Functional antihypertensive protein food containing high-activity ACE inhibitory peptide

Abstract

According to the functional antihypertensive protein food containing the high-activity ACE inhibitory peptide, the docking condition of short peptides and ACE active centers is analyzed through artificial intelligence, peptide chain structures with high ACE inhibitory ability can be efficiently screened out, then analysis is conducted according to the sequence characteristics of the peptide chain structures, specific peptide structures with higher effects are found, and the functional antihypertensive protein food containing the high-activity ACE inhibitory peptide is obtained. And designing corresponding high-selectivity protease to obtain the functional antihypertensive protein food. Protein food with specific functions can be obtained through the processes of interaction simulation, sequence analysis, feature extraction, enzymolysis optimization and the like. The technology is applied to treatment of hypertension, and heart and kidney physical injuries are effectively improved. An improved enzymolysis process is added on the basis of the traditional food production process, so that the release of endogenous functional peptide chains in soybean milk can be realized, and the blood pressure can be reduced. Therefore, the compound can show better safety and low antigenicity, and provides a powerful scientific basis for developing green and natural medicinal and edible antihypertensive health-care food.

Description

Technical Field

[0001] The present invention relates to the field of chemical and bioengineering technology, and in particular to a highly active ACE inhibitory peptide containing tryptophan at the C-terminus, a preparation and application method thereof, and a functional blood pressure-lowering protein food. The present invention specifically relates to docking simulation between the human ACE active site and a small peptide, sequence analysis of 168,000 small peptide molecules, and structural improvement of a highly selective protease. Background Art

[0002] Hypertension, considered a major "hidden killer" that endangers human health, is showing a trend of continued growth and younger prevalence. The development of hypertension is closely related to imbalances in the two main blood pressure regulating systems: the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). In clinical studies, renin-angiotensin converting enzyme (ACE) inhibitors have been shown to significantly reduce the relative mortality rate (approximately 10%) and cardiovascular mortality rate (approximately 12%) in patients with hypertension.

[0003] However, the side effects of taking chemical drugs such as lisinopril, alacepril and captopril, such as dry cough, headache, loss of taste and hyperkalemia, cannot be ignored. In contrast, food-borne ACE inhibitory peptides (ACEIPs) are safe, mild in effect and easily absorbed, and are gradually being promoted as an alternative treatment for hypertension. Soy protein is a high-quality source of ACEIPs because it is inexpensive, has low allergenicity, and can be used as an industrial by-product. In addition, different functional peptides have unique amino acid sequence characteristics due to their specific biological activities. However, there is currently a lack of complete analysis of the ACEIPs sequence and a lack of processing methods for the corresponding proteins.

[0004] Currently, researchers have isolated, purified, and identified peptides with ACE inhibitory activity from a variety of animal and plant-derived foods, including dairy products, eggs, meat, fish, soybeans, and wheat. These food-derived ACE inhibitory peptides not only exhibit significant biological activity but also demonstrate good safety and low antigenicity, providing a strong scientific basis for the development of green, natural, medicinal and edible blood pressure-lowering health foods. However, other blood pressure-lowering peptide products on the market suffer from various issues, including unclear mechanisms of action, high levels of impurities, and unusual flavors.

[0005] Therefore, it is necessary to address the problems of significant side effects of current traditional antihypertensive products and the imperfect development of new ACE inhibitors, and to solve the problem of strong side effects and great harm to health of traditional drugs. Summary of the Invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0007] On the one hand, a C-terminal tryptophan-containing high-activity ACE inhibitory peptide is provided for its role in lowering blood pressure.

[0008] On the other hand, a highly active ACE inhibitory peptide containing tryptophan at the C-terminus is provided, comprising one or more of the following peptide segments:

[0009] PDFKNPLGW peptide, whose amino acid sequence is: H-Pro-Asp-Phe-Lys-Asn-Pro-Leu-Gly-Trp-OH;

[0010] The amino acid sequence of the SEYPPLGRF peptide is: H-Ser-Glu-Tyr-Pro-Pro-Leu-Gly-Arg-Phe-OH;

[0011] The ALGAGRLF peptide segment has the following amino acid sequence: H-Ala-Leu-Gly-Ala-Gly-Arg-Leu-Phe-OH;

[0012] The FGSPAGPF peptide fragment has the following amino acid sequence: H-Phe-Gly-Ser-Pro-Ala-Gly-Pro-Phe-OH;

