A twelve-peptide with ACE inhibitory activity and its screening method and application

Abstract

The present invention provides a twelve peptide with ACE inhibitory activity and a screening method and application thereof, belonging to the field of polypeptide technology. The present invention isolates a twelve peptide from wheat fermentation broth, and through sequencing analysis, the amino acid sequence of the twelve peptide is Pro-Tyr-Ser-Ala-Gly-Gln-Leu-Ser-Ala-Cys-Leu-GLn (PYSAGQLSACLQ). The twelve peptide of the present invention has the ability to inhibit ACE enzyme activity in vitro, and animal experiments also show that the twelve peptide has an effect close to that of the mainstream antihypertensive drug captopril, and has a high application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides, and in particular relates to a twelve-peptide having ACE inhibitory activity and a screening method and application thereof. Background Art

[0002] Hypertension is divided into primary hypertension and secondary hypertension. Clinically, the main feature is increased arterial blood pressure. As the disease worsens, it often causes damage to organs such as the heart, brain, and kidneys, and functional or organic changes. It is one of the major diseases affecting human health.

[0003] Currently, the drugs used to lower blood pressure mainly include angiotensin-converting enzyme inhibitors, angiotensin II antagonists, calcium antagonists, beta-receptor blockers, diuretics, etc., but these drugs have side effects, and long-term use will bring hidden dangers. In addition, the high prices of most antihypertensive drugs will bring additional financial pressure to patients with hypertension.

[0004] At present, the prior art reports that polypeptides derived from animal proteins or plant proteins have in vitro ACE enzyme activity inhibition, for example, patent publication number CN107383168A discloses a thick-shelled mussel-derived antihypertensive peptide, patent publication number CN113943346A discloses a spirulina-derived antihypertensive active peptide, patent publication number CN101045747A discloses a tetrapeptide obtained by treating royal jelly material with trypsin, and patent publication number CN105779536A discloses a short peptide derived from almonds, all of which have angiotensin converting enzyme inhibitory effects. However, there are no reports on antihypertensive peptides derived from cereals.

[0005] Although the patent with publication number CN112868881A discloses an active wheat polypeptide separated by hydrolysis from wheat bran gluten, which can inhibit the activity of cell matrix metalloproteinases, increase skin elasticity, accelerate cell proliferation, promote cell metabolism, activate skin, resist free radicals, delay aging, etc., it is widely used in the field of anti-aging medicine and health food. For another example, the patent with publication number CN102524514A discloses an active wheat germ polypeptide that can effectively reduce fat accumulation in the body and has a preventive effect on nutritional obesity. At present, no reports on the antihypertensive activity of wheat polypeptides have been found. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a twelve peptide with ACE inhibitory activity, which is derived from fermented wheat and has in vitro ACE enzyme inhibitory activity and hypotensive effect, enriching the polypeptide source of antihypertensive drugs.

[0007] The present invention provides a twelve peptide having ACE inhibitory activity, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] The present invention provides a hypotensive peptide composition with ACE inhibitory activity, comprising the dodecapeptide and at least one of the following polypeptides: a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 to SEQ ID NO: 4.

[0009] Preferably, the molar ratio of the dodecapeptide to the polypeptide is 1-100:1-100.

[0010] Preferably, the molar ratio of the dodecapeptide to the polypeptide is 1-5:4-10.

[0011] The present invention provides the use of the dodecapeptide or the antihypertensive peptide composition in the preparation of antihypertensive drugs.

[0012] The present invention provides the use of the dodecapeptide or the antihypertensive peptide composition in the preparation of ACE enzyme inhibitors.

[0013] The present invention provides an ACE enzyme inhibitor, comprising the dodecapeptide or the antihypertensive peptide composition and auxiliary materials.

[0014] The present invention provides a blood pressure lowering polypeptide drug, comprising the dodecapeptide or the blood pressure lowering peptide composition and medically acceptable adjuvants.

[0015] The invention provides the use of wheat in fermentation production of a blood pressure reducing polypeptide containing the dodecapeptide.

[0016] Preferably, the fermentation production temperature is 20-35° C.; the fermentation production time is 24-96 hours; and the mass ratio of the wheat raw material to the water in the system is 1:(5-20).

