Fermented wheat antihypertensive peptide and preparation method thereof, antihypertensive peptide combination and application thereof

Abstract

The present invention provides a fermented wheat antihypertensive peptide and a preparation method thereof, an antihypertensive peptide composition and application, and belongs to the field of polypeptide technology. The present invention separates and purifies peptide segments with significant antihypertensive activity from the fermentation broth after wheat is fermented by yeast. After sequencing analysis, the sequence of the antihypertensive peptide is Val-Leu-Gly-Phe-Gly-Thr-Phe (VLGFGTF). The antihypertensive peptide of the present invention has the ability to inhibit ACE enzyme activity in vitro, and animal experiments also show that the antihypertensive peptide has an effect close to that of the mainstream antihypertensive drug captopril.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptides, and in particular relates to a fermented wheat antihypertensive peptide and a preparation method thereof, a antihypertensive peptide composition and application thereof. Background Art

[0002] Hypertension is the most common chronic disease in the world, characterized by increased arterial blood pressure of systolic pressure ≥140mmHg and diastolic pressure ≥90mmHg. It is also the main predisposing factor for various cardiovascular and cerebrovascular diseases. Nowadays, there are many kinds of synthetic antihypertensive drugs, but these drugs will cause certain side effects. Therefore, edible proteins from natural sources are increasingly attracting attention, most of which do not produce side effects. Many scholars have proved that antihypertensive peptides can be derived from food, among which plant protein has great potential for preparing ACE inhibitory peptides.

[0003] Many cereals themselves do not have antihypertensive activity, but through fermentation and enzymatic hydrolysis, the proteins therein are decomposed to release peptides with antihypertensive activity. For example, the patent with publication number CN106434815A discloses the preparation of soybean antihypertensive peptides using membrane separation-electrodialysis technology. Another example is the patent with publication number CN103052717A, which discloses a method for industrial production of antihypertensive active peptides using corn germ protein as raw material. This natural peptide can avoid the side effects of "pril" drugs. However, the types of antihypertensive peptides from plant sources reported so far are limited, and the antihypertensive activity is also uneven. It is urgent to develop new plant antihypertensive peptides. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a fermented wheat antihypertensive peptide, which enriches the types of plant-derived antihypertensive peptides and has a higher activity in inhibiting ACE enzyme.

[0005] The invention provides a fermented wheat antihypertensive peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0006] The present invention provides a method for preparing the fermented wheat antihypertensive peptide, comprising the following steps:

[0007] The wheat raw material is fermented under the action of yeast to obtain fermentation liquid;

[0008] enriching components smaller than 3 kDa from the fermentation broth;

[0009] Purifying the components smaller than 3 kDa by chromatographic column to collect the components that inhibit ACE enzyme activity in vitro;

[0010] 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.

[0011] Preferably, the inoculation amount of the yeast is 4% to 12%; the fermentation temperature is 20 to 35° C., the fermentation time is 24 to 96 hours, and during the fermentation period, the mass ratio of the wheat raw material to the water in the system is 1:(5 to 20).

[0012] Preferably, the chromatographic column comprises a Sephadex G-15 gel chromatographic column or a Phenomenex SEC YarraSec-2000 Prep chromatographic column;

[0013] The specification of the Sephadex G-15 gel chromatography column is 3.7 cm×100 cm; when the Sephadex G-15 gel chromatography column is used for purification, the elution flow rate is 4 mL / min; the component having in vitro ACE enzyme inhibition activity is a component having an in vitro ACE enzyme inhibition rate of not less than 50%;

[0014] The specification 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.

[0015] Preferably, the eluent separated by the C18 chromatographic column comprises mobile phase A and mobile phase B;

[0016] The mobile phase A is ultrapure water containing 0.1% formic acid by volume, and the mobile phase B is acetonitrile containing 0.1% formic acid by volume; the flow rate of the eluent is 350 nL / min;

[0017] The separation gradient is: 0-5min, 6% by volume mobile phase B, 94% by volume mobile phase A;

[0018] 5-57min, 6% to 12% by volume mobile phase B, 88% to 94% by volume mobile phase A;

[0019] 57-71min, 12% to 29% by volume mobile phase B, 71% to 88% by volume mobile phase A;

[0020] 71-72min, 29% to 40% by volume mobile phase B, 60% to 71% by volume mobile phase A;

[0021] 72-80min, 40% to 95% by volume of mobile phase B, 5% to 60% by volume of mobile phase A.

