CN115947781A - Fermented wheat antihypertensive peptide, preparation method thereof, antihypertensive peptide combination and application - Google Patents

Abstract

The invention provides fermented wheat antihypertensive peptide, a preparation method thereof, an antihypertensive peptide composition and application, and belongs to the technical field of polypeptide. The wheat can be separated and purified from the fermentation liquor after being fermented by yeast to obtain a peptide segment with obvious blood pressure reducing activity, and the sequence of the blood pressure reducing peptide is Val-Leu-Gly-Phe-Gly-Thr-Phe (VLGFGTF) through sequencing analysis. The antihypertensive peptide has the capability of inhibiting the activity of ACE enzyme in vitro, and animal experiments also show that the antihypertensive peptide has the effect close to that of the mainstream antihypertensive drug captopril.

Description

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

Technical Field

The invention belongs to the technical field of polypeptides, and particularly relates to fermented wheat antihypertensive peptide, a preparation method thereof, an antihypertensive peptide composition and application.

Background

Hypertension is the most common chronic disease in the world, is mainly characterized by the increase of arterial blood pressure with systolic pressure more than or equal to 140mmHg and diastolic pressure more than or equal to 90mmHg, and is also the most main induction factor of various cardiovascular and cerebrovascular diseases. Nowadays, a variety of synthesized antihypertensive drugs are available, but all of them cause certain side effects. Thus, edible proteins of natural origin are of increasing interest, most of which do not produce side effects. Many scholars have demonstrated that antihypertensive peptides can be derived from food, with the potential for producing ACE inhibitory peptides from vegetable proteins.

Many cereals do not have hypotensive activity per se, but proteins therein are decomposed by fermentation and enzymatic hydrolysis to release peptide fragments having hypotensive activity, and for example, patent publication No. CN106434815A discloses the preparation of soybean hypotensive peptides by membrane separation-electrodialysis technology. For another example, patent publication No. CN103052717A discloses a method for industrially producing antihypertensive active peptide from corn germ protein as a raw material. The natural peptide segment can avoid the side effect of the pril drugs. However, the types of antihypertensive peptides derived from plants reported at present are limited, and the antihypertensive activity is also uneven, and development of new plant antihypertensive peptides is urgently needed.

Disclosure of Invention

In view of the above, the present invention aims to provide a fermented wheat antihypertensive peptide, which enriches the variety of antihypertensive peptides derived from plants and has high activity of inhibiting ACE enzyme.

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

The invention provides a preparation method of fermented wheat antihypertensive peptide, which comprises the following steps:

fermenting wheat raw material under the action of yeast to obtain fermentation liquor;

enriching less than 3kDa components from the fermentation broth;

purifying the fraction of less than 3kDa by a chromatographic column, and collecting fractions inhibiting ACE enzyme activity in vitro;

and separating the component with the in vitro ACE enzyme activity inhibition function by a C18 chromatographic column and sequencing by mass spectrometry to obtain the fermented wheat antihypertensive peptide.

Preferably, the inoculation amount of the yeast is 4-12%; the fermentation temperature is 20-35 ℃, the fermentation time is 24-96 h, and the mass ratio of the wheat raw material to the water in the system is 1: (5-20).

Preferably, 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 chromatographic column is 3.7cm multiplied by 100cm; when the Sephadex G-15 gel chromatographic column is used for purification, the elution flow rate is 4mL/min; the component for inhibiting the activity of the ACE enzyme in vitro is a component for inhibiting the ACE enzyme in vitro by not less than 50 percent;

the specification of the Phenomenex SEC Yarra Sec-2000 Prep chromatographic column is 21.20mm multiplied by 300mm, and the elution flow rate is 10mL/min when the Phenomenex SEC Yarra Sec-2000 Prep chromatographic column is used for purification.