[0017] The present invention provides a twelve peptide with ACE inhibitory activity, and the amino acid sequence is shown in SEQ ID NO: 1. The present invention isolates and screens a twelve peptide from wheat yeast fermentation broth, and is tested by an in vitro ACE inhibitory activity evaluation system, and has a strong ACE enzyme inhibitory activity, and the inhibition rate can reach 98.58%, which is similar to the inhibitory activity of captopril. It was verified by in vivo experiments on spontaneously hypertensive rats that the twelve peptide can effectively lower blood pressure values, which indicates that the twelve peptide has a good blood pressure lowering effect. It can be seen that the twelve peptide provided by the present invention fills the gap in the prior art of separating antihypertensive peptides from wheat, and at the same time has good ACE enzyme inhibitory activity and antihypertensive activity, enriches the types of antihypertensive polypeptides, and provides active substances for the preparation of ACE enzyme inhibitors and antihypertensive drugs, and has a high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the result of separation of antihypertensive peptides from fermented wheat. The numbers below indicate the numbers of the collected fractions.

[0019] Figure 2 The results are the comparison of the in vitro ACE enzyme inhibition activities of the dodecapeptide prepared by the present invention and captopril;

[0020] Figure 3 This is a line graph showing the changes in blood pressure in SHR rats during four weeks of infusion of antihypertensive peptides. DETAILED DESCRIPTION

[0021] The present invention provides a twelve peptide having ACE inhibitory activity, and the amino acid sequence is shown in SEQ ID NO: 1 (PYSAGQLSACLQ).

[0022] In the present invention, the screening and separation method of the dodecapeptide preferably comprises the following steps:

[0023] The wheat raw material is fermented under the action of yeast to obtain fermentation liquid;

[0024] enriching components smaller than 3 kDa from the fermentation broth;

[0025] Purifying the components smaller than 3 kDa by chromatographic column to collect the components that inhibit ACE enzyme activity in vitro;

[0026] The components capable of inhibiting ACE enzyme activity in vitro are separated by C18 chromatography column and sequenced by mass spectrometry to obtain fermented wheat antihypertensive peptides.

[0027] In the present invention, the wheat raw material is preferably in the form of crushed whole wheat. The preparation method of the wheat is preferably to screen and clean the wheat, dry it, and crush it. The particle size of the wheat flour is preferably 5 to 100 meshes, more preferably 10 meshes. The present invention has no special restrictions on the type of wheat, and the grains of wheat varieties well known in the art can be used. The crushed wheat is preferably sterilized before fermentation. The present invention has no special restrictions on the sterilization method, and a sterilization method well known in the art can be used, such as high-pressure steam sterilization.

[0028] In the present invention, the inoculation amount of yeast is preferably 2% to 8%, more preferably 4%. The yeast liquid concentration is preferably (1 to 10)×10 8CFU / mL. The fermentation temperature is preferably 20-35°C, more preferably 22-32°C, and most preferably 28°C. The fermentation time is preferably 24-96h, more preferably 28-90h, further preferably 35-85h, and most preferably 48h. During the fermentation, the mass ratio of the wheat raw material to the water in the system is preferably 1:(5-20), more preferably 1:8-16, and most preferably 1:10. The present invention conducts an orthogonal experiment through four factors and three levels, and the results show that different combinations of fermentation conditions have a significant effect on the ACE enzyme inhibition rate of the fermentation broth. When the inoculation amount of the yeast is 2%, the fermentation temperature is 20°C, the fermentation time is 24h, and the mass ratio of the wheat raw material to the water in the system is 1:5, the ACE inhibition rate of the fermentation broth is only 23.6%; when the inoculation amount of the yeast is 2%, the fermentation temperature is 28°C, the fermentation time is 48h, and the mass ratio of the wheat raw material to the water in the system is 1:10, the ACE inhibition rate of the fermentation broth is only 48.9%; when the inoculation amount of the yeast is 4%, the fermentation temperature is 20°C, the fermentation time is 48h, and the mass ratio of the wheat raw material to the water in the system is 1:20, the ACE inhibition rate of the fermentation broth is only 41.3%; when the inoculation amount of the yeast is 4% , when the fermentation temperature is 28℃, the fermentation time is 96h, and the mass ratio of wheat raw material to water in the system is 1:5, the ACE inhibition rate of the fermentation liquid is only 45.5%; when the inoculation amount of the yeast is 4%, the fermentation temperature is 35℃, the fermentation time is 24h, and the mass ratio of wheat raw material to water in the system is 1:10, the ACE inhibition rate of the fermentation liquid is only 40.2%; when the inoculation amount of the yeast is 8%, the fermentation temperature is 35℃, the fermentation time is 48h, and the mass ratio of wheat raw material to water in the system is 1:5, the ACE inhibition rate of the fermentation liquid is only 43.2%; when the inoculation amount of the yeast is 8%, the fermentation temperature is 28℃, the fermentation time is 24h, and the mass ratio of wheat raw material to water in the system is 1:20, the ACE inhibition rate of the fermentation liquid reaches 70.2%. However, too high a water ratio will increase the cost of subsequent separation and purification, so the material-liquid ratio is set to 1:5 for comprehensive consideration.