[0013] The STPLHLW peptide has the following amino acid sequence: H-Ser-Thr-Pro-Leu-His-Leu-Trp-OH;

[0014] The DHPPASW peptide fragment has the following amino acid sequence: H-Asp-His-Pro-Pro-Ala-Ser-Trp-OH;

[0015] The SWGEDW peptide has the following amino acid sequence: H-Ser-Trp-Gly-Glu-Asp-Trp-OH;

[0016] The QMPTEW peptide fragment has the following amino acid sequence: H-Gln-Met-Pro-Thr-Glu-Trp-OH;

[0017] The GDGLKW peptide has the following amino acid sequence: H-Gly-Asp-Gly-Leu-Lys-Trp-OH;

[0018] The GKGLW peptide has the following amino acid sequence: H-Gly-Lys-Gly-Leu-Trp-OH;

[0019] The amino acid sequence of the EWEGF peptide is: H-Glu-Trp-Glu-Gly-Phe-OH;

[0020] The GLGPF peptide has the following amino acid sequence: H-Gly-Leu-Gly-Pro-Phe-OH;

[0021] The LLEW peptide has the following amino acid sequence: H-Leu-Leu-Glu-Trp-OH;

[0022] or

[0023] The amino acid sequence of the LGKW peptide is: 1H-Leu-Gly-Lys-Trp-OH.

[0024] On the other hand, a highly active ACE inhibitory peptide containing tryptophan at the C-terminus is also provided, including the following peptide segments:

[0025] The GDGLKW peptide has the following amino acid sequence: H-Gly-Asp-Gly-Leu-Lys-Trp-OH;

[0026] as well as,

[0027] The amino acid sequence of the GKGLW peptide is: H-Gly-Lys-Gly-Leu-Trp-OH.

[0028] On the other hand, a method for preparing a highly active ACE inhibitory peptide containing tryptophan at the C-terminus is also provided, the method comprising the following steps:

[0029] Step 1: Perform enzymatic separation and purification on various common food protein sources, analyze their endogenous ACE inhibitory peptides, and determine that soybeans are the raw material for obtaining the highly active ACE inhibitory peptide containing tryptophan at the C-terminus;

[0030] Step 2: Using different commercial proteases, the optimal hydrolytic protease for preparing highly active ACE inhibitory peptides was selected based on the degree of hydrolysis, peptide content, and ACE inhibition rate;

[0031] Step 3: Using ultrafiltration and gel chromatography technology, the soybean protein is hydrolyzed with the hydrolyzing protease to screen the peptide component with the highest ACE inhibition rate;

[0032] Step 4: Based on the blood pressure reduction experiment, the highly active ACE inhibitory peptide containing tryptophan at the C-terminus is screened out.

[0033] Preferably, the hydrolytic protease in step 2 comprises alkaline protease and chymotrypsin.

[0034] Preferably, the enzymatic hydrolysis conditions in step three are: time 2 h, enzyme-substrate ratio 2000 U / g, and substrate concentration 5 mg / mL.

[0035] On the other hand, a method for applying a high-activity ACE inhibitory peptide containing tryptophan at the C-terminus is also provided. The high-activity ACE inhibitory peptide containing tryptophan at the C-terminus is used to prepare a functional blood pressure-lowering protein food.

[0036] On the other hand, a blood pressure-lowering soy milk is provided, which is prepared by adding a highly active ACE inhibitory peptide containing tryptophan at the C-terminus to soy milk and enzymatically hydrolyzing it for at least 2 hours.

[0037] The beneficial effects brought about by the technical solution provided by the embodiment of the present invention include at least:

[0038] The present invention uses artificial intelligence to analyze the docking of short peptides with the ACE active center. The present invention can efficiently screen out peptide chain structures with strong ACE inhibition, and then analyze their sequence characteristics to find specific peptide structures with stronger effects, design corresponding highly selective proteases, and obtain functional blood pressure-lowering protein foods.