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

the mobile phase A is ultrapure water containing formic acid with the volume concentration of 0.1 percent, and the mobile phase B is acetonitrile containing formic acid with the volume concentration of 0.1 percent; the flow rate of the eluent is 350nL/min;

the separation gradient was: 0-5min, mobile phase B with the volume percentage of 6 percent and mobile phase A with the volume percentage of 94 percent;

5-57min, mobile phase B with the volume percentage of 6-12 percent and mobile phase A with the volume percentage of 88-94 percent;

57-71min, mobile phase B with the volume percentage of 12-29 percent and mobile phase A with the volume percentage of 71-88 percent;

71-72min, mobile phase B with the volume percentage of 29-40 percent and mobile phase A with the volume percentage of 60-71 percent;

72-80min, mobile phase B with the volume percentage of 40-95 percent and mobile phase A with the volume percentage of 5-60 percent.

The invention provides a fermented wheat antihypertensive peptide composition, which comprises the antihypertensive peptide and polypeptide; the polypeptide comprises at least one of: the amino acid sequence is shown as the peptide segment shown in SEQ ID NO. 2-SEQ ID NO. 3.

Preferably, the molar ratio of the antihypertensive peptide to the polypeptide is 1-100.

The invention provides an application of the antihypertensive peptide, the antihypertensive peptide obtained by the preparation method or the antihypertensive peptide composition in preparing antihypertensive drugs.

The invention provides application of the antihypertensive peptide, the antihypertensive peptide obtained by the preparation method or the antihypertensive peptide composition in preparation of an ACE enzyme inhibitor.

The invention provides an ACE enzyme inhibitor, which comprises the antihypertensive peptide or the antihypertensive peptide composition of claim 6 or 7 and auxiliary materials.

The invention provides a fermented wheat antihypertensive peptide, the amino acid sequence of which is shown in SEQ ID NO. 1. The polypeptide with the blood pressure reducing effect is separated from the wheat fermentation liquor fermented by the yeast for the first time, and the in-vitro ACE enzyme inhibition activity of the polypeptide reaches more than 90%. Spontaneous hypertension rat experiments prove that the polypeptide has good in-vivo blood pressure rise inhibition property and shows good blood pressure reduction effect from the 2 nd week of intragastric administration. Therefore, the antihypertensive peptide provided by the invention can be used for inhibiting the activity of ACE enzyme in vitro and preparing a medicine for reducing blood pressure, enriches the variety of antihypertensive medicines and greatly improves the medicinal value of wheat.

Drawings

FIG. 1 shows the separation of antihypertensive peptides from fermented wheat, the numbers below indicate the numbers of the fractions collected;

FIG. 2 is a bar graph showing the in vitro ACE enzyme inhibitory activity of fermentation supernatants of wheat fermented by different species of microorganisms;

FIG. 3 is a line graph showing the change in blood pressure in SHR rats in four weeks after the infusion of antihypertensive peptides.

Detailed Description

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)).

The invention provides a preparation method of fermented wheat antihypertensive peptide, which comprises the following steps:

fermenting wheat raw material under the action of saccharomycetes to obtain fermentation liquor;

enriching components of less than 3kDa from the fermentation broth;

purifying the fraction of less than 3kDa by using a chromatographic column, and collecting the fraction inhibiting the ACE enzyme activity in vitro;

and separating the component with the in vitro ACE enzyme activity inhibition function by a C18 chromatographic column and sequencing by mass spectrometry to obtain the fermented wheat antihypertensive peptide.

The invention ferments wheat material under the action of yeast to obtain fermentation liquor.

In the present invention, the form of the wheat raw material is preferably pulverized whole wheat. The preparation method of the wheat flour is preferably obtained by screening and cleaning wheat, drying and crushing. The grain size of the wheat flour is preferably 5 to 100 mesh, and more preferably 10 to 50 mesh. The wheat variety is not particularly limited, and the grains of the wheat variety well known in the field can be adopted. The wheat flour is preferably subjected to a sterilization treatment before fermentation. The method of sterilization in the present invention is not particularly limited, and a sterilization method known in the art, for example, an autoclave sterilization method, an ultraviolet irradiation sterilization method, and the like may be used.

