Preparation method and application of ACE inhibitory peptide from white mullet

Abstract

The invention discloses a preparation method and application of a white mullet ACE inhibitory peptide, and relates to the field of biotechnology. The white mullet ACE inhibitory peptide is extracted from white mullet meat, and the amino acid sequence is: Phe-Pro-His-His-Asp-Arg-Phe (FPHHDRF). The preparation method of the white mullet ACE inhibitory peptide comprises the steps of: enzymatically hydrolyzing the white mullet meat to obtain an enzymatic solution, ultrafiltration separation of ultrafiltration components with a molecular weight cutoff of less than 3000Da, further separation by gel chromatography, and further separation and purification of the chromatographic components by high performance liquid chromatography to obtain a polypeptide. The polypeptide in the invention has ACE inhibitory activity and can act on some chronic diseases.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a preparation method and application of a white mullet ACE inhibitory peptide. Background Art

[0002] ACE (angiotensin converting enzyme) mainly increases blood pressure by converting angiotensin I into angiotensin II. ACE inhibitory peptides reduce the production of angiotensin II, causing blood vessels to contract, thereby achieving the effect of lowering blood pressure. Natural ACE inhibitory peptides are mainly derived from animal and plant foods. Among aquatic organisms, many types of ACE inhibitory peptides have been found in fish, ginseng, shrimp, and shellfish, which have shown significant antihypertensive effects in hypertension, such as ACE inhibitory peptides found in tilapia, sea cucumbers, Antarctic krill, and green clams. Hypertension is a chronic disease and an important factor affecting cardiovascular and cerebrovascular diseases. ACE inhibitory peptides derived from food are closely related to hypertension and are an effective alternative treatment option for relieving hypertension.

[0003] Fish are rich in protein. Yong'an White Mullet (Opniocepnalus argus var Kimnra), also known as white snakehead, is a specialty fish species in Neijiang, Sichuan. It is a very rare and high-quality fish among freshwater fish in my country. It is richer in protein than ordinary fish and contains amino acids, calcium, iron and other elements required by the human body. After consumption, it can supplement a variety of nutrients needed by the human body. In 2022, the annual output of Neijiang Yong'an White Mullet will be about 500 tons, mainly sold to Sichuan, Chongqing and other places. It has a large market demand, and has developed a series of deep-processing products such as polypeptide instant powder, polypeptide frozen slices, flavored white mullet, and white mullet noodles. The discovery of ACE inhibitory peptides can provide theoretical support for the development of white mullet health foods and increase the market value of white mullet. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of a white mullet ACE inhibitory peptide. The white mullet ACE inhibitory peptide has ACE inhibitory activity and can be used in the preparation of blood pressure regulating products.

[0005] The present invention is achieved through the following technical solutions:

[0006] A white mullet ACE inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] Preferably, the white mullet ACE inhibitory peptide is extracted from white mullet meat.

[0008] The preparation method of white mullet ACE inhibitory peptide comprises the following steps:

[0009] Homogenization of white mullet was performed to simulate human in vitro digestion to prepare white mullet enzymatic hydrolysate;

[0010] The enzymatic hydrolysate is separated by an ultrafiltration tube with an ultrafiltration component having a molecular weight cut-off less than 3000Da;

[0011] The ultrafiltration fractions were chromatographed using gel chromatography;

[0012] The components obtained after chromatography are separated and purified.

[0013] The present invention also provides a white mullet ACE inhibitory peptide obtained by the above-mentioned white mullet ACE inhibitory peptide preparation method.

[0014] The invention also provides application of the white mullet ACE inhibitory peptide in food.

[0015] The above-mentioned white mullet ACE inhibitory peptide can also be synthesized using amino acids as raw materials by solid phase synthesis, which is well known in the art.

[0016] At the same time, the above-mentioned white mullet ACE inhibitory peptide has a high ACE inhibitory activity and is a polypeptide with a certain blood pressure lowering function.

[0017] Therefore, the white mullet ACE inhibitory peptide can be used in blood pressure lowering foods.