[0029] In the present invention, the enrichment method is preferably carried out by ultrafiltration, specifically, the fermentation broth is centrifuged to collect the supernatant, the filtrate is collected by an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and the powder is collected by freeze drying.

[0030] In the present invention, when the chromatographic column is purified, it is preferred to elute with pure water at a flow rate of 4 to 10 mL / min, and collect a tube of eluate every 2 to 20 min. The collected eluates are tested by the in vitro ACE enzyme inhibition activity evaluation system, and the eluate with an in vitro ACE enzyme inhibition rate of 50% is further separated and sequenced. When purifying the chromatographic column, the component less than 3 kDa is preferably prepared with pure water to prepare a loading solution. The concentration of the loading solution is preferably 50 mg / mL. The chromatographic column preferably includes a Sephadex G-15 gel chromatographic column or a Phenomenex SEC Yarra Sec-2000 Prep chromatographic column. The specifications of the Sephadex G-15 gel chromatographic column are 3.7 cm × 100 cm; when the Sephadex G-15 gel chromatographic column is used for purification, the elution flow rate is 4 mL / min. The size of the Phenomenex SEC Yarra Sec-2000 Prep chromatographic column is 21.20 mm×300 mm. When the Phenomenex SEC Yarra Sec-2000 Prep chromatographic column is used for purification, the elution flow rate is 10 mL / min. In the embodiment of the present invention, the eluate fractions numbered 24, 25 and 26 are further separated and sequenced.

[0031] In the present invention, the specifications of the C18 chromatographic column are preferably 15cm×150μm, 3μm. When the C18 chromatographic column is separated, it is preferably loaded by sandwich method, and the loading volume is 5nl. The conditions of the mass spectrometry analysis are preferably ion source parameters: mass spectrometry conditions are: ion source parameters: spray voltage: 2.1kV; drift tube temperature: 250℃; ion source: EASY-Spray source; DP energy: 100; full scan resolution: 70000FWHM; scanning range: 350-1800m / z; secondary mass spectrometry resolution: 17500FWHM; automatic gain control target: 5e6; intensity threshold: 5.00E+03; fragmentation method: HCD; NCE: 29%; Top N: 20. In an embodiment of the present invention, the dodecapeptide is derived from the eluate fraction numbered 25, and the polypeptide activity is tested by an in vitro ACE inhibitory activity evaluation system. The results show that the ACE enzyme inhibition rate of the dodecapeptide is 98.58%, which is similar to the inhibitory activity of the classic ACE inhibitor captopril, and is superior to other polypeptide components isolated from the same batch.

[0032] The present invention provides a hypotensive peptide composition with ACE inhibitory activity, comprising the dodecapeptide and at least one of the following polypeptides: a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 to SEQ ID NO: 4.

[0033] In the present invention, the molar ratio of the dodecapeptide to the polypeptide is preferably 1-100:1-100, more preferably 1-5:4-10, and most preferably 1:0.01. The in vitro ACE enzyme inhibition rate of the polypeptide shown in SEQ ID NO: 2 is 67.85% or more, the in vitro ACE enzyme inhibition rate of the polypeptide shown in SEQ ID NO: 3 is 79.3% or more, and the in vitro ACE enzyme inhibition rate of the polypeptide shown in SEQ ID NO: 4 is 87.3% or more.

