阅读说明：本技术 一种枸杞ace抑制肽以及衍生多肽和应用、混合物 (Wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture ) 是由 靳艳 庄晶玲 叶明亮 于 2019-08-29 设计创作，主要内容包括：本发明涉及一种利用枸杞蛋白通过发酵法制备血管紧张素转换酶(angiotensin-converting enzyme,ACE)抑制活性的多肽。多肽SLSLPNFHP,其氨基酸序列为Ser-Leu-Ser-Leu-Pro-Asn-Phe-His-Pro,分子量为1011.12Da。其具有良好的ACE抑制活性,具有开发成为调节血压的功能性食品或者是药物的良好应用前景。(The invention relates to a polypeptide for preparing angiotensin-converting enzyme (ACE) inhibitory activity by utilizing medlar protein through a fermentation method. The amino acid sequence of the polypeptide SLSLPNFHP is Ser-Leu-Ser-Leu-Pro-Asn-Phe-His-Pro, and the molecular weight is 1011.12 Da. The product has good ACE inhibitory activity and good application prospect in development of functional food or medicine for regulating blood pressure.)

1. An ACE inhibitory peptide of wolfberry, which is characterized in that: the polypeptide SLSLPNFHP has a sequence table SEQ ID NO: 1, amino acid sequence; the amino acid sequence of the polypeptide is Ser-Leu-Ser-Leu-Pro-Asn-Phe-His-Pro.

2. A derivative polypeptide of SLSLPNFHP wherein the polypeptide of claim 1 is the polypeptide set forth in SEQ ID NO: 1 by substituting, deleting and/or adding one or more than 2 amino acids of the amino acid sequence shown in the claim 1, and the derivative polypeptide has the same function with the small peptide of the claim 1.

3. Use of a peptide according to claim 1 or 2 for the preparation of an angiotensin-converting enzyme (ACE) inhibitor and/or a blood pressure lowering medicament.

4. The use according to claim 3, wherein the Angiotensin Converting Enzyme (ACE) inhibitor and/or antihypertensive drug comprises poly SLSLPNFHP as active ingredient, and optionally pharmaceutically acceptable carriers or adjuvants.

5. Use of the polypeptide of claim 1 or 2 as an active ingredient in an in vitro angiotensin-converting enzyme (ACE) inhibitor, or as an active ingredient in a functional food having blood pressure lowering effect.

6. The use according to claim 5, wherein the inhibitor or functional food further comprises a pharmaceutically acceptable carrier or excipient.

7. A mixture as angiotensin converting enzyme inhibitor, antihypertensive medicine or functional food with blood pressure lowering effect is prepared from SLSLPNFHP powder as active component, and optionally pharmaceutically acceptable carrier or adjuvant.

Technical Field

The invention relates to a polypeptide SLSLPNFHP with angiotensin-converting enzyme (ACE) inhibitory activity prepared from medlar protein by a fermentation method, and application thereof in functional foods and medicines for regulating blood pressure.

Background

Hypertension is one of the most common cardiovascular and cerebrovascular diseases, and is the most important risk factor for cardiovascular diseases such as stroke and coronary heart disease. With the rapid development of social economy and the change of the living dietary structure of people, the incidence of hypertension also increases year by year, and the body health of residents in China is seriously influenced. The prevention and treatment of hypertension are major problems facing the present society.

ACE is widely present in the human body as a multifunctional enzyme, and functions to regulate blood pressure by acting on the Renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS) in the body. Therefore, ACE inhibitors are widely used as a blood pressure lowering drug. However, the chemically synthesized western medicine antihypertensive drug has toxic and side effects on human bodies, particularly great burden is caused on liver and kidney tissues due to drug absorption, liver and kidney injury is caused, symptoms are repeated after drug withdrawal, and the curative effect after healing is not very ideal.

