sample was desalted and retained on the pre-column and then separated by the analytical column. The analytical column specification was C18 reverse phase chromatography column (AcclaimPepMap RSLC, 75μm×25cm C18-2μm ), the gradient used in the experiment was that the mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 30 min. The mass spectrometer used the ThermoFisher Q Exactive system (ThermoFisher, USA) combined with the nanospray Nano Flex ion source (ThermoFisher, USA), the spray voltage was 1.9 kV, and the ion transfer tube heating temperature was 275 ° C. The mass spectrometer scanning mode was in the information-dependent acquisition working mode (DDA, Data Dependent Analysis), the primary mass spectrometer scanning resolution was 70000, the scanning range was 100-1500 m / z, and the maximum injection time was 100 ms. A maximum of 20 charges of 1 were collected in each DDA cycle. + To 3 + The secondary spectrum of the mass spectrometer was obtained, and the maximum injection time of the secondary mass spectrometer ions was 50 ms. The collision chamber energy (high energy collision induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 6 seconds. The original raw spectrum files collected by mass spectrometry were processed and searched using PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada) software. The database was the target species protein database downloaded by Uniprot, and the search parameters were set as follows: the mass tolerance of the primary mass spectrometer was 10 ppm, and the secondary mass spectrometer was 0.05 Da. The variable modifications were Oxidation (M), Acetylation (Protein N-term), Deamidation (NQ), Pyro-glu from E, and Pyro-glu from Q.

[0085] The results of mass spectrometry identification showed a total of 62 peptides, as shown in the table below:

[0086] Table 2 Mass spectrometry identification results of <1KD soybean peptide II

[0087]

[0088]

[0089]

[0090] Experimental Example 3 Screening of antihypertensive peptides based on molecular docking technology

[0091] First, the peptides identified by LC-MS / MS were preliminarily screened according to Peptide Ranker, and peptides with predicted bioactivity scores>0.9 were screened out, and molecular docking was performed with cholesterol esterase. The smaller the "binding_energy" value, the better the docking effect, and the higher the cholesterol-lowering potential. The peptides with predicted bioactivity scores>0.9 are shown in Table 2:

[0092] Table 3<1KD soybean peptide II peptide activity prediction table

[0093]

[0094]

[0095] The crystal structure of human angiotensin converting enzyme (PDB number: 1O8A) was obtained from the RCSB database (http: / / www.rcsb.org / ). The semi-flexible molecular docking scores of the above peptides with biological activity prediction scores higher than 0.90 and human angiotensin converting enzyme were performed using AutoDock 4.2.6 software as shown in the following table.

[0096] Table 4 Molecular docking score of potential antihypertensive peptides and human angiotensin converting enzyme

[0097] Peptides binding_energy / Kcal FWf 0.6 VPFGPWW 0.32 HHPPF 0.71 FPFPRPP 0.72 VpVP -1.84 RWPDFP 0.54 VPFFP -1.23 HkDJ -0.64 PFPRPP 0.72 PIPFPRP -0.34 VPFGPWW -0.32 PFPRPR 0.87 FPFPRPPH 3.11 FVFMMI -0.52 RPIPFP -0.38 RPIPFPR -0.46 GEQPRPFPFPRP 1.49 QFPFPRPPH 1.52 WNPNNKPF -0.73 KPW -0.63

[0098] The peptide VFPPPW with the strongest binding ability to human angiotensin converting enzyme was screened out, and the molecular docking diagram is shown in Figure 3 VFPPPW was prepared by Fmoc solid phase synthesis and was designated as soybean antihypertensive peptide 1. The purity of the peptide was determined to be greater than 95% by high performance liquid chromatography and mass spectrometry.

[0099] Experimental Example 4 Different Examples of <1KD Soy Peptide and Soy Antihypertensive Peptide 1 in Vivo Antihypertensive Effects

[0100] Spontaneously hypertensive model rats (SHR), 60, male, 10-12 weeks old, weighing 210-230 g; normotensive rats (WKY), 10, male, 10-12 weeks old, weighing 210-230 g. The temperature of the breeding environment was maintained at (25±1)℃, the humidity was maintained at 55%±5%, the light / dark cycle was 12 hours per day, and the lighting time was from 8:00 am to 20:00 pm. Rats could eat standard feed and tap water at will, and the breeding environment was disinfected regularly. Before the experiment, SHR and WKY had a one-week environmental adaptation period. After the adaptation period, the weight and blood pressure of the rats were measured, and they were divided into different groups according to the measurement results to ensure the uniformity of the baseline data of the rats in each group.