[0034] In the present invention, the method for preparing the antihypertensive peptide composition preferably comprises synthesizing each polypeptide and the dodecapeptide by an artificial synthesis method, and then mixing them according to a ratio.

[0035] In view of the fact that the dodecapeptide and the antihypertensive peptide composition have good in vitro ACE enzyme inhibitory activity and the antihypertensive peptide has good in vivo antihypertensive activity, the present invention provides the use of the dodecapeptide or the antihypertensive peptide composition in the preparation of antihypertensive drugs.

[0036] In view of the good in vitro ACE enzyme inhibitory activity of the dodecapeptide and the antihypertensive peptide composition, the present invention provides the use of the dodecapeptide or the antihypertensive peptide composition in the preparation of ACE enzyme inhibitors.

[0037] The present invention provides an ACE enzyme inhibitor, comprising the dodecapeptide or the antihypertensive peptide composition and auxiliary materials.

[0038] The present invention has no special limitation on the auxiliary materials, and auxiliary materials of enzyme inhibitors well known in the art can be used.

[0039] The present invention provides a blood pressure lowering polypeptide drug, comprising the dodecapeptide or the blood pressure lowering peptide composition and medically acceptable adjuvants.

[0040] The present invention has no special restrictions on the medically acceptable excipients, and the excipients of polypeptide antihypertensive drugs well known in the art can be used.

[0041] The invention provides the use of wheat in fermentation production of antihypertensive active polypeptides including the dodecapeptide.

[0042] In the present invention, Rhizopus, Mucor racemosus, Mucor tall, Mucor lussima, Monascus, Acetobacter, Lactobacillus, Streptococcus lactis, Streptococcus thermophilus and Saussurea spp. are inoculated into the crushed wheat as fermentation agents for fermentation, and the obtained fermentation broth has in vitro inhibition of ACE enzyme activity, and the twelve peptides with the sequence shown in SEQ ID NO: 1 can be detected, but the inhibition rate of each microorganism is not as good as Candida. The yeast is preferably Saccharomyces cerevisiae, Pichia pastoris, Candida, Brettanomyces and Lussima zygosaccharomyces, and Candida is most preferably. In the embodiments of the present invention, it is also verified that the wheat after crushing is enzymatically hydrolyzed by multiple proteases, among which the enzymatic hydrolysis products of papain, bromelain, ficin and neutral protease all show in vitro inhibition of ACE enzyme activity, and twelve peptides can be detected, but the inhibition rate is generally lower than the microbial fermentation effect.

[0043] The following is a detailed description of a twelve peptide with ACE inhibitory activity provided by the present invention, a screening method and an application thereof, in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] A method for screening conditions for wheat fermentation using yeast

[0046] 1) Wheat pretreatment: After the wheat is screened and cleaned, it is fully dried, mechanically crushed, and sterilized with high-temperature steam to obtain sterilized wheat flour.

[0047] 2) Yeast activation method: prepare yeast activation medium (YPD medium: glucose 20g / L, yeast extract 10g / L, peptone 20g / L), sterilize, inoculate yeast solution at 5% inoculation amount, activate at 180r / min for 48h, and obtain activated yeast solution. The yeast is Candida catenulate.

[0048] 3) Fermentation method: The concentration is 10 8 CFU / mL yeast was inoculated into a mixture of wheat and water (the mass ratio of wheat flour to water was 1:5-20) at an inoculation rate of 2%-8%, and fermented at 20-35°C and 180r / min for 24-96h. The specific parameter combinations are shown in Table 1.

[0049] Table 1 Factor level table of orthogonal experiment of yeast fermentation of wheat L9(3 4 )

[0050]

[0051] 4) Evaluation method of in vitro ACE inhibitory activity of fermentation broth: Take the sample to be tested and adjust the pH value to 8.3 with borate buffer. After centrifugation at 10000g, take 50μL of supernatant and 50μL of borate buffer (pH=8.3) with a concentration of 6.5mM hippuryl-histidyl-leucine composition, mix well and add 10μL of ACE enzyme dissolved in the above borate buffer to start the reaction. After 30min, 10% trichloroacetic acid is added to the entire reaction system for inactivation, and the amount of hippuric acid product is analyzed by liquid phase after filtration through a 0.22μm filter membrane. Repeat the above reaction with borate buffer instead of sample as a control group. Liquid phase assay conditions are as follows: Hypersil GOLD (50×2.1mm, 1.9μm), mobile phase is a 5% acetonitrile aqueous solution containing 0.1 trichloroacetic acid, flow rate 0.3ml / min; UV detection wavelength 228nm; column temperature: 35℃; injection volume: 3μl. The test results are shown in Table 2.