In the present invention, the inoculation amount of the yeast is preferably 2% to 8%, more preferably 4%. The concentration of the bacterial liquid of the yeast strain is preferably (1-10) multiplied by 10 ⁸ CFU/mL. The yeast is preferably candida. The temperature of the fermentation is preferably 20 to 35 ℃, more preferably 22 to 32 ℃, and most preferably 28 ℃. The fermentation time is preferably 24 to 96 hours, more preferably 28 to 90 hours, even more preferably 35 to 85 hours, and most preferably 48 hours. During the fermentation period, the mass ratio of the wheat raw material to the water in the system is preferably 1: (5 to 20), more preferably 1. The results of four-factor three-level orthogonal experiments show that the combination of different fermentation conditions has obvious influence on the ACE enzyme inhibition rate of the fermentation liquor, and the yeast strain is inoculatedThe seed amount is 2%, the fermentation temperature is 20 ℃, the fermentation time is 24h, and the mass ratio of the wheat raw material to the water in the system is 1: when 5 hours, the ACE inhibition rate of the fermentation liquor is only 23.6%; the inoculation amount of the yeast strain is 2%, the fermentation temperature is 28 ℃, the fermentation time is 48h, and the mass ratio of the wheat raw material to the water in the system is 1: when 10 hours, the ACE inhibition rate of the fermentation liquor is only 48.9%; the inoculation amount of the yeast strain is 4%, the fermentation temperature is 20 ℃, the fermentation time is 48h, and the mass ratio of the wheat raw material to the water in the system is 1: at 20 hours, the ACE inhibition rate of the fermentation liquor is only 41.3 percent; the inoculation amount of the yeast strain is 4%, the fermentation temperature is 28 ℃, the fermentation time is 96h, and the mass ratio of the wheat raw material to the water in the system is 1: when 5 hours, the ACE inhibition rate of the fermentation liquor is only 45.5%; the inoculation amount of the yeast strain is 4%, the fermentation temperature is 35 ℃, the fermentation time is 24h, and the mass ratio of the wheat raw material to the water in the system is 1: when 10 hours, the ACE inhibition rate of the fermentation liquor is only 40.2%; the inoculation amount of the yeast strain is 8%, the fermentation temperature is 35 ℃, the fermentation time is 48h, and the mass ratio of the wheat raw material to the water in the system is 1: when 5 hours, the ACE inhibition rate of the fermentation liquor is only 43.2%; the inoculation amount of the yeast strain is 8%, the fermentation temperature is 28 ℃, the fermentation time is 24h, and the mass ratio of the wheat raw material to the water in the system is 1: at 20 hours, the ACE inhibition rate of the fermentation liquor reaches 70.2%. However, the cost of subsequent separation and purification is increased due to an excessively high water ratio, so that the feed-liquid ratio is set to be 1:5.

after the fermentation liquor is obtained, the invention enriches the components less than 3kDa from the fermentation liquor.

In the invention, the enrichment method is preferably completed by adopting an ultrafiltration method, and specifically, the fermentation liquor is centrifuged, then supernatant is collected, a filtrate is collected through an ultrafiltration tube with the molecular weight cutoff of 3kDa, and powder is collected through freeze drying.

After the component less than 3kDa is obtained, the component less than 3kDa is subjected to chromatographic column purification, and the component inhibiting the ACE enzyme activity in vitro is collected.

In the present invention, the fraction of less than 3kDa is preferably used to prepare the loading solution with pure water. The concentration of the loading solution is preferably 50mg/mL. The chromatographic column preferably 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 chromatographic column is 3.7cm multiplied by 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 SEC Yarra Sec-2000 Prep chromatographic column is 21.20mm multiplied by 300mm, and the elution flow rate is 10mL/min when the Phenomenex SEC Yarra Sec-2000 Prep chromatographic column is used for purification. In the embodiment of the invention, the components which can inhibit ACE enzyme in vitro with the inhibiting rate of not less than 50 percent are collected.