[0018] The present invention also provides a product containing the above-mentioned white mullet ACE inhibitory peptide.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] In the present invention, a white mullet ACE inhibitory peptide is provided, and its amino acid sequence is FPHHDRF. The white mullet ACE inhibitory peptide is extracted from white mullet meat, and a method for obtaining the white mullet ACE inhibitory peptide is provided. The white mullet ACE inhibitory peptide can be obtained according to a specific preparation method, or it can be synthesized by solid phase synthesis according to a disclosed amino acid sequence. The white mullet ACE inhibitory peptide has a high ACE inhibitory activity, so the white mullet ACE inhibitory peptide can be used in chronic diseases such as hypertension, which broadens the application range of the white mullet ACE inhibitory peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the chromatogram of the ultrafiltration fraction with a molecular weight below 3000Da after being separated by Sephadex G-15 gel chromatography;

[0022] Figure 2 The results of different ACE inhibitory activities of components 3kd, B2, and B3;

[0023] Figure 3 is the HPLC chromatogram of chromatography group B2;

[0024] Figure 4 is the secondary mass spectrum of the sequence FPHHDRF;

[0025] Figure 5 This is the liquid chromatogram for purity verification of the sequence FPHHDRF after synthesis;

[0026] Figure 6 The results are for the different ACE inhibitory activities of FPHHDRF and captopril. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and accompanying drawings. It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. In the embodiment, those who do not indicate specific conditions are carried out according to the conditions recommended by normal conditions or manufacturers. Those who do not indicate manufacturers for reagents or instruments used are conventional products that can be obtained by commercial purchase.

[0028] The invention provides a white mullet ACE inhibitory peptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0029] The white mullet ACE inhibitory peptide is extracted from white mullet, especially from the meat of white mullet.

[0030] The second embodiment of the present invention provides a method for extracting ACE inhibitory peptides from white mullet, comprising the following steps:

[0031] Step 1: Remove the skin and bones of the white mullet, leaving only the fish meat, add ultrapure water at a mass ratio of 1: (3-5) and homogenize using a homogenizer, adjust the solution to pH 2.0 with HCl, then add 3-4% pepsin, simulate gastric digestion enzymatic hydrolysis under 37°C water bath conditions, adjust the solution to pH 5.33 with 0.9M NaHCO3, and then adjust it to pH 7.5 with 1M sodium hydroxide, add 3-4% pancreatic enzyme, simulate intestinal digestion under 37°C water bath conditions for 2.5h, and finally place the solution in boiling water for 10 minutes to terminate digestion. The sample is cooled to room temperature and centrifuged at 8000xg, 4°C using a refrigerated centrifuge for 20 minutes, and the supernatant is freeze-dried into powder to obtain white mullet crude peptide powder;

[0032] Step 2: Dissolve the lyophilized powder in distilled water, filter with a 3000Da ultrafiltration tube, retain component B with a molecular weight below 3000Da, freeze-dry and store at -18°C;

[0033] Step 3: The ultrafiltration component B is prepared into a 20-30 mg / mL solution and filtered through a 0.22 μm aqueous microporous filter membrane, and chromatographically separated through Sephdex G-15 dextran gel, and the separated peak components are collected respectively, and freeze-dried for later use;

[0034] Step 4: The components with ACE inhibitory activity obtained after chromatography were separated and purified by RP-HPLC, and then the components were identified by LC-MS / MS to obtain the amino acid sequence as follows:

[0035] Phe-Pro-His-His-Asp-Arg-Phe(FPHHDRF).

[0036] In a preferred embodiment, in step 3, the sample loading amount of the Sephadex G-15 gel chromatography method is 2-4 mL. To ensure the chromatography effect, the preferred sample loading amount is 4 mL, the elution flow rate is 1-2 mL / min, and the detection wavelength is 220 nm.

[0037] In the following specific embodiments of the present invention, the ultrafiltration components are chromatographed by Sephadex G-15 gel chromatography, and antioxidant assays are performed on each component. Components with ACE inhibitory activity are selected, and further separated by RP-HPLC, and then identified by LC-MS / MS.