[0052] Table 2 Orthogonal experiment results

[0053]

[0054]

[0055] The results showed that the yield of antihypertensive peptides could be maximized when the inoculation amount was 8%, the fermentation temperature was 28°C, the fermentation time was 24h, and the solid-liquid ratio (wheat flour: water) was 1:20. However, too high a water ratio would increase the cost of subsequent separation and purification, so the solid-liquid ratio was set at 1:5 after comprehensive consideration.

[0056] Example 2

[0057] A preparation method of fermented wheat hypotensive component

[0058] Wheat pretreatment: Weigh a certain amount of wheat, wash it with clean water and dry it, grind it with a grinder, and then sterilize the crushed wheat under high pressure at 121°C for 20 minutes.

[0059] Yeast activation: prepare yeast activation medium (YPD medium: glucose 20g / L, yeast extract 10g / L, peptone 20g / L), sterilize, inoculate Candida solution, activate at 28°C, shake at 180r / min, for 48h;

[0060] Inoculate yeast for fermentation: 8 CFU / mL yeast was added into a mixture of wheat flour and water in a ratio of 1:5 at a ratio of 8%, and fermented at 28°C and 180 r / min for 24 h to obtain wheat fermentation product;

[0061] Separation of antihypertensive components: The supernatant was separated by centrifugation of wheat fermentation, and ultrafiltration was performed using an ultrafiltration tube with a molecular weight cutoff of 3kDa, and components with a molecular weight less than 3kDa after ultrafiltration were collected. The components less than 3kDa were freeze-dried to obtain a powder, and then a 50mg / ml loading solution was prepared with pure water, the loading volume was 10ml, and a Sephadex G-15 gel chromatography column (3.7*100cm) was used for purification. The eluent was pure water, the flow rate was 4mL / min, and the eluate was collected. Every 80mL was collected as a portion until no components with obvious ultraviolet absorption flowed out. A total of 27 fractions were obtained, and the eluate in the collection tube was subjected to in vitro ACE enzyme activity inhibition detection. The detection method is the same as described in Example 1. The results of in vitro ACE enzyme inhibition rate of the eluate in each section are shown in Figure 1 .

[0062] The results showed that fractions 24, 25 and 26 had high ACE enzyme inhibitory activity, reaching 80±4.5%, 91.5±8.3% and 88±2.1%, respectively, indicating that these three collected components contained antihypertensive peptides. These three components were used for subsequent fine separation and sequence determination.

[0063] Example 3

[0064] The eluate in the collection tube in Example 2 was subjected to fine separation and sequence determination, respectively, and the specific methods are as follows:

[0065] The peptide sequences in the purified fractions were identified using a liquid chromatography-mass spectrometry platform (Thermo, EASY-nLC 1000System Q-Exactive HFX).

[0066] Chromatographic conditions: C18 column (EASY-Spray column, 15cm×150μm, 3μm), mobile phase A: 100% ultrapure water, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid; sample volume 5nL (1μg peptide), sandwich method loading. Loading Pump flow rate 350nL / min, 15min. Separation flow rate 350nL / min, separation gradient: 0-5min, 6%B; 5-57min, 6-12%B; 57-71min, 12-29%B; 71-72min, 29-40%B; 72-80min, 40-95%B.

[0067] The mass spectrometry conditions were as follows: ion source parameters: spray voltage: 2.1 kV; drift tube temperature: 250°C; ion source: EASY-Spray source; DP energy: 100; full scan resolution: 70000 FWHM; scanning range: 350-1800 m / z; secondary mass spectrometry resolution: 17500 FWHM; automatic gain control target: 5e6; intensity threshold: 5.00E+03; fragmentation mode: HCD; NCE: 29%; Top N: 20.