[0022] The invention provides a fermented wheat antihypertensive peptide composition, comprising the antihypertensive peptide and a polypeptide; the polypeptide comprises at least one of the following: a peptide segment with an amino acid sequence as shown in SEQ ID NO: 2 to SEQ ID NO: 3.

[0023] Preferably, the molar ratio of the antihypertensive peptide to the polypeptide is 1-100:1-100.

[0024] The present invention provides the use of the antihypertensive peptide, the antihypertensive peptide obtained by the preparation method or the antihypertensive peptide composition in the preparation of antihypertensive drugs.

[0025] The present invention provides use of the antihypertensive peptide, the antihypertensive peptide obtained by the preparation method or the antihypertensive peptide composition in the preparation of ACE enzyme inhibitors.

[0026] The present invention provides an ACE enzyme inhibitor, comprising the antihypertensive peptide or the antihypertensive peptide composition according to claim 6 or 7 and excipients.

[0027] The present invention provides a fermented wheat antihypertensive peptide, the amino acid sequence of which is shown in SEQ ID NO: 1. The present invention is the first to isolate a polypeptide with antihypertensive effect from wheat fermentation broth fermented by yeast, and the polypeptide has an in vitro inhibition of ACE enzyme activity of more than 90%. And it has been verified by experiments on spontaneously hypertensive rats that the polypeptide has a good property of inhibiting blood pressure increase in vivo, and shows a good blood pressure lowering effect starting from the second week of intragastric administration. Therefore, the antihypertensive peptide provided by the present invention can be used to inhibit ACE enzyme activity in vitro and prepare blood pressure lowering drugs, enriching the types of antihypertensive drugs and greatly improving the medicinal value of wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the result of separation of antihypertensive peptides from fermented wheat. The numbers below indicate the numbers of the collected fractions.

[0029] Figure 2 The bar graph shows the in vitro inhibition of ACE activity of fermentation supernatants of wheat fermented by different types of microorganisms.

[0030] 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

[0031] The invention provides a fermented wheat antihypertensive peptide, the amino acid sequence of which is shown in SEQ ID NO: 1 (Val-Leu-Gly-Phe-Gly-Thr-Phe (VLGFGTF)).

[0032] The present invention provides a method for preparing the fermented wheat antihypertensive peptide, comprising the following steps:

[0033] The wheat raw material is fermented under the action of yeast to obtain fermentation liquid;

[0034] enriching components smaller than 3 kDa from the fermentation broth;

[0035] Purifying the components smaller than 3 kDa by chromatographic column to collect the components that inhibit ACE enzyme activity in vitro;

[0036] 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.

[0037] The invention ferments wheat raw materials under the action of yeast to obtain fermentation liquid.

[0038] In the present invention, the wheat raw material is preferably in the form of crushed whole wheat. The preparation method of the wheat flour is preferably to screen and clean the wheat, dry it, and crush it. The particle size of the wheat flour is preferably 5-100 mesh, more preferably 10-50 mesh. 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 wheat flour 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, ultraviolet irradiation sterilization, etc.

[0039] In the present invention, the inoculation amount of the yeast is preferably 2% to 8%, more preferably 4%. The bacterial concentration of the yeast strain is preferably (1 to 10)×10 8CFU / mL. The yeast is preferably Candida. 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 is subjected to a four-factor three-level orthogonal experiment, 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 strain 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 strain 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 strain 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 strain 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 strain 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 strain 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 strain 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 based on comprehensive considerations.

[0040] After obtaining the fermentation broth, the present invention enriches components less than 3 kDa from the fermentation broth.

[0041] 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.

