Walnut meal protein active peptide and application thereof

Abstract

The invention discloses a walnut meal protein active peptide, which is a pentapeptide with an amino acid sequence of Phe-Asp-Trp-Leu-Arg. The walnut meal protein active peptide has the function of inhibiting angiotensin converting enzyme activity, and its half inhibition concentration (IC 50 ) value is 8.02 μg / mL. The walnut meal protein active peptide of the present invention is prepared by hydrolyzing walnut meal protein, a by-product of walnut oil processing, by conventional enzymatic hydrolysis. The raw material is a by-product of food processing; and it has a simple structure, strong activity, and high safety. It can also be obtained by artificial synthesis. The present invention provides a reference for the development of foods, health products and synthetic hypertension therapeutic drugs with ACE inhibitory function, and has broad application prospects and very important significance.

Description

Technical Field

[0001] The invention belongs to the technical field of food-derived protein active peptides, and specifically relates to an active peptide derived from walnut meal and its ability to inhibit angiotensin converting enzyme activity, and belongs to the field of high-value development of low-byproduct resources. Background Art

[0002] Bioactive peptides are specific protein fragments that can exist naturally in organisms or be produced by protein degradation during food processing. Bioactive peptides in food sources generally refer to two categories: one is bioactive peptides that can effectively promote body functions or states to improve human health, and the other is bioactive peptides that can give food a savory and mellow taste.

[0003] Hypertension is a major risk factor for the development of cardiovascular and cerebrovascular diseases. Angiotensin-converting enzyme (ACE) is a key enzyme in the pathogenesis of hypertension. At present, the number of patients with hypertension in my country has increased dramatically, and the age of the patients is getting younger and younger. Inhibiting ACE is an important and effective means of medical intervention for hypertension. However, long-term use of antihypertensive drugs has certain side effects. In this context, some food-derived protein bioactive peptides with strong ACE inhibitory activity are considered to be safe means to prevent and control the occurrence and development of hypertension. Therefore, the research and development of food-derived protein bioactive peptides has also become a hot topic in global research.

[0004] Walnut is one of the four major nuts in the world. It has extremely high nutritional value and has always been considered the best food for warming the lungs and kidneys, promoting heart and brain health, and strengthening the body. Walnut kernels are rich in oil, and the unsaturated fatty acid content in the oil is high, which is an important material basis for the economic benefits of walnut industrialization. A large amount of walnut meal is produced in the production of walnut oil. Because the protein in walnut meal can be as high as 50% or more, a small part is developed into walnut protein powder, but most of it is still used as animal feed. Walnut meal should be effectively developed and become a new economic benefit point for the walnut industry. It has been reported that walnut meal hydrolysate has ACE inhibitory activity. 201910228464.0 discloses a walnut meal polypeptide with ACE inhibitory activity and a preparation method thereof. Two peptides are obtained by using traditional technologies such as protein extraction, enzymatic hydrolysis, chromatographic separation and purification, but the IC50 value of its ACE inhibitory activity cannot be determined, and the strength of its ACE inhibitory activity cannot be quantified. Although the currently disclosed traditional development technology can clearly determine the sequence of the peptide, it has the disadvantages of requiring pretreatment of raw materials, long experimental cycle, large funding, and some highly active fragments may be ignored. Summary of the invention

[0005] The present invention makes full use of the method of bioinformatics, with the help of a bioactive peptide information library, from the perspectives of the molecular weight, bioactivity level, polarity, ADMET (absorption, distribution, metabolism, excretion and toxicity) and other properties of the bioactive peptides, combined with high-throughput mass spectrometry information, to screen peptides with potential ACE inhibitory activity from walnut meal products hydrolyzed by conventional enzymatic hydrolysis technology, and then combine molecular docking to determine the peptides that are stably bound to ACE (PDB ID: 1O8A), and step-by-step screening to obtain a pentapeptide FDWLR that has both ACE inhibitory activity and is non-toxic, soluble in water, and absorbed by the human intestine.

[0006] The pentapeptide FDWLR obtained by screening was chemically synthesized to obtain a synthetic peptide with a purity of 98%. The ability of the pentapeptide FDWLR to inhibit ACE was determined by the kit method. Its IC 50 The value is 8.02 μg / mL, or 10.9 μM.

[0007] The walnut meal protein active peptide of the invention is a pentapeptide with the ability to inhibit ACE, is derived from walnut meal, and has an amino acid sequence of Phe-Asp-Trp-Leu-Arg (FDWLR).

[0008] The composition (or effective ingredient) of the angiotensin converting enzyme inhibitor described in the present invention is the above-mentioned walnut meal protein active peptide, and one or more drugs or food acceptable excipients can also be added to improve the absorption effect of drugs and foods or facilitate use, so as to prepare suitable dosage forms, such as capsules or pills, powders, tablets, granules, oral liquids and injections, that is, suitable dosage forms for use in pharmacy, or suitable edible methods in the food field; it can be used to prepare functional foods, health products and drugs for the treatment or auxiliary treatment of hypertension.