After obtaining the eluate, the invention separates the eluate by a C18 chromatographic column and performs mass spectrometry to obtain the antihypertensive peptide.

In the present invention, the specification of the C18 column is preferably 15cm × 150 μm,3 μm. In the fine separation, the sample is preferably loaded by a sandwich method, and the loading volume is 5nl.

In the present invention, the conditions of the mass spectrometry are preferably ion source parameters: spraying voltage: 2.1kV; temperature of the drift tube: 250 ℃; an ion source: EASY-Spray source; DP energy: 100, respectively; full scan resolution: 70000FWHM; scanning range: 350-1800m/z; second-order mass spectrum resolution: 17500FWHM; automatic gain control target: 5e6; intensity threshold value: 5.00E +03; the cracking mode is as follows: HCD; NCE:29 percent; top N:20.

in the invention, the prepared antihypertensive peptide is subjected to in vitro detection of ACE enzyme inhibitory activity. The result shows that the ACE enzyme inhibition rate of the antihypertensive peptide is more than 90%. Meanwhile, a rat model of essential hypertension is taken as a target, and the antihypertensive peptide shows the function of reducing blood pressure after being perfused for 2 weeks.

The invention provides a fermented wheat antihypertensive peptide composition, which comprises the antihypertensive peptide and polypeptide; the polypeptide comprises at least one of: the amino acid sequence is shown as the peptide segment shown in SEQ ID NO. 2-SEQ ID NO. 3.

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

In the present invention, the molar ratio of the antihypertensive peptide to the polypeptide is preferably 1 to 100, more preferably 3 to 85;2 to 90, more preferably 5 to 70, still more preferably 7 to 50, and most preferably 1 to 30, and most preferably 1. The molar ratio of the peptide fragments in the polypeptide is preferably 1.

In view of the fact that the antihypertensive peptide and the antihypertensive peptide composition have good in vitro ACE enzyme inhibitory activity and in vivo antihypertensive activity, the invention provides the application of the antihypertensive peptide, the antihypertensive peptide obtained by the preparation method or the antihypertensive peptide composition in preparing antihypertensive drugs.

The formulation and preparation of the hypotensor are not particularly limited in the present invention, and the formulation and preparation method of the hypotensor known in the art can be adopted.

In view of the fact that the antihypertensive peptide and the antihypertensive peptide composition have good in-vitro ACE enzyme inhibitory activity, the invention provides the application of the antihypertensive peptide, the antihypertensive peptide obtained by the preparation method or the antihypertensive peptide composition in preparing an ACE enzyme inhibitor. The dosage form and preparation method of the ACE enzyme inhibitor are not particularly limited in the present invention, and the dosage form and preparation method of the enzyme inhibitor well known in the art may be adopted.

The invention provides an ACE enzyme inhibitor which comprises antihypertensive peptide or the antihypertensive peptide composition and auxiliary materials.

The auxiliary materials are not particularly limited in the present invention, and those of enzyme inhibitors well known in the art may be used.

The fermented wheat antihypertensive peptide provided by the invention, the preparation method thereof, the antihypertensive peptide composition and the application are described in detail in the following with reference to the examples, but the fermented wheat antihypertensive peptide, the preparation method, the antihypertensive peptide composition and the application are not to be construed as limiting the scope of the invention.

Example 1

Method for screening conditions of fermenting wheat by yeast strain

1) Pretreatment of wheat: the wheat is screened and cleaned, fully dried, mechanically crushed and sterilized by high-temperature steam to obtain sterilized wheat flour.