[0068] The results showed that: by performing data analysis using PEAKS Studio 10.0, a total of 18 peptides were obtained. Various peptides were determined from the purified product (Table 3), among which peptide 1 was the antihypertensive peptide 1 to be protected by the present invention.

[0069] Example 4

[0070] The peptide segments separated and identified in Example 3 were artificially synthesized to obtain pure products, and the antihypertensive activity was tested. The specific method is as follows:

[0071] 1. Weigh 2.0 g of blank Wang resin into a clean and dry reaction tube, add 15 mL of DMF, and activate at room temperature for about 30 minutes.

[0072] 2. Filter off the solvent, add 1mmol of 5-fold molar excess of the first amino acid at the C-terminus, 5-fold molar excess of DMAP, 5-fold molar excess of DIC, and DMF as solvent, and react at room temperature for 3h. After the reaction is completed, wash with DMF 4 to 6 times, 5-6mL each time. Then add appropriate amount of pyridine and acetic anhydride in a volume ratio of 1:1, and react for 30min. After the reaction is completed, wash with DMF 4 to 6 times, 5-6mL each time.

[0073] 3. Remove the solvent by filtration, add 10mL of 20% piperidine DMF solution to the resin, stir under N2 for 10min, filter out the solution, add 10mL of 20% piperidine DMF solution, stir under N2 for 5min, filter out the solution, repeat this operation twice, wash with DMF 4 times, methanol 2 times, 5-6mL each time. And use ninhydrin method to detect the removal effect.

[0074] 4. Weigh 3 times the molar excess of the second amino acid at the C-terminus, 3 times the molar excess of HBTU and 3 times the molar excess of HOBT into a reaction tube, add an appropriate amount of DMF solution to completely dissolve it, then add 10 times the molar excess of (pure) DIEA, react at room temperature for 40 minutes, and wash with DMF 4 to 6 times, 5-6 mL each time. Take a small amount of resin and detect it with ninhydrin detection reagent. After it turns colorless, add 10 mL of 20% piperidine DMF solution to remove Fmoc, and do it twice, 10 minutes and 5 minutes respectively, then wash it with DMF 4 times and methanol 2 times, 5-6 mL each time. Take out a small amount of resin and detect it with ninhydrin detection reagent. After it turns blue, continue to synthesize the next amino acid, and repeat the above steps until the entire amino acid sequence is synthesized.

[0075] The synthesized peptides were respectively diluted to 10 mg / mL, and the in vitro ACE enzyme inhibitory activity of each newly synthesized peptide was tested using the method described in Example 1. The results are shown in Table 3.

[0076] The results showed that among the prepared peptides, 11 peptides had the ability to inhibit ACE enzyme activity, among which peptide 1 had the best in vitro ACE enzyme inhibition activity, indicating that wheat fermentation produced antihypertensive peptides. The results of the in vitro ACE enzyme inhibition activity comparison between peptide 1 and the positive ACE enzyme inhibitor captopril are shown in Figure 2 .

[0077] Table 3 Peptide sequences detected by mass spectrometry and in vitro ACE inhibitory activity of peptides

[0078]

[0079]

[0080]

[0081] Example 5

[0082] Method for preparing antihypertensive peptides by fermenting wheat with different types of yeast

[0083] Liquid culture medium was used to activate Saccharomyces cerevisiae, Pichia pastoris, Candida, Brettanomyces and Zygosaccharomyces rouxii to obtain five different types of yeast seed liquid. Each yeast seed liquid was diluted to 10 8CFU / mL, inoculated into a mixture of crushed wheat and water (1:5) at a ratio of 4%, fermented at 28°C, 180r / min for 24h, and obtained wheat fermentation; then the fermented mixture was centrifuged to obtain the supernatant, diluted 5 times, and the in vitro antihypertensive activity of the fermentation was determined by the in vitro ACE inhibitory activity evaluation method, and the liquid mass spectrometry system in Example 3 was used to set the mass spectrometry selective scanning and parallel reaction detection conditions (Table 4) with the quasi-molecular ion peak and the charge-to-mass ratio of the antihypertensive peptide (SEQ ID NO: 1). Detect whether there is antihypertensive peptide 1 in the fermentation product.