[0009] Advantages and technical effects of the present invention:

[0010] 1. The present invention uses computers and the Internet's gradually mature bioinformatics database or analysis software, combined with high-throughput mass spectrometry analysis, to screen peptides in a complex enzymatic hydrolysis system; the screening is based on all short peptides that can be measured in the enzymatic hydrolysis system, which not only improves the probability of successful screening, but also avoids the defect that some highly active fragments have not been discovered. The present invention does not require laborious and time-consuming experimental operations such as protein purification, hydrolyzate separation and purification;

[0011] 2. The walnut meal protein active peptide provided by the present invention has extremely high inhibitory activity of angiotensin converting enzyme, and its half inhibition concentration (IC 50 ) value is only 8.02μg / mL, and it is non-toxic, soluble in water, and easily absorbed by the human intestine;

[0012] 3. The active peptide of the present invention can be synthesized by artificial chemistry and is suitable for industrial production and market promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the ligand-protein molecule surface generated according to electrostatic potential energy;

[0014] Figure 2 is the three-dimensional structure of the docking posture;

[0015] Figure 3 is a local diagram of the docking posture;

[0016] Figure 4 It is a two-dimensional structural diagram of the docking posture. DETAILED DESCRIPTION

[0017] The technical scheme of the present invention is further described in detail below through examples, but the content of the present invention is not limited thereto. The methods in the present examples are conventional methods unless otherwise specified, and the materials and reagents used are obtained from commercial sources unless otherwise specified;

[0018] Example 1: Preparation of walnut meal protein active peptide FDWLR

[0019] 1. Obtain walnut meal from a walnut oil production plant, crush it, and determine its crude protein content using the Kjeldahl method;

[0020] 2. Disperse the crushed walnut meal in food-grade water at a weight-volume ratio of 3:40 g:mL, adjust the pH to 9.5 with 6mol / LHCL and 2mol / L NaOH, add alkaline protease at an enzyme: substrate mass ratio of 1:15 based on crude protein content, and hydrolyze in a 50°C constant temperature water bath oscillator for 4 hours; adjust the pH of the hydrolysis mixture to 2.0-2.5, add pepsin at an enzyme: substrate mass ratio of 1:15, and oscillate in a 37°C water bath for 1 hour; adjust the pH of the hydrolysis mixture to 7.8-8.2, add trypsin at an enzyme: substrate mass ratio of 1:15, and oscillate in a 37°C water bath for 2 hours, place the hydrolysis mixture in a boiling water bath for 15 minutes to inactivate the enzyme, cool to room temperature, centrifuge at 5000 rpm for 20 minutes, and take the supernatant for vacuum freeze-drying and storage.

[0021] 3. High-throughput mass spectrometry analysis of enzymatic hydrolysates

[0022] The lyophilized enzymatic hydrolysate was accurately weighed and dissolved in ultrapure water to prepare a 1 mg / mL solution. The solution was filtered through a 0.22 μm water filter membrane and the filtrate was placed in a 1.5 mL injection bottle. The HPLC-Q-Orbitrap-MS 2 )Identification of peptides in hydrolysates.

[0023] Chromatographic conditions: Infinitylab Poroshell 120EC-C18 (1.9 μm, 2.1×100 mm) column; column temperature 30°C; mobile phase A is acetonitrile containing 0.1% formic acid (v / v), mobile phase B is ultrapure water containing 0.1% formic acid (v / v). Injection volume 2 μL; flow rate 0.20 mL / min; elution gradient: 0-1 min 5% A, A increased from 5% to 10% in 1 min-2.5 min, A increased from 10% to 25% in 2.5 min-12.5 min, A increased from 25% to 52.5% in 12.5 min-20 min, A increased from 52.5% to 95% in 20 min-22 min, A stabilized at 95% in 22 min-24 min, A decreased from 95% to 5% in 24 min-25 min, and maintained at 5% in 25 min-30 min.

[0024] The mass spectrometry parameters were: spray voltage 3.2 kV, capillary temperature 350 °C, dryer temperature 350 °C, data acquisition range 200-3000 m / z. The resolution of Full MS was 70000, and the dd MS 2 The resolution is 35000.

[0025] Mass spectrometry data were analyzed using de novo sequencing by Peak Studio 8.0 software, and peptides with an average local confidence (ALC) ≥ 85% were selected for further analysis.

[0026] The results are shown in Table 1:

[0027] Table 1 UPLC-Q-Orbitrap-MS 2 Identified peptide information in the mixture of walnut meal hydrolyzed by protease technology

[0028]

[0029]

[0030]

[0031]

[0032] 4. Screening of bioactive peptides

[0033] The peptides screened above were evaluated for biological activity using the Peptide Ranker program (http: / / disilldeep.ucd.ie / PeptideRankert). It is generally believed that peptides with a score higher than 0.5 have potential biological activity. The present invention selects peptides with a score higher than 0.8 for further bioinformatics analysis.