2) The yeast strain activation method comprises the following steps: preparing yeast activation culture medium (YPD culture medium: glucose 20g/L, yeast extract 10g/L, peptone 20 g/L), sterilizing, inoculating yeast liquid according to inoculum size of 5%, and activating at 180r/min for 48h to obtain activated yeast liquid. Wherein the yeast is Candida (Candida catenulate).

3) The fermentation method comprises the following steps: to a concentration of 10 ⁸ The CFU/mL yeast is inoculated into a mixture (the mass ratio of wheat flour to water is 1.

TABLE 1 Yeast fermentation wheat orthogonal test factor horizon L9 (3) ⁴ )

4) The evaluation method of the in vitro ACE inhibitory activity of the fermentation liquor comprises the following steps: a sample to be tested was adjusted to pH 8.3 with a borate buffer, 50. Mu.L of the supernatant and 50. Mu.L of a borate buffer of a equacyl-histidyl-leucine composition (pH = 8.3) at a concentration of 6.5mM were centrifuged at 10000g, and after mixing well, 10. Mu.L of ACE enzyme dissolved with the above borate buffer was added to initiate the reaction. After 30min, 10% trichloroacetic acid is added into the whole reaction system for inactivation, and the product hippuric acid is filtered by a filter membrane of 0.22 mu m and used for liquid phase analysis. The above reaction was repeated using borate buffer instead of the sample as a control. The liquid phase measurement conditions were as follows: hypersil GOLD (50X 2.1mm,1.9 μm) with a mobile phase of 5% acetonitrile in water containing 0.1 trichloroacetic acid at a flow rate of 0.3ml/min; the ultraviolet detection wavelength is 228nm; column temperature: 35 ℃; sample introduction amount: 3 μ l. The results are shown in Table 2.

TABLE 2 results of orthogonal experiments

The results show that at an inoculum size of 8% fermentation temperature of 28 ℃ and a fermentation time of 24h, a feed-to-liquid ratio (wheat flour: water) of 1. However, the cost of subsequent separation and purification is increased due to an excessively high water ratio, so that the feed-liquid ratio is set to be 1:5.

example 2

Preparation method of fermented wheat antihypertensive component

Pretreatment of wheat: weighing a certain amount of wheat, washing with clear water and drying, pulverizing with a grinder, and autoclaving the pulverized wheat at 121 deg.C for 20 min.

Activation of yeast: preparing yeast activation medium (YPD medium: glucose 20g/L, yeast extract 10g/L, peptone 20 g/L), sterilizing, inoculating Candida catenulata (Candida catenulate) bacterial liquid, activating for 48h at 28 deg.C with shaking bed 180 r/min;

inoculating yeast for fermentation: to a concentration of 10 ⁸ Inoculating 8% of CFU/mL yeast into wheat flour and water according to the proportion of 1:5, fermenting for 24 hours at 28 ℃ under the condition of 180r/min to obtain a wheat fermentation product;

separation of the depressurization component: fermenting and centrifuging the wheat to separate supernatant, performing ultrafiltration by using an ultrafiltration tube with the molecular weight cutoff of 3kDa, and collecting the components with the molecular weight less than 3kDa after ultrafiltration. Freeze-drying the fraction less than 3kDa to obtain powder, preparing 50mg/mL sample solution with a sample volume of 10mL with pure water, purifying with Sephadex G-15 gel chromatography column (3.7X 100cm), collecting eluate with pure water flow rate of 4mL/min, collecting eluate, collecting one part per 80mL until no component with obvious ultraviolet absorption flows out, to obtain 27 fractions, and detecting in vitro ACE enzyme activity inhibition of the eluate in the collection tube, wherein the detection method is as described in example 1. The in vitro ACE enzyme inhibition results for each zone eluate are shown in table 3 and figure 1.

The results show that: fractions 6, 7 and 8 had higher ACE enzyme inhibitory activity, reaching 92.1 ± 3.5%,89.6 ± 7.2% and 91 ± 1.3%, respectively, indicating that the three collected fractions contained antihypertensive peptides.

