An ACE inhibitory peptide derived from cooking wine residue, its preparation method and application

Abstract

This invention belongs to the field of resource utilization technology of cooking wine residue, and discloses an ACE-inhibiting peptide derived from cooking wine residue. To improve the economic value of cooking wine residue and develop an oligopeptide with antihypertensive effects, this invention uses cooking wine residue as raw material and obtains an antihypertensive peptide monomer through various separation and purification methods such as water extraction, membrane filtration, and high-performance liquid chromatography. The amino acid sequence is WCYND, and the molecular weight is 699.73 Da. It exhibits good activity, can be artificially synthesized, and is safe and non-toxic. Its ACE-inhibiting activity has been measured, and this antihypertensive oligopeptide has an inhibitory effect on angiotensin-converting enzyme in vitro, which can be applied in the field of antihypertensive treatment and is beneficial for the high-value comprehensive utilization of cooking wine residue.

Description

Technical Field

[0001] This invention relates to the field of resource utilization technology of cooking wine residue, and more specifically, to an ACE inhibitory peptide derived from cooking wine residue, its preparation method, and its application. Background Technology

[0002] Hypertension is a disease characterized by elevated arterial pressure. Normal adult blood pressure is defined as systolic pressure ≤140 mmHg and diastolic pressure ≤90 mmHg. Hypertension is diagnosed when an adult's systolic pressure is ≥160 mmHg and diastolic pressure is ≥95 mmHg. Hypertension is one of the most common and serious chronic health problems worldwide. Early symptoms are often subtle, but later stages can lead to cerebral hemorrhage or sudden heart attacks, severely endangering health. Research shows that many systems in the body are closely related to blood pressure regulation, and antihypertensive drugs can act on any link affecting blood pressure regulation, thus lowering blood pressure. The RAS system has been confirmed as a key target for treating hypertension. ACE inhibitors can reduce mortality in hypertensive patients. Chemically synthesized drugs such as captopril and lisinopril are used clinically to treat hypertension, but they may have some side effects. In contrast, natural bioactive peptides are more environmentally friendly and safer, thus becoming a research hotspot in recent years. Besides a healthy daily diet that increases the intake of fruits, vegetables, and high-quality protein to lower blood pressure, the bioactive peptide fragments contained in protein also greatly benefit human health.

[0003] Rice is one of the world's most important crops, and the nutritional value of rice protein is comparable to that of animal proteins such as eggs, fish, shrimp, and beef. However, my country's level of development and comprehensive utilization of rice resources is not high, and its research level lags behind that of developed countries, resulting in the economic value of rice not being fully realized. Cooking wine is one way to utilize rice, and the production process generates a large amount of fermentation substrate, also known as cooking wine lees. Currently, apart from a few manufacturers drying it and selling it as animal feed, most manufacturers sell it as animal feed at low prices, and some manufacturers directly discharge it as waste, which wastes protein resources and pollutes the environment. Cooking wine lees are rich in various amino acids, vitamins, and trace elements, making them highly nutritious. Several studies have shown that polypeptides with antihypertensive activity can be isolated and extracted from cooking wine lees, and due to their non-toxic side effects, they have become a focus of research in the field of antihypertensive therapy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned problems existing in the prior art and to provide an oligopeptide extracted from cooking wine residue.

[0005] A second objective of this invention is to provide applications of the aforementioned oligopeptides.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] An oligopeptide extracted from cooking wine residue, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] This invention uses cooking wine residue as raw material and obtains a blood pressure-lowering oligopeptide through various separation and purification methods. The oligopeptide has the amino acid sequence WCYND and a molecular weight of 699.73 Da. It has the characteristics of high purity, good activity, artificial synthesis capability, and safety and non-toxicity.

[0009] The results of the ACE inhibitory activity assay showed that the antihypertensive peptide has a significant inhibitory effect on ACE in vitro, which can be applied to the field of blood pressure reduction and is beneficial to the comprehensive utilization of cooking wine residue.