[0084] The results showed that wheat supernatants fermented by various yeasts had the ability to inhibit ACE activity in vitro, among which the wheat supernatant grown by Candida had the highest inhibition of ACE activity. At the same time, antihypertensive peptide 1 was detected in all yeast fermentation products, indicating that yeast fermentation of wheat can produce antihypertensive peptides (Table 4).

[0085] Table 4 Mass spectrometry peptide detection parameters

[0086]

[0087]

[0088] Table 5 Results of different yeast fermentation of wheat to produce antihypertensive peptides

[0089] Yeast Types ACE enzyme inhibitory activity (%) Whether antihypertensive peptide 1 was detected Saccharomyces cerevisiae 27.61±4.11 + Pichia pastoris 23.14±2.91 + Candida 46.05±1.23 + Brettanomyces 13.21±3.25 + Zygosaccharomyces rouxii 18.75±2.11 +

[0090] Example 6

[0091] Method for preparing antihypertensive active products containing dodecapeptide by fermenting wheat with different kinds of microorganisms

[0092] Rhizopus, Monascus, Monascus and Acetobacter were inoculated at a ratio of 2% into a mixture of crushed wheat and water (1:5), and fermented at 28°C, 180r / min for 24 hours; Lactobacillus, Streptococcus lactis, Streptococcus thermophilus and snow lotus freeze-dried powder were inoculated at a ratio of 2% into a mixture of crushed wheat, water and milk (1:3:2), and fermented at 37°C for 24 hours, and the snow lotus group was fermented at 28°C, 180r / min for 24 hours; then all the fermented materials were centrifuged to obtain the supernatant, and after diluting 5 times, the in vitro antihypertensive activity of the fermented materials was determined by the in vitro ACE inhibitory activity evaluation method, and the liquid quality conditions in Example 5 were used to detect whether the fermented products contained antihypertensive peptide 1.

[0093] The results showed that wheat fermented by various microorganisms had ACE inhibitory activity, and antihypertensive peptide 1 was detected in all yeast fermentation products, indicating that wheat fermented by various microorganisms can produce the target antihypertensive peptide (Table 6).

[0094] Table 6 Results of different microbial fermentation of wheat to produce antihypertensive peptides

[0095]

[0096]

[0097] Example 7

[0098] A method for producing antihypertensive peptides by fermenting wheat with protease.

[0099] Mix crushed wheat and water in a ratio of 1:5, use a homogenizer to fully break up the mixture, then add 50U / mL of papain, bromelain, ficin, neutral protease, pepsin, trypsin and chymosin at a ratio of 1%, and fully react at 37°C, 300r / min for 4h; then centrifuge to obtain the supernatant, and use the in vitro ACE inhibitory activity evaluation method to determine the in vitro antihypertensive activity of the fermentation product, and use the liquid quality conditions in Example 5 to detect whether the fermentation product contains antihypertensive peptide 1.

[0100] The results showed that wheat fully fermented with papain, bromelain, ficin and neutral protease had the ability to inhibit ACE activity, and antihypertensive peptide 1 was detected, indicating that wheat fermented with these proteases can produce the target antihypertensive peptide. Pepsin, trypsin and chymosin did not produce antihypertensive peptides.

[0101] Table 7 Results of different proteases fermenting wheat to produce antihypertensive peptides

[0102] Protease Type ACE enzyme inhibitory activity (%) Whether antihypertensive peptide 1 was detected Papain 22.01±3.25 + Bromelain 12.35±4.33 + Ficin 9.87±3.65 + Neutral protease 25.61±6.30 + Pepsin / - Trypsin / - Chymosin / -

[0103] Example 8

[0104] Effect of wheat fermented antihypertensive peptide on blood pressure in spontaneously hypertensive rats

[0105] Spontaneously hypertensive rats (SHR), male, healthy and clean grade, weighing 200-250g, a total of 32 rats, tested environmental conditions, temperature range 25℃, relative humidity range 70%, standard feed feeding, free access to food and water. SHR rats were adapted in the animal room for one week, and were randomly divided into 4 groups, 8 rats in each group, according to the measured basal blood pressure and body weight, which were a control group gavaged with normal saline, a positive control group gavaged with the commonly used antihypertensive drug captopril, an experimental group gavaged with the antihypertensive peptide 1 protected by the present invention, and a negative control group gavaged with unfermented wheat, wherein the unfermented wheat was obtained by centrifuging the crushed wheat and water, and taking the supernatant and freeze-drying. Each rat was gavaged with 2mL of liquid, wherein the concentration of the captopril group was 10mg / kg; the concentration of the experimental group and the unfermented wheat group was 100mg / mL, and the perfusion was once a day for 4 consecutive weeks. After the continuous perfusion, the blood pressure of the rats was measured once a week, and the blood pressure was measured by the rat tail artery systolic pressure method.