Example 3

Preparation method of wheat antihypertensive peptide

The eluate in the collection tube remaining in example 2 was subjected to fine separation and sequence determination, respectively, as follows:

the peptide sequence of the purified fraction was identified using a liquid chromatography-mass spectrometry platform (Thermo, EASY-nLC 1000System Q-active HFX).

The chromatographic conditions are as follows: c18 column (EASY-Spray column, 15cm. Times.150. Mu.m, 3. Mu.m), mobile phase A:100% ultrapure water, 0.1% formic acid; and (3) mobile phase B:100% acetonitrile, 0.1% formic acid; the loading volume was 5nL (1. Mu.g polypeptide), and loading was performed by the sandwich method. The Loading Pump flow rate was 350nL/min,15min. The separation flow rate is 350nL/min, and the separation gradient is as follows: 0-5min,6% by volume B;5-57min, 6-12%; 57-71min,12-29% B;71-72min,29-40% of B;72-80min,40-95% by weight B.

The mass spectrum conditions are as follows: ion source parameters: spray voltage: 2.1kV; drift tube temperature: 250 ℃; an ion source: EASY-Spray source; DP energy: 100, respectively; full scan resolution: 70000FWHM; scanning range: 350-1800m/z; second-order mass spectrum resolution: 17500FWHM; automatic gain control target: 5e6; intensity threshold value: 5.00E +03; a cracking mode: HCD; NCE:29 percent; top N:20.

the results show that: a total of 39 peptide fragments were determined from the purified product by data analysis of PEAKS Studio 10.0 (Table 3), wherein peptide fragment 1 is the antihypertensive peptide to be protected according to the present invention.

Example 4

The peptide fragment synthesis method separated and identified in the embodiment 3 is used for obtaining a pure product and detecting the antihypertensive activity, and the specific method is as follows:

1. 2.0g of blank Wang resin is weighed into a clean and dry reaction tube, 15mL of DMF is added, and the mixture is activated for about 30min at room temperature.

2. The solvent was filtered off with suction, and 1mmol of the first C-terminal amino acid in 5-fold molar excess, DMAP in 5-fold molar excess, DIC in 5-fold molar excess, and DMF were added as solvent and reacted at room temperature for 3 hours. After the reaction is finished, washing the reaction product with DMF for 4 to 6 times, and 5 to 6mL each time. Then adding a proper amount of pyridine and acetic anhydride with the volume ratio of 1. After the reaction is finished, washing the reaction product with DMF for 4 to 6 times, and 5 to 6mL each time.

3. Suction filtration to remove solvent, 1 mL 20% piperidine DMF solution into the resin, N2 stirring for 10min filtering solution, then adding 10mL 20% piperidine DMF solution, N2 blowing stirring for 5min filtering solution, repeat this operation twice, using DMF washing 4 times, methanol washing 2 times, each time 5-6mL. And the removal effect is detected by an indantrione method.

4. Weighing 3 times molar excess of the second amino acid at the C terminal, 3 times molar excess of HBTU and 3 times molar excess of HOBT in a reaction tube, adding a proper amount of DMF solution to completely dissolve the second amino acid, then adding 10 times molar excess of (pure) DIEA, reacting for 40min at room temperature, and washing for 4-6 times with DMF, wherein each time is 5-6mL. Detecting a small amount of resin with ninhydrin detection reagent, adding 10mL of 20% piperidine DMF solution to remove Fmoc after the resin is colorless, performing Fmoc twice for 10min and 5min respectively, and then washing with DMF for 4 times and methanol for 2 times, wherein each time is 5-6mL. And taking out a small amount of resin, detecting the resin by using a ninhydrin detection reagent, continuously synthesizing the next amino acid after detecting the resin to be blue, and repeating the steps until all amino acid sequences are synthesized.