[0034] 5. ADMET property prediction

[0035] The peptides screened above were translated into simplified molecular linear input specification format through PepSMI (https: / / www.novoprolabs.com / tools / convert-peptide-to-smiles-string), and the ADMET properties of the peptides were predicted using AdmetSAR (http: / / lmmd.ecust.edu.cn / admetsar1). The peptides with solubility, easy absorption, metabolizability and low toxicity were further subjected to bioinformatics analysis;

[0036] The results are shown in Table 2:

[0037] Table 2 ADMET prediction using SMILES encoding of peptides

[0038]

[0039] 6. Molecular docking

[0040] The crystal structure of ACE (PDB ID: 1O8A) from the RCSB protein database (http: / / www.rcsb.org) was used as a research template for molecular docking, and the above-screened peptides were subjected to docking analysis. The "Build Protein" tool in SYBYL-X 2.0 software was used to build the structure of the peptide. Before the docking analysis, the ACE protein was dehydrated and hydrogenated, and the molecular docking was performed using the Surflex-Dock tool in SYBYL-X 2.0 software. The optimal binding conformation of the peptide and ACE was determined based on the consistency scoring function (C-Score) and the total score (T-Score), the binding stability of the peptide and ACE was determined, the interaction between the peptide and the ACE active site was constructed, and the peptide FDWLR that was stably bound to ACE (PDB ID: 1O8A) was obtained.

[0041] According to the crystal structure of ACE (PDB ID: 1O8A), the ACE molecule contains zinc ions (Zn[Ⅱ]) and three main active pockets S1, S2 and S1'. The S1 pocket contains Ala354, Glu384 and Tyr523 amino acid residues, the S2 pocket contains Gln281, His353, Lys511, His513 and Tyr520 amino acid residues, and the S1' pocket contains Glu162 amino acid residues. The molecular docking results of FDWLR and ACE are shown in Figure 1-4, Table 3, It can be seen from the figure that FDWLR forms a total of 8 hydrogen bonds with Asn66, Asn85, Arg124, Ser355, Ala356, Glu384 and His387 amino acid residues of ACE. Among them, FDWLR forms two hydrogen bonds with Tyr62 and Ala356 amino acid residues respectively. Glu384 is a key amino acid residue in the S1 pocket, and FDWLR forms one hydrogen bond with it, with a bond distance of There are 12 hydrophobic interactions between FDWLR and the amino acid residues Arg124, Ser517, Asn66, Tyr360, Trp357, His383, Ala354, His353, Ser355, Leu139, Glu143 and Val518 of ACE. FDWLR can form hydrophobic interactions with the amino acid residue Ala354 in the S1 pocket and the amino acid residue His353 in the S2 pocket. The effective interaction between FDWLR and the active site of ACE contributes to its ACE inhibitory activity.

[0042] Zn(Ⅱ) in the active site of ACE interacts with three amino acid residues of ACE (His383, His387 and Glu411) to form a tetrahedral structure. Although FDWLR cannot directly coordinate with Zn(Ⅱ), it can form hydrogen bonds with His387 amino acid residue and hydrophobic interactions with His383 amino acid residue. It has been observed that hydrogen bonds between ACE inhibitory peptides and Zn(Ⅱ) ligands may affect the tetrahedral structure of Zn(Ⅱ) complexes, thereby inhibiting ACE activity. The formation of hydrophobic interactions between ACE inhibitory peptides and His38 amino acid residues may also lead to enhanced ACE inhibitory activity. In summary, FDWLR exerts ACE inhibitory activity by changing the conformation of ACE.

[0043] Table 3 Interaction between pentapeptide FDWLR and ACE (PDB ID: 1O8A)

[0044]

[0045] Example 2: Half inhibition rate of walnut meal protein active peptide FDWLR inhibiting ACE (PDB ID: 108A)

[0046] The peptide walnut meal protein active peptide FDWLR obtained by screening in Example 1 was synthesized at Shanghai Jiepeptide Biotechnology Co., Ltd., and then a series of active peptide aqueous solutions with gradient concentrations were prepared. The ability of the peptide to inhibit ACE was determined using an ACE inhibitor activity detection kit. The experimental operation was carried out according to the instructions of the kit, and the peptide concentration that achieved a half inhibition rate (IC 50 value); half inhibitory concentration (IC50 ) value was only 8.02 μg / mL, or 10.9 μM.

[0047] In conclusion, walnut meal protein active peptide FDWLR has application prospects in the treatment of hypertension.

Claims

1. A walnut meal protein active peptide, whose amino acid sequence is Phe-Asp-Trp-Leu-Arg.

2. Use of the walnut meal protein active peptide according to claim 1 in the preparation of drugs for treating hypertension.

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