[0101] SHR rats were divided into 6 groups, each with 10 rats, including hypertension model control group, <1KD soybean peptide Ⅰ, <1KD soybean peptide Ⅱ, <1KD soybean peptide Ⅲ, soybean antihypertensive peptide 1 (VFPPPW). Blank control group WKY rats were blank control group with 10 rats.

[0102] Table 5 Oral gavage dosage table for each group

[0103] Group <![CDATA[Gavage administration / mg·kg -1 <!-- 11 -->]]> Blank control group An equal volume of saline Hypertension model control group An equal volume of saline <1KD soybean peptide Ⅰ Prepare 500mg / kg by dissolving in normal saline <1KD soybean peptide Ⅱ Prepare 500mg / kg by dissolving in normal saline <1KD soybean peptide Ⅲ Prepare 500mg / kg by dissolving in normal saline Soybean hypotensive peptide 1 (VFPPPW) Prepare 20mg / kg by dissolving in normal saline Positive control group 10 mg / kg captopril

[0104] The rats were fed with a normal diet and given the drug by gavage once a day according to the dosage gavage table for 8 consecutive weeks. The body weight, food intake, blood pressure and heart rate of the rats were collected every week during the experiment.

[0105] When measuring the blood pressure and heart rate of rats, in order to ensure the accuracy of the measured data, the rats to be tested need to be placed in a warm box (37°C) in advance and left to stand for about 15 minutes. After the rats are quiet and their blood pressure is stable, the systolic blood pressure, diastolic blood pressure and heart rate of SHR and WKY rats are monitored using the BP-2006A small animal non-invasive blood pressure meter. The rats are fixed in the heating hood, and a blackout cloth is put on the outer layer of the heating hood to prevent the rats from having a stress response that affects the accuracy of the test data. The rats are placed in the heating hood and covered with a blackout cloth. The temperature of the heating hood is set to 37-39°C to ensure that the tail artery of each rat is fully dilated during the measurement. The sphygmomanometer airbag cuff is put on 3 cm from the tail root of the fixed rat, and the "pulse signal sensor" in the airbag cuff is kept directly above the tail artery. When the sensor shows that the pulse wave is in a stable wave shape, blood pressure measurement begins. The system was connected to a computer, and the test results were recorded using BP98AWU software, recording three to five readings with heart rate changes <10 beats / min and blood pressure changes <20 mmHg, and the measurements were repeated three times for each rat to obtain the average value.

[0106] Each group was observed by gavage for 8 weeks. During the experiment, the animals in each group showed normal growth, development, and activity, and were in good mental state, with no signs of poisoning or death.

[0107] Table 6 Changes in body weight of rats in each group

[0108]

[0109] During the experiment, the animals in each group were active and in good spirits. The weight of the rats in each group increased with the increase of age, and the growth and development were normal. There was no difference in weight between the experimental groups at the same age. <1KD soybean peptides Ⅰ, Ⅱ, Ⅲ and the screened soybean antihypertensive peptide 1 (VFPPPW) are non-toxic and have no side effects, and have no effect on the growth and development of rats.

[0110] Table 7 Systolic blood pressure (SBP) of rats in each group after 8 weeks of intervention

[0111]

[0112]

[0113] Table 7 shows the weekly systolic blood pressure test values ​​of rats in each group. In the 0th week of intervention, the systolic blood pressure of the hypertension model group, <1KD soybean peptide I, <1KD soybean peptide II, <1KD soybean peptide III, soybean antihypertensive peptide 1 (VFPPPW), and positive control group were significantly higher than those of the blank control group (P < 0.01), and the hypertension model was established.

[0114] After 8 weeks of intervention, compared with the 0th week of each group, the systolic blood pressure of <1KD soybean peptide Ⅰ, <1KD soybean peptide Ⅱ, <1KD soybean peptide Ⅲ, soybean antihypertensive peptide 1 (VFPPPW), and the positive control group were significantly reduced, indicating that <1KD soybean peptide and the screened antihypertensive short peptide (VFPPPW) have significant effects on lowering systolic blood pressure in vivo. And <1KD soybean peptide Ⅱ and soybean antihypertensive peptide 1 (VFPPPW) have extremely significant differences in systolic blood pressure compared with the 0th week (P < 0.01), which is consistent with the results of angiotensin converting enzyme inhibition rate activity test.

[0115] Table 8 Diastolic blood pressure (DBP) of rats in each group after 8 weeks of intervention

[0116]

[0117] Table 8 shows the weekly diastolic blood pressure test values ​​of rats in each group. In the 0th week of intervention, the diastolic blood pressure of the hypertension model group, <1KD soybean peptide I, <1KD soybean peptide II, <1KD soybean peptide III, soybean antihypertensive peptide 1 (VFPPPW), and positive control group were significantly higher than those of the blank control group (P < 0.01), and the hypertension model was established.