The synthesized peptide fragments were separately diluted to 10mg/mL and the in vitro ACE enzyme inhibitory activity assay was performed on each newly synthesized peptide fragment using the method described in example 1. The results are shown in Table 3.

The results show that: the prepared peptide fragments generally have the capacity of inhibiting the activity of ACE enzyme, namely the peptide fragments belong to antihypertensive peptides, wherein the peptide fragment 1 has the optimal activity of inhibiting the ACE enzyme in vitro.

TABLE 3 peptide sequences and in vitro ACE enzyme inhibitory Activity of peptide fragments detected by Mass Spectrometry

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Example 5

Respectively activating Saccharomyces cerevisiae, hansenula anomala, hansenula cerealis, hansenula sordida, hansenula anomala, saccharomyces cerevisiae, pichia pastoris, zygosaccharomyces bailii, candida, bacillus subtilis, rhizopus and Monascus ruber, and diluting 10 kinds of yeasts and Bacillus subtilis to a constant volume of 10 ⁸ CFU/mL, inoculating 4% of the mixture into a mixture of crushed wheat and water (1; the fermented mixture was then centrifuged to obtain a supernatant, which was diluted 5-fold with buffer, and the in vitro hypotensive activity of the fermentate was determined using the in vitro ACE inhibitory activity evaluation method, while using the liquid system of liquid in example 3, mass selective scanning and parallel reaction detection conditions were set with the peak of the peptide sequence 1 excimer ion and the ratio of the ion charge to mass in the hypotensive peptide sequence (table 4). Detecting whether the fermentation product contains antihypertensive peptide 1.

Table 4 mass spectrometry peptide fragment detection parameters:

the results show that: the wheat products fermented by the microorganisms under certain conditions all have in vitro ACE enzyme inhibition activity, wherein the Candida J14-4 inhibition activity is the strongest (figure 2); liquid chromatography and mass spectrometry showed that antihypertensive peptides were detected in all the microbial fermentations except for Hansenula anomala DTM9 and Candida L9-6.

Example 6

Experiment of influence of wheat fermented antihypertensive peptide on blood pressure of primary hypertension rats

Primary hypertension rats (SHR), male, healthy, clean grade, weight 200-250g, 32 total, test environmental conditions, temperature range 25 deg.C, relativeHumidity range of 70%, standard feed for feeding, free eating and drinkingWater (W). The SHR rats are adapted to an animal room for one week, and are randomly divided into 4 groups according to the measured basic blood pressure and body weight, wherein each group comprises 8 rats which are respectively a control group of gavage normal saline, a positive control group of gavage common antihypertensive drug captopril, an experimental group of the antihypertensive peptide protected by the invention, and a negative control group of unfermented wheat after gavage, wherein the unfermented wheat is obtained by crushing wheat, fully mixing with water, centrifuging, taking supernatant, cooling, and drying. Each rat is perfused with 2mL of liquid, wherein the administration concentration of the captopril group is 10mg/kg; the gavage concentration of the experimental group and the unfermented wheat group was 100mg/mL, and the administration was performed once a day for 4 weeks. After the end of continuous perfusion, the blood pressure of the rats was measured once a week by the tail artery systolic pressure method.

The results are shown in FIG. 3. The results show that: the blood pressure of the negative control group of the gavage normal saline and the blood pressure of the experimental group of the unfermented wheat are not obviously reduced, the blood pressure of the positive control group fed with captopril is obviously reduced, the blood pressure is stable from the third week to the fourth week, and the blood pressure is reduced by 21mmgh. The blood pressure of the experimental group filled with the antihypertensive peptide begins to decrease until the third week, and the blood pressure decrease is similar to that of the antihypertensive drug captopril until the fourth week. In addition, the blood pressure of rats gavaged with unfermented wheat samples was not significantly improved. The results show that the antihypertensive peptide of the invention also has good effect of reducing blood pressure in vivo.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, after understanding the above summary of the invention, those skilled in the art may make various modifications and alterations without departing from the principle of the present invention, and such modifications and alterations should also be considered as protection scope of the present invention. Meanwhile, the above embodiments do not limit the present invention, and modifications, equivalent substitutions, and improvements, etc., made within the spirit and principle of the present invention are included in the scope of the present invention.