[0039] The present invention develops a complete system for treating disease pathway targets. Through processes such as interaction simulation, sequence analysis, feature extraction, and enzymatic optimization, protein foods with specific functions can be obtained. The present invention applies this technology to the treatment of hypertension, effectively improving cardiac and renal pathological damage. By adding an improved enzymatic hydrolysis process to the traditional food production process, the release of endogenous functional peptide chains in soy milk can be achieved, thereby lowering blood pressure. Therefore, it can demonstrate good safety and low antigenicity, providing a strong scientific basis for the development of green, natural, medicinal and edible blood pressure-lowering health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0041] Figure 1 Hydrolysis degree (sub-figure A) and peptide content (sub-figure B) of soybean protein hydrolysates at different enzymatic hydrolysis times;

[0042] Figure 2 Effects of different factors on enzymatic hydrolysis products (Sub-figure A: enzyme addition amount, Sub-figure B: enzyme addition time, Sub-figure C: substrate concentration);

[0043] Figure 3 Amino acid content of soybeans and their hydrolysates from different proteases;

[0044] Figure 4 Gel filtration elution curve;

[0045] Figure 5 In vitro digestion stability of ACE inhibitory peptides (sub-figure A: GKGLW peptide, sub-figure B: GDKGLW peptide);

[0046] Figure 6 Acute hypotensive effects of soybean oligopeptides on systolic blood pressure (sub-figure A) and diastolic blood pressure (sub-figure B) in rats;

[0047] Figure 7 3D docking results of five peptides with ACE (sub-figure A: ACE three-dimensional structure, sub-figure B: LGKW peptide, sub-figure C: GKGLW peptide, sub-figure D: GDGLKW peptide, sub-figure E: DHPPASW peptide, sub-figure F: EWEGF peptide). DETAILED DESCRIPTION

[0048] The technical solution of the present invention is described below in conjunction with the accompanying drawings.

[0049] In the embodiments of the present invention, words such as "exemplarily" and "for example" are used to indicate examples, illustrations, or explanations. Any embodiment or design described as an "exemplary" in the present invention should not be interpreted as being preferred or advantageous over other embodiments or designs. Rather, the use of the word "exemplary" is intended to present concepts in a concrete manner. Furthermore, in the embodiments of the present invention, "and / or" can mean both or either of the two.

[0050] In the embodiments of the present invention, the terms "image" and "picture" may be used interchangeably. It should be noted that, when the distinction between them is not emphasized, their intended meanings are the same. The terms "of," "corresponding," and "corresponding" may be used interchangeably. It should be noted that, when the distinction between them is not emphasized, their intended meanings are the same.

[0051] In the embodiments of the present invention, sometimes a subscript such as W1 may be mistakenly written as a non-subscript form such as W1. When the difference is not emphasized, the meanings to be expressed are the same.

[0052] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0053] The sources of the reagents and the preparation equipment of this embodiment can be provided by the laboratory or purchased on the market.

[0054] Currently, researchers have isolated, purified, and identified peptides with ACE inhibitory activity from a variety of animal and plant-derived foods, including dairy products, eggs, meat, fish, soybeans, and wheat. These food-derived ACE inhibitory peptides not only exhibit significant biological activity but also demonstrate good safety and low antigenicity, providing a strong scientific basis for the development of green, natural, edible and medicinal health foods that can lower blood pressure.

[0055] Based on the above foundation, the present invention proposes a targeted step-by-step dual enzymatic hydrolysis technology, utilizing the substrate-specific synergistic effect of alkaline protease and chymotrypsin to precisely cut the hydrophobic and aromatic amino acid-rich regions in soy protein, significantly improving the ACE inhibition rate (71.98%). From the soybean enzymatic hydrolysis products, highly active ACE inhibitory peptides GKGLW and GDGLKW containing tryptophan (Trp) at the C-terminus were screened and identified, and molecular docking and kinetic simulations revealed the competitive inhibition mechanism of Trp embedded in the ACE catalytic cavity through hydrogen bonding, hydrophobic interactions, and π-π stacking.

[0056] Specifically, the present invention provides a functional blood pressure-lowering protein food containing a highly active ACE inhibitory peptide, specifically involving the following contents:

[0057] To ensure zero chemical additives in food, the present invention directly adds highly selective proteases during the production process. For example, in the production of blood pressure-lowering soy milk, an average of 2000 IU / g of tryptophan protease is added per 100 mL of soy milk to hydrolyze endogenous proteins in the food. After a 2-hour reaction, the maximum efficiency of extracting blood pressure-lowering peptides is achieved. Soy milk produced through this process can be consumed directly to lower blood pressure.

[0058] The present invention provides a method for producing a functional blood pressure-lowering protein food. Taking blood pressure-lowering soy milk as an example, the production method includes the following steps:

[0059] 1. Enzymatic separation, purification and identification of various common food protein sources were carried out, and their endogenous ACE inhibitory peptides were analyzed, thus confirming that soybeans are a very high-quality raw material for obtaining endogenous functional antihypertensive peptides.