[0010] Therefore, the present invention also provides the use of the oligopeptide in the preparation of functional products that inhibit the activity of angiotensin-converting enzyme ACE.

[0011] The present invention also provides a method for preparing the above-mentioned oligopeptide, which involves enzymatically hydrolyzing cooking wine residue and then separating and purifying it through multiple separations with the aim of improving the inhibitory activity of angiotensin-converting enzyme.

[0012] Preferably, the enzyme used in the preparation method for enzymatic hydrolysis is a neutral protease.

[0013] More preferably, the preparation method includes the following steps:

[0014] S1. Mix the raw material of cooking wine residue with water, add neutral protease, and enzymatically hydrolyze in a water bath at 46℃ for 3 hours. After stopping the enzymatic hydrolysis and cooling to room temperature, centrifuge. Retain the supernatant and determine its ACE inhibition rate;

[0015] S2. The sample with high ACE inhibitory activity obtained in S1 was passed through 0.45μm and 0.2μm microporous membranes, 3000Da spiral wound membranes and 1000Da ultrafiltration cup membranes, respectively, and the ACE inhibitory activity of the retentate and permeate was measured.

[0016] S3. The highly ACE-inhibiting active component obtained in S2 was treated with macroporous adsorption resin and eluted with ethanol of different concentrations. The ACE inhibition rate was measured separately. The highly active component was then separated again using a Sephadex G-25 chromatographic column, and the absorbance was monitored. The ACE inhibition rate of the obtained component was then measured again.

[0017] S4. The fraction with high ACE inhibitory activity obtained in S3 was separated by high performance liquid chromatography (HPLC) using a C18 column (20 mm × 450 mm, 10 μm). The HPLC elution conditions were as follows: 0-20 min, 10-30% B; 20-50 min, 30-57% B; 50-60 min, 57-90% B; 60-62 min, 90-10% B; 62-70 min, 10% B. The fractions were collected based on the peak shape of the chromatogram. The ACE inhibition rate was used as the criterion for further subdivision.

[0018] S5. The highly active ACE component obtained in S4 was analyzed by analytical high-performance liquid chromatography (HPLC). The HPLC elution conditions were as follows: 0-30 min, 5-35% B; 30-50 min, 35-45% B; 50-50.1 min, 45-70% B; 50.1-60 min, 90% B. The monomer peaks were collected and the ACE inhibition rate was determined to obtain highly active ACE-inhibiting oligopeptides.

[0019] S6. The highly active monomers obtained in S5 were identified using a UPLC-LTQ-Orbitrap-Velos Pro mass spectrometer combined with a PPSQ amino acid sequencer. The EASY-nLC conditions were as follows: elution conditions: 0-60 min, 15-50% B; 50-70 min, 50-100% B; 70-75 min, 100% B; flow rate: 300 nl / min; mobile phase: phase A was 0.1% (v / v) formic acid aqueous solution, and phase B was pure acetonitrile solution. The mass spectrometry conditions were as follows: injection flow rate: 20 μl / min; injection volume: 5 μl; positive ion mode, ESI electrospray ionization source, spray voltage: 2.0 kV; collision mode: CID; scan range: 350-2000 m / z; acquisition time: 75 min. The peptide structure was determined using Proteome Discoverer software.

[0020] The present invention also provides the application of the oligopeptide in the preparation of functional products for lowering blood pressure.

[0021] The present invention also provides an inhibitor of angiotensin-converting enzyme ACE, the active ingredient of which is an oligopeptide with an amino acid sequence as shown in SEQ ID NO: 1.