Claims (10)

1. A fermented wheat antihypertensive peptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A method for preparing fermented wheat antihypertensive peptides according to claim 1, which comprises the following steps:

fermenting wheat raw material under the action of yeast to obtain fermentation liquor;

enriching less than 3kDa components from the fermentation broth;

purifying the fraction of less than 3kDa by a chromatographic column, and collecting fractions inhibiting ACE enzyme activity in vitro;

and separating the component with the in vitro ACE enzyme activity inhibition function by a C18 chromatographic column and sequencing by mass spectrometry to obtain the fermented wheat antihypertensive peptide.

3. The method according to claim 2, wherein the yeast is inoculated in an amount of 4% to 12%; the fermentation temperature is 20-35 ℃, the fermentation time is 24-96 h, and the mass ratio of the wheat raw material to the water in the system is 1: (5-20).

4. The method of claim 3, wherein the chromatographic column comprises a SephadexG-15 gel chromatographic column or a Phenomenex SEC Yarra Sec-2000 Prep chromatographic column;

the specification of the Sephadex G-15 gel chromatographic column is 3.7cm multiplied by 100cm; when the Sephadex G-15 gel chromatographic column is adopted for purification, the elution flow rate is 4mL/min; the component for inhibiting the activity of the ACE enzyme in vitro is a component for inhibiting the ACE enzyme in vitro by not less than 50 percent;

the specification of the Phenomenex SEC Yarra Sec-2000 Prep chromatographic column is 21.20mm multiplied by 300mm, and the elution flow rate is 10mL/min when the Phenomenex SEC Yarra Sec-2000 Prep chromatographic column is used for purification.

5. The preparation method according to any one of claims 2 to 4, wherein the eluent for the C18 chromatographic column separation comprises a mobile phase A and a mobile phase B;

the mobile phase A is ultrapure water containing formic acid with the volume concentration of 0.1 percent, and the mobile phase B is acetonitrile containing formic acid with the volume concentration of 0.1 percent; the flow rate of the eluent is 350nL/min;

the separation gradient was: 0-5min,6% mobile phase B by volume percentage, and 94% mobile phase A by volume percentage;

5-57min, mobile phase B with the volume percentage of 6-12 percent and mobile phase A with the volume percentage of 88-94 percent;

57-71min, mobile phase B with the volume percentage of 12-29 percent and mobile phase A with the volume percentage of 71-88 percent;

71-72min, mobile phase B with the volume percentage of 29-40 percent and mobile phase A with the volume percentage of 60-71 percent;

72-80min, mobile phase B with the volume percentage of 40-95 percent and mobile phase A with the volume percentage of 5-60 percent.

6. A fermented wheat antihypertensive peptide composition, comprising the antihypertensive peptide and polypeptide of claim 1; the polypeptide comprises at least one of: the amino acid sequence is shown as the peptide segment shown in SEQ ID NO. 2-SEQ ID NO. 3.

7. The fermented wheat antihypertensive peptide composition of claim 6, wherein the molar ratio of the antihypertensive peptide to the polypeptide is 1-100.

8. Use of the antihypertensive peptide of claim 1, the antihypertensive peptide obtained by the production method of any one of claims 2 to 5, or the antihypertensive peptide composition of claim 6 or 7 for the production of antihypertensive drugs.

9. Use of the peptide according to claim 1, the peptide according to any one of claims 2 to 5, or the peptide composition according to claim 6 or 7 for the preparation of an ACE enzyme inhibitor.

10. An ACE enzyme inhibitor comprising the antihypertensive peptide of claim 1 or the antihypertensive peptide composition of claim 6 or 7 and an auxiliary.

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