[0038] Determination of ACE Inhibitory Activity The release of hippuric acid was measured by HPLC.

[0039] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0040] Example 1

[0041] A method for extracting, separating and purifying ACE inhibitory peptide from white mullet, the specific steps are as follows:

[0042] Step 1: Remove the skin and bones of the white mullet, leaving only the fish meat, add ultrapure water at a mass ratio of 1:4 and homogenize using a homogenizer, adjust the solution to pH 2.0 with HCl, then add 4% pepsin, simulate gastric digestion enzymolysis under 37°C water bath conditions, adjust the solution to pH 5.33 with 0.9M NaHCO3, and then adjust it to pH 7.5 with 1M sodium hydroxide, add 4% pancreatin, simulate intestinal digestion under 37°C water bath conditions for 2.5h, and finally place the solution in boiling water for 10 minutes to terminate digestion. The sample was cooled to room temperature and centrifuged at 8000xg, 4°C using a refrigerated centrifuge for 20 minutes, and the supernatant was freeze-dried into powder to obtain white mullet crude peptide powder;

[0043] Step 2: Dissolve the lyophilized powder in distilled water, filter with a 3000Da ultrafiltration tube, retain component B with a molecular weight below 3000Da, freeze-dry and store at -18°C;

[0044] Step 3: Prepare the ultrafiltration component B into a 25 mg / mL solution and filter it through a 0.22 μm aqueous microporous filter membrane, and perform chromatography separation through Sephdex G-15 dextran gel. The sample volume of the gel chromatography is 4 mL, the elution flow rate is 1 mL / min, and the detection wavelength is 220 nm. The separated peak components are collected and freeze-dried for later use. The chromatographic spectrum results are shown in Figure 1 As shown, three separated components were obtained and labeled with B1, B2, and B3 in sequence;

[0045] Step 4: The B1, B2, and B3 components obtained after chromatography were assayed for ACE inhibitory activity, and the samples were dissolved in pH 8.3, 0.1 mol / L borate buffer (containing 0.3 mol / L NaCl). 50 μL of 5 mmol / L hippuryl-histidyl-leucine (HHL) was added to 50 μL of white mullet polypeptide samples of different concentrations, incubated at 37°C for 6 min, and then 50 μL of 0.1U / mL ACE solution dissolved in pH 8.3, 0.1 mol / L borate buffer (containing 0.3 mol / L NaCl) was added to initiate the reaction. After further incubation at 37°C for 30 min, 100uL of 1 mol / L HCl was added to terminate the reaction. The hippuric acid content released by ACE was determined by HPLC at 228 nm;

[0046] Chromatographic conditions: chromatographic column: ODS-AP 300A, mobile phase: acetonitrile (0.1% TFA): water (0.1% TFA) = 25:75 (volume ratio), isocratic elution; flow rate 1 mL / min; temperature 30°C; injection volume 10 μL; detection wavelength: 228 nm. ACE inhibitory activity was calculated according to the following formula:

[0047]

[0048] Where:

[0049] P——ACE inhibitory activity;

[0050] A1——peak area of ​​sample solution after reaction;

[0051] A0——peak area after borate buffer replaces sample solution;

[0052] The blank control group of samples was replaced by borate buffer; the reaction solution was centrifuged and the amount of hippuric acid produced was detected by HPLC;

[0053] Results Figure 2 The horizontal axis represents each component, and the vertical axis represents the concentration of each component required to reduce ACE inhibition IC50 by 50%. Component B1 has no ACE inhibition activity, and components B2 and B3 have better effects on ACE inhibition activity. Component B2 is selected for the next step of separation and purification;

[0054] Step 5: Use liquid chromatography to separate and purify the B2 ultrafiltration fraction. Liquid phase conditions: analytical column: C18, 3μm, 100A; mobile phase A: water (0.1% TFA); mobile phase B: ACN (0.1% TFA); flow rate: 1mL / min; gradient elution: 0min (2% B), 0-18min (2%-10% B), 18-36min (10%-20% B), 36-55min (20%-30% B), 55-70min (30%-40% B); sample volume 10μL, the results are as follows Figure 3 ;