[0022] The present invention also provides a blood pressure lowering product, the active ingredient of which is an oligopeptide with an amino acid sequence as shown in SEQ ID NO: 1.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides an ACE-inhibiting peptide derived from cooking wine residue. To improve the economic value of cooking wine residue and develop an oligopeptide with antihypertensive effects, this invention uses cooking wine residue as raw material and obtains an antihypertensive peptide monomer through various separation and purification methods such as water extraction, membrane filtration, and high-performance liquid chromatography. The amino acid sequence is WCYND, and the molecular weight is 699.73 Da. It exhibits good activity, can be artificially synthesized, and is safe and non-toxic. Its ACE-inhibiting activity has been determined, showing that this antihypertensive oligopeptide inhibits angiotensin-converting enzyme in vitro, and can be applied in the field of antihypertensive treatment. Furthermore, it is beneficial for the high-value comprehensive utilization of cooking wine residue. Attached Figure Description

[0025] Figure 1 Comparison of ACE inhibition rates between enzymatically hydrolyzed crude extract and commercially available antihypertensive peptides;

[0026] Figure 2 The ACE inhibitory activity of the membrane filtration component was compared with that of a commercially available antihypertensive peptide.

[0027] Figure 3 The ACE inhibitory activity of eluted fractions with different concentrations of ethanol was compared with that of commercially available antihypertensive peptides;

[0028] Figure 4 The ACE inhibitory activity of macroporous adsorption resin-separated components was compared with that of commercially available antihypertensive peptides.

[0029] Figure 5 To compare the ACE inhibitory activity of the prepared liquid-phase separated components with that of commercially available antihypertensive peptides;

[0030] Figure 6 To analyze the ACE inhibitory activity of the liquid-phase separated monomers and compare it with commercially available antihypertensive peptides;

[0031] Figure 7 The images show the primary mass spectrum and PPSQ sequencing results for monomeric F1. Detailed Implementation

[0032] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] Example 1

[0034] The isolation and purification of ACE inhibitory peptides includes the following steps:

[0035] S1. Mix the cooking wine residue with water, and add 0.3% neutral protease at pH 7.2. Incubate the mixture in a 46℃ water bath for 3 hours, then stop the enzymatic hydrolysis by placing the mixture at 90℃ for 10 minutes. After the hydrolysate cools to room temperature, centrifuge at 4000 rpm for 20 minutes. Retain the supernatant and determine its ACE inhibition rate; it is 40.33%. Figure 1 As shown.

[0036] S2. The enzymatically hydrolyzed crude extract was passed sequentially through 0.45μm and 0.2μm microporous membranes. The 0.2μm permeate was then separated in a 3000Da spiral wound membrane and a 1000Da ultrafiltration cup membrane (the ultrafiltration cup was used at room temperature throughout the process, and the pressure in the sealed space was maintained at 0.1-0.22MPa). The ACE inhibitory activity of the retentate and permeate was measured separately. The results showed that the 1000Da permeate had the highest inhibitory activity, reaching 56.26%. Figure 2 As shown.

[0037] S3. The 1000 Da ultrafiltration permeate was eluted with 10%, 20%, 35%, 50%, and 70% ethanol through a macroporous adsorption resin. After measuring the ACE inhibition rate, the sample eluted with 10% ethanol showed the highest inhibition activity, reaching 58.85%. Subsequently, the 10% ethanol eluent was separated again using a Sephadex G-25 column. The results showed that M4 had the highest inhibition activity among all components, at 63.29%. Figure 3 , Figure 4 As shown.

[0038] S4. After filtering M4 through a 0.45 μm filter membrane, the fraction was separated by preparative high-performance liquid chromatography (HPLC). Preparation conditions: ① Column: 20 mm × 450 mm, C18 packing material (10 μm). Macherey Nagel (France); ② Mobile phase: Pump A was ultrapure water, Pump B was acetonitrile; ③ Flow rate: 10 ml / min; ④ Injection volume: 5 ml; ⑤ Detection wavelength: 214 nm. The highest ACE inhibitory activity was found in fraction D3 at 67.07%, followed by further subdivision of D3. Results are as follows... Figure 5 As shown.

[0039] S5. Component analysis of D3 was performed using analytical high-performance liquid chromatography (HPLC). Separation conditions: ① Column: ECOSILC18 (260mm × 4.6mm, 5μm); ② Mobile phase: Pump A: ultrapure water, Pump B: acetonitrile; ③ Flow rate: 1ml / min; ④ Injection volume: 20μl; ⑤ Detection wavelength: 214nm. ACE inhibitory activity assay showed that the highest activity of monomer F1 was 77.57%, which is higher than that of commercially available antihypertensive peptides. Subsequently, the F1 monomer peak was collected and its structure was analyzed. Results are as follows... Figure 6 As shown.