[0060] Through the study of the human blood pressure regulation mechanism, it can be found that the interaction between the three amino acid residues at the C-terminus and N-terminus of the peptide and the S1, S1′ and S2′ hydrophobic subsites of ACE and zinc ions is the key factor determining its inhibitory activity. As one of the top ten major soybean producing countries in the world, my country has abundant soybean resource reserves. The amino acid composition of soy protein is highly similar to that of animal protein and meets the ideal plant protein standard recommended by FAO / WHO. Its absorption effect is better than that of amino acids, and its endogenous peptide ACE half-inhibitory concentration is low (Table 1), which can meet the blood pressure reduction needs. Therefore, the present invention selects soybean as a blood pressure-lowering food raw material.

[0061]

[0062] Table 1 ACE inhibitory peptides from different food sources

[0063] 2. Preparation and characterization of soybean ACE inhibitory peptides. Using different commercial proteases, the optimal protease was selected based on degree of hydrolysis, peptide content, and ACE inhibition rate. The enzymatic hydrolysis efficacy and substrate preference of the different enzymes were further analyzed based on the molecular weight distribution, SDS-PAGE, and amino acid content of the hydrolyzed products. The preparation conditions of the soybean antihypertensive peptide were further optimized using single-factor and response surface experiments based on substrate concentration, enzyme dosage, and hydrolysis time.

[0064] Using soy protein isolate as raw material, alkaline protease was screened out as the optimal hydrolase (Table 2):

[0065]

[0066] Table 2 Basic information of protease

[0067] The enzymatic hydrolysis product had the highest degree of hydrolysis (23.98%) and peptide content (22.65%). The enzymatic hydrolysis conditions were optimized by single factor experiment, and the optimal conditions ( Figure 1 、 Figure 2 ) for 2 h, enzyme-substrate ratio 2000 U / g, substrate concentration 5 mg / mL, and step-by-step double enzymatic hydrolysis ( Figure 3 ) strategy (alkaline protease + chymotrypsin), a highly active ACE inhibitory peptide was prepared by directed double enzymatic hydrolysis technology, which significantly improved the ACE inhibition rate (increased by 18.56%). Figure 1 As shown, response surface analysis showed that enzymatic hydrolysis time had the greatest impact on the inhibition rate. After verifying the optimal process conditions, a stable and efficient enzymatic hydrolysis product was obtained. Molecular weight distribution and amino acid composition analysis showed that alkaline protease preferentially cleaves hydrophobic amino acids (Leu, Val), while chymotrypsin targets aromatic amino acids (Trp, Phe). The two synergistically release highly active peptides.

[0068] This study used soybean as raw material to prepare highly active ACE inhibitory peptides through a targeted dual enzymatic hydrolysis technique, and systematically explored their preparation process, separation and purification, in vivo antihypertensive effect, and molecular mechanism of action. Table 3 shows:

[0069]

[0070] Table 3 ACE inhibition rate and IC of GFC components 50 value

[0071] The study found that stepwise enzymatic hydrolysis with alkaline protease and chymotrypsin could achieve an ACE inhibition rate of over 70%, and screened out 14 peptide sequences with high ACE inhibition: PDFKNPLGW, SEYPPLGRF, ALGAGRLF, FGSPAGPF, STPLHLW, DHPPASW, SWGEDW, QMPTEW, GDGLKW, GKGLW, EWEGF, GLGPF, LLEW, and LGKW. The amino acid sequences are as follows:

[0072] H-Pro-Asp-Phe-Lys-Asn-Pro-Leu-Gly-Trp-OH;

[0073] H-Ser-Glu-Tyr-Pro-Pro-Leu-Gly-Arg-Phe-OH;

[0074] H-Ala-Leu-Gly-Ala-Gly-Arg-Leu-Phe-OH;

[0075] H-Phe-Gly-Ser-Pro-Ala-Gly-Pro-Phe-OH;

[0076] H-Ser-Thr-Pro-Leu-His-Leu-Trp-OH;

[0077] H-Asp-His-Pro-Pro-Ala-Ser-Trp-OH;

[0078] H-Ser-Trp-Gly-Glu-Asp-Trp-OH;

[0079] H-Gln-Met-Pro-Thr-Glu-Trp-OH;

[0080] H-Gly-Asp-Gly-Leu-Lys-Trp-OH;

[0081] H-Gly-Lys-Gly-Leu-Trp-OH;

[0082] H-Glu-Trp-Glu-Gly-Phe-OH;

[0083] H-Gly-Leu-Gly-Pro-Phe-OH;

[0084] H-Leu-Leu-Glu-Trp-OH;

[0085] H-Leu-Gly-Lys-Trp-OH.