[0106] Results Figure 3 . The results showed that the blood pressure of the negative control group and the experimental group of unfermented wheat, which were gavaged with normal saline, did not decrease significantly, while the blood pressure of the positive control group gavaged with captopril showed a significant downward trend, and the blood pressure stabilized from the third week to the fourth week, decreasing by 21 mmgh. The blood pressure of the experimental group infused with antihypertensive peptides began to decrease in the third week, and the blood pressure decrease in the fourth week was similar to that of the antihypertensive drug captopril. The above results show that the antihypertensive peptides of the present invention also have a good blood pressure lowering effect in vivo.

[0107] The above is only a preferred embodiment of the present invention. It should be pointed out that after understanding the above invention content, those skilled in the art can make some improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. At the same time, the above embodiments do not limit the present invention. Modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims

1. A dodecapeptide having ACE inhibitory activity, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. A hypotensive peptide composition having ACE inhibitory activity, characterized in that: The method comprises the twelve peptide according to claim 1 and at least one of the following polypeptides: a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 to SEQ ID NO:

4.

3. The antihypertensive peptide composition having ACE inhibitory activity according to claim 2, characterized in that: The molar ratio of the dodecapeptide to the polypeptide is 1-100:1-100.

4. The antihypertensive peptide composition having ACE inhibitory activity according to claim 2 or 3, characterized in that: The molar ratio of the dodecapeptide to the polypeptide is 1-5:4-10.

5. Use of the dodecapeptide according to claim 1 or the antihypertensive peptide composition according to any one of claims 2 to 4 in the preparation of antihypertensive drugs.

6. An ACE enzyme inhibitor, characterized in that The method comprises the dodecapeptide according to claim 1 or the antihypertensive peptide composition according to any one of claims 2 to 4 and auxiliary materials.

7. A blood pressure lowering polypeptide drug, characterized in that: The invention comprises the dodecapeptide according to claim 1 or the antihypertensive peptide composition according to any one of claims 2 to 4 and a medically acceptable auxiliary material.

8. Use of wheat in fermentation production of a hypotensive polypeptide containing the twelve peptide according to claim 1, wherein the method for screening and separating the twelve peptide comprises the following steps: The wheat raw material is fermented under the action of yeast to obtain fermentation liquid; The fermentation temperature is 20-35°C; the fermentation time is 24-96h; the mass ratio of the wheat raw material to the water in the system is 1:(5-20); enriching components smaller than 3 kDa from the fermentation broth; Purifying the components smaller than 3 kDa by chromatographic column to collect the components that inhibit ACE enzyme activity in vitro; During purification on the chromatographic column, pure water is used for elution at a flow rate of 4 to 10 mL / min, and a tube of eluate is collected every 2 to 20 minutes; Separating the component with in vitro ACE enzyme activity through a C18 chromatographic column and performing mass spectrometry sequencing to obtain a fermented wheat antihypertensive peptide; The specifications of the C18 chromatographic column are 15 cm × 150 μm, 3 μm; when the C18 chromatographic column is separated, the sample is loaded by sandwich method, and the loading volume is 5 nl; The conditions for the mass spectrometry sequencing are: ion source parameters: mass spectrometry conditions are: ion source parameters: spray voltage: 2.1 kV; drift tube temperature: 250°C; ion source: EASY-Spray source; DP energy: 100; full scan resolution: 70000 FWHM; scan range: 350-1800 m / z; secondary mass spectrometry resolution: 17500 FWHM; automatic gain control target: 5e6; intensity threshold: 5.00E+03; fragmentation mode: HCD; NCE: 29%; Top N: 20.

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