[0042] After obtaining the component smaller than 3 kDa, the present invention purifies the component smaller than 3 kDa by a chromatographic column, and collects the component that inhibits ACE enzyme activity in vitro.

[0043] In the present invention, the component less than 3kDa is preferably prepared with pure water to prepare a loading solution. The concentration of the loading solution is preferably 50mg / mL. The chromatographic column preferably includes a Sephadex G-15 gel chromatographic column or a Phenomenex SECYarra Sec-2000 Prep chromatographic column. The specification of the Sephadex G-15 gel chromatographic column is 3.7cm×100cm; when the Sephadex G-15 gel chromatographic column is used for purification, the elution flow rate is 4mL / min. The specification of the Phenomenex SECYarra Sec-2000 Prep chromatographic column is 21.20mm×300mm, and when the Phenomenex SEC YarraSec-2000 Prep chromatographic column is used for purification, the elution flow rate is 10mL / min. In an embodiment of the present invention, components with an in vitro inhibition rate of not less than 50% of ACE enzyme inhibition are collected.

[0044] After obtaining the eluate, the present invention separates the eluate through a C18 chromatographic column and performs mass spectrometry sequencing to obtain the antihypertensive peptide.

[0045] In the present invention, the specification of the C18 chromatographic column is preferably 15 cm×150 μm, 3 μm. During the fine separation, the sandwich method is preferably used for sample loading, and the sample loading volume is 5 nl.

[0046] In the present invention, the conditions for the mass spectrometry analysis are preferably 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.

[0047] In the present invention, the prepared antihypertensive peptide was tested for ACE enzyme inhibition activity in vitro. The results showed that the ACE enzyme inhibition rate of the antihypertensive peptide was more than 90%. At the same time, a primary hypertension rat model was used as a subject, and the antihypertensive peptide showed a blood pressure lowering effect after oral administration for 2 weeks.

[0048] The invention provides a fermented wheat antihypertensive peptide composition, comprising the antihypertensive peptide and a polypeptide; the polypeptide comprises at least one of the following: a peptide segment with an amino acid sequence as shown in SEQ ID NO: 2 to SEQ ID NO: 3.

[0049] In the present invention, the in vitro ACE enzyme inhibition rate of the peptide segment with the amino acid sequence as shown in SEQ ID NO: 2 to SEQ ID NO: 3 reaches more than 86%.

[0050] In the present invention, the molar ratio of the hypotensive peptide to the polypeptide is preferably 1-100:1-100, more preferably 3-85; 2-90, further preferably 5-70:8-79, further preferably 7-50:1-30, and most preferably 1:0.01. The molar ratio of each peptide segment in the polypeptide is preferably 1:1.

[0051] In view of the good in vitro ACE enzyme inhibitory activity and in vivo antihypertensive activity of the antihypertensive peptide and the antihypertensive peptide composition, the present invention provides the use of the antihypertensive peptide, the antihypertensive peptide obtained by the preparation method or the antihypertensive peptide composition in the preparation of antihypertensive drugs.

[0052] The present invention has no special restrictions on the dosage form and preparation of the antihypertensive drug, and the dosage form and preparation method of the antihypertensive drug known in the art can be adopted.

[0053] In view of the good in vitro ACE enzyme inhibitory activity of the antihypertensive peptide and the antihypertensive peptide composition, the present invention provides the use of the antihypertensive peptide, the antihypertensive peptide obtained by the preparation method or the antihypertensive peptide composition in the preparation of ACE enzyme inhibitors. The present invention has no special restrictions on the dosage form and preparation method of the ACE enzyme inhibitor, and the dosage form and preparation method of the enzyme inhibitor known in the art can be used.

[0054] The present invention provides an ACE enzyme inhibitor, comprising the antihypertensive peptide or the antihypertensive peptide composition and auxiliary materials.

[0055] 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.

[0056] The fermented wheat antihypertensive peptide and its preparation method, antihypertensive peptide composition and application provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] Method for screening conditions of yeast strains for wheat fermentation

[0059] 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.

[0060] 2) Yeast strain 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.