[0118] After 8 weeks of intervention, compared with the 0th week of each group, the diastolic blood pressure of <1KD soybean peptide Ⅰ, <1KD soybean peptide Ⅱ, <1KD soybean peptide Ⅲ, soybean antihypertensive peptide 1 (VFPPPW), and the positive control group were significantly reduced, indicating that <1KD soybean peptide and the screened antihypertensive short peptide (VFPPPW) have significant effects on lowering diastolic blood pressure in vivo. And soybean antihypertensive peptide 1 (VFPPPW) has a very significant difference in systolic blood pressure compared with the 0th week (P < 0.01), and the effect is equivalent to that of the positive control group. It shows that the antihypertensive peptide 1 (VFPPPW) screened by mass spectrometry identification, activity screening and molecular docking has a significant effect on lowering blood pressure and can be used for the development of antihypertensive drugs.

[0119] Table 9 Heart rate of rats in each group after 8 weeks of intervention

[0120]

[0121] The heart rate of rats is related to blood pressure. Generally speaking, the higher the blood pressure, the higher the heart rate. As shown in Table 9, after 8 weeks of intervention, the heart rates of <1KD soybean peptide I, <1KD soybean peptide II, <1KD soybean peptide III, soybean antihypertensive peptide 1 (VFPPPW), and the positive control group were significantly lower than those of the hypertension model control group (P < 0.01), which was consistent with the results of systolic and diastolic blood pressure.

[0122] It can be seen from the above experimental results that the <1KD soybean peptide I, <1KD soybean peptide II, and <1KD soybean peptide III prepared by three preparation methods, namely, nattokinase enzymatic hydrolysis of soybean protein isolate method, Bacillus subtilis liquid fermentation soybean protein hydrolysate method, and Bacillus subtilis solid fermentation soybean method, all have high angiotensin converting enzyme inhibition rate activity, significant antihypertensive effect in vivo, can be used to develop health foods, and the process is simple and controllable, which is conducive to commercial application. The soybean antihypertensive peptide 1 (polypeptide sequence VFPPPW) screened out by mass spectrometry identification and molecular biology methods has an in vivo antihypertensive effect comparable to that of positive drugs, and can be used for the development of antihypertensive drug raw materials. The soybean antihypertensive peptides provided in this application and their preparation methods, as well as the screened single-sequence short peptides, can be used for the development of food, health products and medicines for people with hypertension.

[0123] The lipid-lowering polypeptides and their screening methods and applications provided in the embodiments of the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the methods and core ideas of the present application. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A soybean antihypertensive peptide, characterized in that: The soybean antihypertensive peptide includes one or more of soybean antihypertensive peptide I with a molecular weight of less than 1000Da, soybean antihypertensive peptide II with a molecular weight of less than 1000Da, and soybean antihypertensive peptide III with a molecular weight of less than 1000Da; Among them, the soybean antihypertensive peptide I with a molecular weight of less than 1000Da is prepared by the nattokinase enzymatic hydrolysis of soybean protein isolate, the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by the Bacillus subtilis liquid fermentation of soybean protein hydrolysate, and the soybean antihypertensive peptide III with a molecular weight of less than 1000Da is prepared by the Bacillus subtilis solid fermentation of soybeans.

2. The soybean antihypertensive peptide according to claim 1, characterized in that The soybean antihypertensive peptide includes the soybean antihypertensive peptide II with a molecular weight less than 1000Da.

3. The soybean antihypertensive peptide according to claim 2, characterized in that The soybean antihypertensive peptide II with a molecular weight less than 1000 Da includes a polypeptide with an amino acid sequence of VFPPPW.

4. The soybean antihypertensive peptide according to any one of claims 1 to 3, characterized in that: The method for enzymatically hydrolyzing soybean protein isolate with nattokinase comprises: enzymatically hydrolyzing soybean protein isolate with nattokinase, then inactivating the enzyme, separating and drying to obtain soybean antihypertensive peptide I with a molecular weight of less than 1000 Da; and / or, The method of liquid fermentation of soybean protein hydrolysate by Bacillus subtilis comprises: preparing a fermentation medium with glucose, soybean protein hydrolysate, disodium hydrogen phosphate dodecahydrate, disodium hydrogen phosphate monohydrate, magnesium sulfate heptahydrate and calcium chloride as culture medium raw materials, inoculating Bacillus subtilis on the fermentation medium and fermenting, and after the fermentation is completed, centrifuging and filtering to obtain soybean antihypertensive peptide II with a molecular weight of less than 1000Da; and / or, The Bacillus subtilis solid fermentation soybean method comprises: washing, soaking and steaming raw soybeans, inoculating Bacillus subtilis for fermentation, freeze-drying after the fermentation to obtain fermentation powder, mixing the fermentation powder with water and heating to a certain temperature, further enzymolysis by using nattokinase generated during the fermentation process, killing the enzyme after the enzymolysis, and separating to obtain soybean antihypertensive peptide III with a molecular weight of less than 1000Da.