Bioactive peptides are a focus of research in recent years. The natural protein can release peptides with different structures under the action of protease and microorganisms, and researches show that the bioactive peptides show various biological activities such as blood pressure reduction, blood sugar reduction, immunity improvement, antibiosis, antioxidation and the like. The ACE inhibitor prepared by utilizing natural protein has the characteristics of definite function and high safety, so that the ACE inhibitor has wide prospect as functional food or medicine.

The invention relates to an ACE inhibitory active peptide prepared by fermenting medlar protein.

Disclosure of Invention

The object of the present invention is about the ACE inhibitory activity of polypeptide SLSLPNFHP; it has the sequence table of SEQ ID NO: 1, amino acid sequence; has good ACE inhibitory activity and has the function of lowering blood pressure by inhibiting the ACE activity. And has good application prospect in developing functional food or medicine for regulating blood pressure.

In order to achieve the purpose, the invention takes the product polypeptide SLSLPNFHP as a main component on the basis of fully utilizing the medlar protein, inhibits the activity of ACE and regulates blood pressure. On the basis, the polypeptide SLSLPNFHP can be used as the main component of ACE inhibitors, blood pressure regulating medicines and related functional products.

The polypeptide SLSLPNFHP has ACE inhibiting activity and blood pressure lowering activity, has the amino acid sequence of Ser-Leu-Ser-Leu-Pro-Asn-Phe-His-Pro, is single-chain linear structure, is white powder, is easily soluble in water, and has molecular weight of 1011.12 Da; has strong inhibiting effect on ACE activity, and the half inhibitory concentration (IC50) is 26.53 μ M.

The three-dimensional structure of the Protein ACE (PDB code:1o8a) was derived from Protein Data Bank (https:// www.rcsb.org /), predicted for the binding site of polypeptide SLSLPNFHP to ACE using bioinformatics software Peptide (http:// pepsite2. russellab. org.). The presence or absence of interaction between the polypeptide and protein is indicated by the resulting P value and binding site, where a smaller P value indicates a greater probability of binding between the polypeptide and protein, and where a P value >0.5 indicates theoretically no probability of binding between the polypeptide and protein. Binding site means a binding site of a polypeptide to a protein, and if the polypeptide binds to the active site of the protein, it inhibits the protein from interacting with other proteins, thereby inhibiting the activity of the protein.

Polypeptide SLSLPNFHP has a p value of 0.00006553, and binding sites of Trp279 and Gln281^#,His353^#,Ala354^#,Ser355,His383^#,Glu384^#,His387^#,His410,Glu411^#,Phe457,Phe460,Lys511^#,His513^#,Tyr520^#,Arg522,Tyr523^#Therein with^#Marked are important active sites and binding sites of ACE reported in the literature, and the polypeptide SLSLPNFHP has good ACE inhibitory activity.

In addition, the structure-activity relationship of bioactive peptides is related to the ACE inhibitory activity, the polypeptide SLSLPNFHP has the characteristics required by an ACE inhibitor, and the C-terminal tripeptide of the polypeptide has a strong influence on the ACE inhibitory activity. The ACE inhibitory peptide is more suitable for the peptide segment of biological active peptide ZC with hydrophobic amino acids (such as aromatic amino acid and branched chain amino acid) and aromatic amino acid (Trp, Tyr, Phe) or Pro at the tail end, which has stronger ACE inhibitory activity. The C-terminal amino acid of the polypeptide SLSLPNFHP is Pro, which is satisfactory.

The invention has the following advantages:

the invention obtains ACE inhibitory peptide from fermented lycium barbarum protein, the polypeptide SLSLPNFHP has good ACE inhibitory activity, and the half inhibitory concentration (IC50) is 26.53 mu M. Provides more diversified sources for ACE inhibitory peptides and provides more research references for the ACE inhibitory peptides.

Detailed Description

EXAMPLE 1 identification of polypeptide SLSLPNFHP

The medlar protein is subjected to bacillus subtilis fermentation, acid precipitation, ethanol precipitation and LC-MS/MS analysis, and bioactive peptides with ACE inhibitory activity are screened through bioinformatics and structure-activity relationship.