[0055] Step 6: Liquid chromatography-mass spectrometry was used to quantify the purified components ( Figure 3 Liquid phase conditions: analytical column: 150μm id × 150mm, packedwith Acclaim PepMap RPLC C18, 3μm, 100A; mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% ACN; flow rate: 600nL / min; gradient elution: 0min (4%B), 0-2min (4%-8%B), 2-45min (8%-28%B), 45-55min (28%-40%B), 55-56min (40%-95%B), 56-66min (95%B); sample volume 10μL;

[0056] Mass spectrometry conditions: Primary mass spectrometry parameters: Resolution: 70,000; AGCtarget: 3e6; MaximumIT: 100ms; Scanrange: 100 to 1500m / z. Secondary mass spectrometry parameters: Resolution: 17,500; AGCtarget: 1e5; MaximumIT: 50ms; TopN: 20; NCE / steppedNCE: 28. The results are as follows Figure 4 ;

[0057] After identification, the amino acid sequence was obtained as follows:

[0058] Phe-Pro-His-His-Asp-Arg-Phe (FPHHDRF);

[0059] The following table shows the peptide information:

[0060] Table 1 Peptide information

[0061] .

[0062] Example 2

[0063] To further verify the ACE inhibitory activity of the above-mentioned polypeptide FPHHDRF, the sequence was synthesized using solid phase synthesis, and then tested using the method in step 5 to make the purity ≥ 98%. The results are as follows Figure 5 .

[0064] The synthesized peptide FPHHDRF was verified using the method in step 4, and the ACE inhibitory activity of the synthesized peptide sequence and captopril at different concentrations of 0.5, 1, 2, 4, and 8 mg / mL was measured using captopril as a positive control. Figure 6 With the increase of concentration, the ACE inhibitory activity of captopril and polypeptide FPHHDRF became higher. When the concentration was 8 mg / mL, the ACE inhibitory activity of captopril in the control group was 90.29%, and the ACE inhibitory activity of polypeptide FPHHDRF was 60.18%, indicating that FPHHDRF has a strong ACE inhibitory activity.

[0065] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A white mullet ACE inhibitory peptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. The method for preparing the ACE inhibitory peptide of white mullet according to claim 1, characterized in that: The following steps are involved: Homogenization of white mullet was performed to simulate human in vitro digestion to prepare white mullet enzymatic hydrolysate; The enzymatic hydrolysate is separated by an ultrafiltration tube with an ultrafiltration component having a molecular weight cut-off less than 3000Da; The ultrafiltration fractions were chromatographed using gel chromatography; Separating and purifying the components obtained after chromatography; The preparation method of the white mullet enzymatic hydrolysate is as follows: taking white mullet meat, adding ultrapure water at a mass ratio of 1: (3-5) and homogenizing with a homogenizer, adjusting the solution to pH 2.0 with hydrochloric acid, and then adding 3-4% pepsin to simulate gastric digestion enzymatic hydrolysis under 37°C water bath conditions, adjusting the solution to pH 5.33 with 0.9M sodium bicarbonate, and then adjusting the solution to pH 7.5 with 1M sodium hydroxide, adding 3-4% pancreatic enzyme, simulating intestinal digestion for 2.5 hours under 37°C water bath conditions, and finally placing the solution in boiling water for 10 minutes to terminate digestion, cooling the sample to room temperature and centrifuging it at 8000xg, 4°C using a refrigerated centrifuge for 20 minutes, taking the supernatant and lyophilizing it into powder, and dissolving the lyophilized powder in distilled water to obtain the white mullet enzymatic hydrolysate.

3. Use of the white mullet ACE inhibitory peptide as claimed in claim 1 in preparing food.

4. Use of the white mullet ACE inhibitory peptide as claimed in claim 1 in preparing blood pressure lowering food.

5. A product, characterized in that: Contains the white mullet ACE inhibitory peptide according to claim 1.

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