[0040] S6. The peak tip of D3 was collected using analytical HPLC. The results showed that F1 was a target monomer with high ACE inhibitory activity. UPLC-LTQ-Orbitrap-Velos Pro mass spectrometry was used for alignment with Proteome Discoverer (PD) software, and PPSQ amino acid sequencing was performed. From the primary mass spectrum corresponding to the main peak position in the ion chromatogram, its molecular weight was estimated to be approximately 701.49 Da [M+H]. This result was imported into the Uniprot library in the PD software. The searched potential matching sequences were compared with the results provided by PPSQ. The amino acid sequence of F1 was identified as Trp-Cys-Tyr-Asn-Asp (WCYND), with a molecular weight of 699.73 Da. This sequence was confirmed as a novel oligopeptide with ACE inhibitory activity discovered in this patent. The results are as follows: Figure 7 As shown.

[0041] The method for detecting ACE inhibitory activity is as follows:

[0042] 10 μL of ACE solution (0.2 U / mL) was added to the blank group, control group, and sample group, respectively. 10 μL of ACEI was added to the sample group, and 10 μL of buffer solution was added to the blank group and control group. After incubation at 37 °C for 5 min, 30 μL of HHL solution (6.5 mmol / mL, HHL dissolved in 0.1 mol / L borate buffer at pH 8.3, containing 0.3 mol / L NaCl) was added to all groups. After reacting at 37 °C for 1 h, the reaction was stopped by adding 80 μL of 1.0 mol / L HCl to the control group and sample group, respectively.

[0043] Table 1

[0044]

[0045]

[0046] Liquid chromatography conditions: ① Column: ECOSIL C18 (260mm×4.6mm, 5μm); ② Mobile phase: Acetonitrile: Ultrapure water = 25:75 (containing 0.1% (v / v) TFA); ③ Flow rate: 1mL / min; ④ Detection wavelength: 228nm; ⑤ Column temperature: 30℃; ⑥ Injection volume: 20μL.

[0047] Result calculation:

[0048] Principle: HHL rapidly decomposes under the catalysis of ACE to produce hippuric acid (Hip) and a dipeptide (His-Leu, HL). Hippuric acid has a maximum absorption at 228 nm. When an ACEI sample is added, ACE enzyme activity is inhibited, and the amount of hippuric acid produced decreases. Therefore, the inhibition rate of ACEI on ACE activity can be evaluated by measuring the amount of hippuric acid produced using high-performance liquid chromatography.

[0049] The calculation formula is:

[0050] Where: R: the inhibition rate of ACE by the ACEI sample (%);

[0051] A: Peak area of ​​hippuric acid in the control group;

[0052] B: Peak area of ​​hippuric acid in the ACEI-added group;

[0053] A0: Peak area of ​​hippuric acid in the blank tube.

[0054] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. An oligopeptide extracted from cooking wine residue, characterized in that, The amino acid sequence of the oligopeptide is shown in SEQ ID NO:

1.

2. The method for preparing the oligopeptide according to claim 1, characterized in that, The residue from cooking wine is enzymatically hydrolyzed to enhance the inhibitory activity of angiotensin-converting enzyme, and is obtained through multiple separation and purification processes.

3. The method for preparing oligopeptides according to claim 2, characterized in that, The enzyme used for enzymatic hydrolysis is a neutral protease.

4. The use of the oligopeptide according to claim 1 in the preparation of functional products that help lower blood pressure.

5. An inhibitor of angiotensin-converting enzyme ACE, characterized in that, The active ingredient is an oligopeptide with an amino acid sequence as shown in SEQ ID NO:

1.

6. A product with auxiliary blood pressure lowering function, characterized in that, The active ingredient is an oligopeptide with an amino acid sequence as shown in SEQ ID NO: 1.

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