The specific method comprises the following steps:

(1) preparation of matrimony vine protein extract

Taking 10g of dry medlar as a raw material, weighing medlar, adding 100mL of deionized water, soaking for 2 hours, then crushing, ultrasonically extracting for 60min, centrifuging at 5000rpm for 10min, and using 9.6g of precipitate for extracting medlar protein.

Freeze-drying the precipitate, pulverizing again to obtain fructus Lycii powder, adding 192mL of organic solvent extract (n-hexane: ethanol: 2.6:1 (v/v)), stirring at 50 deg.C and 150rpm, extracting for 1 hr, filtering, extracting the filter cake with the same organic solvent extract once, filtering after two times, and freeze-drying to obtain 7.5g of fructus Lycii protein.

(2) Preparation of active peptide of medlar

Adding fructus Lycii protein into 112mL ionized water, adjusting pH to 7.5 with 1M sodium hydroxide solution, inoculating microorganism strain (Bacillus subtilis) with bacteria content of 2 x 10⁸CFU/mL, fermenting at 37 ℃ for 48 hours; after fermentation was completed, centrifugation was carried out at 13000rpm for 15min, and the supernatant was freeze-dried to obtain 1g of crude fermentation extract.

Adding 5mL of 0.01M hydrochloric acid into the fermented crude extract, homogenizing at 2 deg.C for 8min, centrifuging at 12000rpm for 20min, collecting supernatant, adding 3 times of anhydrous ethanol, standing at 4 deg.C for 24 hr, centrifuging at 12000rpm for 20min, collecting supernatant, and lyophilizing to obtain 0.383g of fructus Lycii polypeptide.

Performing mass spectrometry on the lycium barbarum polypeptide by using LTQ Orbitrap Velos: desalting the Lycium barbarum polypeptide, C₁₈The SPE pretreats the column, activated with 2mL acetonitrile, and then washed off with 2mL of 0.1% (v/v) TFA solution. The sample was loaded onto the column after adjusting p H to 2 with 50% (v/v) TFA solution. After desalting with 2mL of 0.1% (v/v) TFA, the eluate was eluted 3 times with 1mL of 80% (v/v) acetonitrile/0.1% (v/v) TFA, and the eluate was freeze-dried and stored at-20 ℃ for mass spectrometry. Adding a proper amount of 0.1% (v/v) formic acid into the freeze-dried sample for redissolving to prepare 0.5 mu g/muAnd (3) carrying out mass spectrometry on the solution of the L by using a linear ion trap electrostatic field Orbitrap mass spectrometer (LTQ Orbitrap Velos), and setting the temperature of an ion transmission capillary tube to be 250 ℃, the electrospray voltage to be 2.2kV and the normalized collision energy to be 35.0%. Both MS and MS/MS were mapped using a data-dependent mode (data-dependent mode). The mass spectrometry scan conditions were set as: and selecting 10 highest-abundance ion peaks from the full scan with each m/z of 400-2000 to perform MS/MS scanning. Samples are analyzed in parallel for three times, and common peptide fragments identified by the three times of analysis are taken for statistics. The collected raw file data was converted to mgf format using Thermo protein discover Daemon (v1.4) and retrieved with Mascot (version 2.3.0, Matrix Science, London, UK) spectrogram in solanaceous plant protein library solanaceous (https:// www.uniprot.org /). The three analyses collectively identify 75 common peptide fragments derived from 14 different proteins, 41.33 percent of the peptide fragments are derived from the protein Fibrillin, the number of amino acids in the peptide fragments is between 8 and 27, the calculated molecular mass is 920.4491 to 2742.4664(Da), and the table I is the mass spectrum identification result of part of the lycium barbarum polypeptides.

table-Mass Spectrometry results for Lycium barbarum Polypeptides

According to the explanation of structure-activity relationship in the invention content, the identified common peptide fragments are screened to obtain the polypeptide with the sequence of SLSLPNFHP, which is derived from the 11S globin seed storage protein 2-like protein.