[0086] 3. Isolation, purification, and stability study of soybean ACE inhibitory peptides. Based on an optimized enzymatic hydrolysis process, the resulting enzymatic hydrolysis products were isolated and purified using ultrafiltration and gel chromatography techniques to screen for peptide components with the highest ACE inhibition rates. Subsequently, UHPLC-Q-Orbitrap-MS / MS was used to identify the peptide sequences and analyze the peptide chain length, number of peptides, and distribution characteristics to identify the optimal ACE inhibitory peptide and clarify its inhibition mode. Further evaluation was conducted on its stability under different temperature, pH conditions, and digestive environments.

[0087] Based on ultrafiltration and gel chromatography technology ( Figure 4 ), the <3kDa component was separated from the hydrolysate, and further purified and separated to obtain G1, G2, and G3 components. Among them, the G3 component had the best function, with an ACE inhibition rate of 69.65% (Table 3) and an IC50 value of 33.73μM, showing the best activity. UHPLC-Q-Orbitrap-MS / MS identified 1752 peptides, of which tetrapeptides to nonapeptides accounted for 83.8%, and the C-terminal tryptophan (Trp)-containing peptides (such as GKGLW and GDGLKW) had the highest activity, with IC50 values ​​of 33.98 and 34.96μM, respectively. Stability experiments showed that the activity of this component was stable under conditions of 20-100℃ and pH2-8, and after simulated gastrointestinal digestion ( Figure 5 ) The inhibition rate did not change significantly. Analysis of the inhibition pattern revealed that both peptides were competitive inhibitors, laying the foundation for subsequent functional verification.

[0088] 4. Antihypertensive effect of soybean oligopeptides on SHR. The antihypertensive effect of the <3kDa oligopeptide fraction obtained by ultrafiltration in spontaneously hypertensive rats (SHR) was evaluated. First, its acute antihypertensive effect was determined, and the changes in body weight and blood pressure of SHR for 35 days were recorded. The lipid metabolism indicators, including total triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), were further analyzed. In addition, oxidative stress-related indicators were measured to evaluate its antioxidant capacity. Finally, hematoxylin-eosin (H&E) staining was used to perform pathological analysis on the kidney, heart and thoracic aorta tissues of SHR to explore its protective effect on target organs.

[0089] Animal experiments have verified the antihypertensive effect of the <3kDa component. After 5 weeks of continuous administration of the high-dose group (500mg / kg), the systolic and diastolic blood pressures of SHR decreased by 40.54mmHg and 26.52mmHg, respectively. Figure 6 ) and significantly reduced organ weights (Table 4).

[0090]

[0091] Table 4 Effects of soybean oligopeptides on rat organs after 35 days of feeding (x±s, n=6)

[0092] Serum biochemical analysis showed that this component reduced the oxidative stress marker MDA (by 40.9%) and increased GSH (by 45.8%) and SOD (by 32.2%) levels, while also regulating lipid metabolism (TC, HDL-C, and LDL-C). Histopathology showed that the high-dose soy peptide group effectively reversed myocardial fibrosis and renal edema, with its multi-target effects (ACE inhibition and anti-oxidation) being the core of its antihypertensive mechanism.

[0093] 5. Computational simulation and mechanistic analysis of the soy peptide-ACE interaction. Molecular docking technology was used to analyze the interaction pattern between the soy ACE inhibitory peptide and key amino acid residues in the ACE active site. Subsequently, molecular dynamics simulations were used to assess the binding stability of the peptide-ACE complex and calculate its binding free energy. Further analysis of the decomposed residue binding energy was performed to identify amino acid residues that play a key role in the peptide-ACE interaction. In addition, the functional role of the C-terminal tryptophan in the peptide-ACE binding process was specifically explored to clarify its contribution to ACE inhibitory activity.