[0061] 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.

[0062] Table 1 Factor level table of orthogonal experiment of yeast fermentation of wheat L9(3 4 )

[0063]

[0064] 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.

[0065] Table 2 Orthogonal experiment results

[0066]

[0067]

[0068] 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.

[0069] Example 2

[0070] A preparation method of fermented wheat hypotensive component

[0071] 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.

[0072] Yeast activation: prepare yeast activation medium (YPD medium: glucose 20g / L, yeast extract 10g / L, peptone 20g / L), sterilize, inoculate Candida catenulate solution, activate at 28°C, shake at 180r / min, for 48h;

[0073] 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;

[0074] 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, and each 80mL was collected as a portion until no component 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, and the detection method was 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 Tables 3 and Figure 1 .

[0075] The results showed that fractions 6, 7 and 8 had high ACE enzyme inhibitory activities, reaching 92.1±3.5%, 89.6±7.2% and 91±1.3%, respectively, indicating that these three collected components contained antihypertensive peptides.

[0076] Example 3

[0077] A preparation method of wheat antihypertensive peptide

[0078] The remaining eluate in the collection tube in Example 2 was subjected to fine separation and sequence determination, respectively, and the specific method is as follows:

[0079] The peptide sequences in the purified fractions were identified using a liquid chromatography-mass spectrometry platform (Thermo, EASY-nLC 1000System Q-Exactive HFX).

[0080] Chromatographic conditions: C18 column (EASY-Spray column, 15cm x 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.

[0081] 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.

[0082] The results showed that: through data analysis using PEAKS Studio 10.0, a total of 39 peptides were obtained. Various peptides were determined from the purified product (Table 3), among which peptide 1 was the antihypertensive peptide to be protected by the present invention.

[0083] Example 4

[0084] The peptide fragment isolated and identified in Example 3 was synthesized to obtain a pure product, and the antihypertensive activity was tested as follows:

[0085] 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.

[0086] 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.

[0087] 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 release effect.

[0088] 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 perform 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.

[0089] 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.

[0090] The results showed that the prepared peptides generally had the ability to inhibit ACE enzyme activity, that is, these peptides were antihypertensive peptides, among which peptide 1 had the best in vitro ACE enzyme inhibitory activity.

[0091] Table 3 Peptide sequences detected by mass spectrometry and in vitro ACE inhibitory activity of peptides

[0092]

[0093]

[0094]

[0095] Example 5

[0096] Activate Saccharomyces cerevisiae, Hansenella anomala, Hansenella cacti, Hansenella anomala, Saccharomyces cerevisiae, Pichia pastoris, Zygosaccharomyces bailii, Candida, Bacillus subtilis, Rhizopus and Monascus, and adjust the volume of 10 yeasts and Bacillus subtilis to 10 8CFU / mL, inoculated into a mixture of crushed wheat and water (1:5) at a ratio of 4%, and Rhizopus and Monascus were inoculated into the same mixture of crushed wheat and water at a bacterial agent mass ratio of 4%, and fermented at 28°C, 180r / min for 24h to obtain wheat fermentation; then the fermented mixture was centrifuged to obtain the supernatant, and after diluting 5 times with buffer, the in vitro antihypertensive activity of the fermentation was determined using the in vitro ACE inhibitory activity evaluation method, and at the same time, 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 of the antihypertensive peptide sequence 1 and the charge-to-mass ratio of the daughter ion. Detect whether there is antihypertensive peptide 1 in the fermentation product.

[0097] Table 4 Mass spectrometry peptide detection parameters:

[0098]

[0099] The results showed that the products of wheat fermented by the above microorganisms under certain conditions all had in vitro ACE inhibitory activity, among which Candida J14-4 had the strongest inhibitory activity ( Figure 2 ); Liquid chromatography-mass spectrometry analysis showed that antihypertensive peptides were detected in all microbial fermentations except for DTM9 and Candida albicans L9-6.