Example 2 detection of ACE inhibitory Activity of polypeptide SLSLPNFHP

The polypeptide SLSLPNFHP is synthesized by Nanjing Jie peptide Biotech, and has an amino acid sequence of Ser-Leu-Ser-Leu-Pro-Asn-Phe-His-Pro, a single-chain linear structure, white powder, water solubility, and molecular weight of 1011.12 Da.

Information of SEQ ID No.1

(a) Sequence characterization

Length: 9 amino acid

Type: amino acids

Chain type: single strand

(b) Molecular type: protein

Description of the sequence:

SEQ ID No.1

SLSLPNFHP

n- (3- (2-furoyl) acryloyl-phenylamyl-glutamyl-glutamic acid (fagg,. lamda.max. about.340 nm. epsilon. about.2270M)^-1cm^-1Molecular weight 399.40) can be enzymatically hydrolyzed by ACE to N- [3- (furan) propenoyl]-2-phenylalanine (FAP,. lamda.max. 340nm,. epsilon. 1512M^-1cm^-1) And glycylglycine (GG, no absorption at 340 nm) and thus can act as a mimetic substrate for ACE. The absorbance value of 1mM FAPGG completely converted to FAP and GG was 0.758, so that the inhibition rate could be calculated from the change value of absorbance at 340 nm.

Reaction system

(1) Buffer solution: 0.1M PBS buffer (pH 8.2, containing 300mM NaCl)

(2) Substrate solution: FAPGG solution was prepared at a concentration of 1.6mM using the above buffer.

(3) Enzyme solution: ACE was formulated as a 0.2U/mL solution using the above buffer solution.

(4) Sample solution: the polypeptide SLSLPNFHP was prepared as a 0.5, 0.1, 0.01mg/mL solution with the above buffers as required for the experiment.

Experiments were performed in 96-well plates. Adding the sample solution, the ACE solution and the substrate solution in sequence according to the second table, mixing uniformly, immediately measuring absorbance at 340nm by using an enzyme-labeling instrument, and recording as OD₀After incubation at 37 ℃ for 30min, the absorbance at 340nm was again determined and recorded as OD₁. The absorbance of each well was measured, and Δ OD was defined as OD₀-OD₁. Each sample was assayed in 3 replicates.

Sample adding method for epibiace inhibitory activity

"-" represents the addition of an equal volume of PBS buffer to the column

ACE inhibition ratio calculation formula:

I＝[(ΔOD_Control-Δ OD_Sample)/(ΔOD_Control-ΔOD_blank)]*100％

Δ OD_controlrepresenting the change of absorbance after the sample solution is replaced by the same amount of buffer solution in the reaction; delta OD_SampleRepresenting the change in absorbance of the sample solution in the reaction; and Δ OD_blankThe change in absorbance of the reaction mixture after the sample solution and the enzyme solution were replaced with the same amount of buffer solution

Absorbance of the assay for inhibitory Activity of Epsantel ACE

TABLE four inhibitory Activity of SLSLPNFHP on ACE at various concentrations

The ACE inhibitory activity was measured at different concentrations of polypeptide SLSLPNFHP as described above. Table three is the absorbance of the experiment for ACE inhibitory activity at different concentrations of SLSLPNFHP, and table four is the inhibitory activity of SLSLPNFHP at different concentrations of ACE, from which the half inhibitory concentration (IC50) of polypeptide SLSLPNFHP was calculated to be 26.53 ± 11.75 μ M.

Sequence listing

<110> institute of chemistry and physics, large connection of Chinese academy of sciences

<120> Chinese wolfberry ACE inhibitory peptide, derivative polypeptide, application and mixture

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ser Leu Ser Leu Pro Asn Phe His Pro

1 5