5. The soybean antihypertensive peptide according to claim 4, characterized in that: In the method for enzymolysis of soy protein isolate by nattokinase: The added amount of nattokinase is 4000-8000 IU / g; and / or, The enzymatic hydrolysis temperature is 50-60°C and the time is 4-6h; and / or, The enzyme inactivation temperature is above 95°C and the time is 10-15 minutes; and / or, The separation is specifically as follows: solid-liquid separation by disc centrifuge, first passing through a 5000Da filter membrane, taking the permeate and then passing through a 1000Da filter membrane, and drying the permeate.

6. The soybean antihypertensive peptide according to claim 4, characterized in that In the Bacillus subtilis liquid fermentation soybean protein hydrolysate method: The fermentation medium comprises the following components in weight percentage: 1%-5% glucose, 5%-15% soybean protein hydrolysate, 1%-3% disodium hydrogen phosphate dodecahydrate, 0.5%-2% sodium dihydrogen phosphate monohydrate, 0.1%-1% magnesium sulfate heptahydrate, and 0.1%-1% CaCl2; and / or, The soybean protein hydrolysate is specifically soybean peptone; and / or, The Bacillus subtilis is Bacillus subtilis CX01, which is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M20221746; and / or, The inoculation amount of Bacillus subtilis is 1%-5%; and / or, The fermentation temperature is 35°C-40°C and the fermentation time is 24h-48h; and / or, The centrifugal separation and filtration specifically includes: taking the supernatant after centrifugal separation for membrane filtration, first passing it through a 5000Da filter membrane after filtration, taking the permeate and then passing it through a 1000Da filter membrane to obtain the permeate.

7. The soybean antihypertensive peptide according to claim 4, characterized in that: In the solid fermentation soybean method using Bacillus subtilis: The raw soybean is small-grain soybean, and the protein content is greater than 35%; and / or, The soaking is specifically: soaking at 5°C to 20°C for 8h-12h; and / or, The cooking is specifically steaming at 105°C-115°C and 0.02MPa for 20-40min; and / or, After the cooking is completed, the mixture is cooled to a temperature of 70-90° C. and then inoculated with Bacillus subtilis; and / or, The inoculation amount of Bacillus subtilis is 3%-8%; and / or, The Bacillus subtilis is Bacillus subtilis CX01, which is deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M20221746; and / or, The fermentation conditions are: fermentation at 35-40°C for 18h-24h; and / or, The baking powder and water are mixed in a solid-liquid mass ratio of 1:(15-25); and / or, The enzymatic hydrolysis temperature is 40-60°C and the time is 2h-4h; and / or, The enzyme inactivation temperature is above 95°C and the time is 10-15 minutes; and / or, The separation is as follows: using a disc centrifuge for solid-liquid separation to obtain the supernatant, first passing it through a 5000Da filter membrane, taking the permeate and then passing it through a 1000Da filter membrane to obtain the permeate.

8. A soybean antihypertensive peptide, characterized in that: The soybean antihypertensive peptide includes a polypeptide with an amino acid sequence of VFPPPW.

9. A method for screening soybean hypotensive peptides, characterized in that: include: Providing a plurality of peptide segments to be screened, wherein the plurality of peptide segments to be screened are determined by mass spectrometry identification of the soybean antihypertensive peptide II having a molecular weight of less than 1000 Da; The probability of biological activity was predicted using the Peptide Ranker database, and peptides with a biological activity prediction score > 0.9 were screened out; The peptides with a predicted biological activity score of >0.9 were subjected to molecular docking screening with angiotensin converting enzyme to obtain soybean antihypertensive peptides; Wherein, the soybean antihypertensive peptide II with a molecular weight of less than 1000Da is the soybean antihypertensive peptide II with a molecular weight of less than 1000Da in the soybean antihypertensive peptide according to any one of claims 1 to 7.

10. Use of the soybean antihypertensive peptide according to claims 1-8 or the soybean antihypertensive peptide screened by the screening method according to claim 9, characterized in that: Used in foods, health foods and medicines to treat high blood pressure or maintain healthy blood pressure levels.

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