[0100] Example 6

[0101] Experimental study on the effect of wheat fermented antihypertensive peptide on blood pressure in rats with essential hypertension

[0102] Essential hypertensive rats (SHR), male, healthy clean grade, weighing 200-250g, total 32 rats, tested in environmental conditions, temperature range 25℃, relative humidity range 70%, fed with standard feed, free access to food and drink water . SHR rats were adapted in the animal room for one week, and randomly divided into 4 groups, 8 rats in each group, according to the measured basal blood pressure and body weight, including a control group gavage with normal saline, a positive control group gavage with captopril, a commonly used antihypertensive drug, an experimental group gavage with the antihypertensive peptide protected by the present invention, and a negative control group gavage with unfermented wheat, wherein the unfermented wheat was obtained by centrifuging the crushed wheat and mixing it with water, and freeze-drying the supernatant. Each rat was gavaged with 2 mL of liquid, wherein the concentration of the captopril group was 10 mg / kg; the gavage concentration of the experimental group and the unfermented wheat group was 100 mg / mL, and the perfusion was performed 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.

[0103] Results Figure 3. The results showed that the blood pressure of the negative control group and the experimental group fed with unfermented wheat did not decrease significantly, while the blood pressure of the positive control group fed 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 drop in the fourth week was similar to that of the antihypertensive drug captopril. In addition, there was no significant improvement in the blood pressure of rats gavaged with unfermented wheat samples. The above results show that the antihypertensive peptides of the present invention also have a good blood pressure lowering effect in vivo.

[0104] 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 fermented wheat antihypertensive peptide, It is characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. A method for preparing the fermented wheat antihypertensive peptide according to claim 1, It is characterized in that The following steps are involved: The wheat raw material is fermented under the action of yeast to obtain fermentation liquid; The yeast inoculation amount is 4% to 12%; the fermentation temperature is 20 to 35 ° C, the fermentation time is 24 to 96 hours, and during the fermentation period, the mass ratio of the wheat raw material to the water in the system is 1: (5 to 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; The chromatographic column comprises a Sephadex G-15 gel chromatographic column or a Phenomenex SEC Yarra Sec-2000 Prep chromatographic column; The specification of the Sephadex G-15 gel chromatography column is 3.7 cm×100 cm; when the Sephadex G-15 gel chromatography column is used for purification, the elution flow rate is 4 mL / min; the component having in vitro ACE enzyme inhibition activity is a component having an in vitro ACE enzyme inhibition rate of not less than 50%; The specification 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; 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 eluent separated by the C18 chromatographic column includes mobile phase A and mobile phase B; The mobile phase A is ultrapure water containing 0.1% formic acid by volume, and the mobile phase B is acetonitrile containing 0.1% formic acid by volume; the flow rate of the eluent is 350 nL / min; The separation gradient was: 0-5 min, 6% by volume mobile phase B, 94% by volume mobile phase A; 5-57 min, 6%~12% by volume mobile phase B, 88%~94% by volume mobile phase A; 57-71 min, 12%-29% by volume mobile phase B, 71%-88% by volume mobile phase A; 71-72 min, 29%-40% by volume mobile phase B, 60%-71% by volume mobile phase A; 72-80 min, 40%~95% by volume of mobile phase B, 5%~60% by volume of mobile phase A.

3. A fermented wheat antihypertensive peptide composition, It is characterized in that It comprises the antihypertensive peptide and polypeptide according to claim 1; the polypeptide comprises at least one of the following: a peptide segment with an amino acid sequence as shown in SEQ ID NO: 2 to SEQ ID NO:

3.

4. The fermented wheat antihypertensive peptide composition according to claim 3, It is characterized in that The molar ratio of the antihypertensive peptide to the polypeptide is 1-100:1-100.

5. Use of the antihypertensive peptide according to claim 1, the antihypertensive peptide obtained by the preparation method according to claim 2, or the antihypertensive peptide composition according to claim 3 or 4 in the preparation of antihypertensive drugs.

6. An ACE enzyme inhibitor, It is characterized in that The invention comprises the antihypertensive peptide according to claim 1 or the antihypertensive peptide composition according to claim 3 or 4 and auxiliary materials.

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