[0094] Based on molecular docking and molecular dynamics simulation ( Figure 7 ), sub-figure BF is the binding simulation of LGKW, GKGLW, EWEGF, GDGLKW, and DHPPASW, and the corresponding binding energies are: -10.99, -12.31, -10.91, -11.57, and -11.04 (unit: kcal / mol). ACE has three main active pockets: S1, S2, and S1′. S1 includes Ala354, Glu384, and Tyr523; S2 consists of 5 residues, Gln281, His353, Lys511, His513, and Tyr520; S1′ only involves residue Glu162, which participates in the charge interaction of the N-terminal amino group of the substrate. In addition, the active center of ACE contains a Zn ion (Zn 2 +), coordinated with His383, His387 and Glu411. Peptides interact with ACE residues through a variety of mechanisms, including van der Waals forces, hydrophobic forces, electrostatic forces, etc., and hydrogen bonds are the main interaction force for the formation of stable peptide-ACE complexes. Among them, GKGLW (-12.31kcal / mol), which has the lowest binding energy, forms five hydrogen bonds with ACE residues Ala354, Tyr523, His387, Ser284 and Gln281. In other words, GKGLW forms two hydrogen bonds with the S1 pocket (Ala354, Tyr523) and one hydrogen bond with the S2 pocket (Gln281). LGKW forms four hydrogen bonds with ACE residues His353, Tyr523, His387 and Asn70, and forms a pi-pi interaction with His383. DHPPASW forms three hydrogen bonds with ACE, but does not involve the active pocket of ACE. The residues involved include Ala356, Asp358 and His387. GDGLKW (-11.04 kcal / mol) forms four hydrogen bonds with ACE, involving residues including Gln403, Arg522, His353 and His387. EWEGF (-10.91 kcal / mol) only forms one hydrogen bond with ACE residue His387. This explains why GKGLW and GDGLKW have a strong inhibitory effect on ACE, because the number of hydrogen bonds can directly reflect or determine the affinity and stability between the ACE receptor and the polypeptide. Studies have confirmed that Trp hinders substrate binding by embedding into the ACE catalytic cavity, providing a molecular basis for peptide design and activity optimization.

[0095] The present invention technically ensures the purity and efficacy of the product through specific production throughout the entire process. It is not only highly effective, but also does not affect the taste of the protein food itself. Animal experiments have shown that such peptides can significantly reduce SHR's SBP (40.54 mmHg reduction) and DBP (26.52 mmHg reduction), and effectively improve cardiac and renal pathological damage. Molecular docking and kinetic simulations revealed that the C-terminal tryptophan-containing peptide binds to the ACE active pocket through hydrogen bonds, hydrophobic interactions, and π-π stacking, illustrating its competitive inhibition mechanism. Short peptides with similar characteristic structures all exhibit ACE inhibition. It can be concluded that by adding an improved enzymatic hydrolysis process to the traditional food production process, the release of endogenous functional peptide chains in soy milk can be achieved, so that it can be consumed directly ( Figure 5 ) can lower blood pressure ( Figure 6 )’s efficacy.

[0096] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. The role of highly active ACE inhibitory peptides containing tryptophan at the C-terminus in lowering blood pressure.

2. The highly active ACE inhibitory peptide containing tryptophan at the C-terminus of claim 1, comprising one or more of the following peptide segments: PDFKNPLGW peptide, whose amino acid sequence is: H-Pro-Asp-Phe-Lys-Asn-Pro-Leu-Gly-Trp-OH; The amino acid sequence of the SEYPPLGRF peptide is: H-Ser-Glu-Tyr-Pro-Pro-Leu-Gly-Arg-Phe-OH; The ALGAGRLF peptide segment has the following amino acid sequence: H-Ala-Leu-Gly-Ala-Gly-Arg-Leu-Phe-OH; The FGSPAGPF peptide fragment has the following amino acid sequence: H-Phe-Gly-Ser-Pro-Ala-Gly-Pro-Phe-OH; The STPLHLW peptide has the following amino acid sequence: H-Ser-Thr-Pro-Leu-His-Leu-Trp-OH; The DHPPASW peptide fragment has the following amino acid sequence: H-Asp-His-Pro-Pro-Ala-Ser-Trp-OH; The SWGEDW peptide has the following amino acid sequence: H-Ser-Trp-Gly-Glu-Asp-Trp-OH; The QMPTEW peptide fragment has the following amino acid sequence: H-Gln-Met-Pro-Thr-Glu-Trp-OH; The GDGLKW peptide has the following amino acid sequence: H-Gly-Asp-Gly-Leu-Lys-Trp-OH; The GKGLW peptide has the following amino acid sequence: H-Gly-Lys-Gly-Leu-Trp-OH; The amino acid sequence of the EWEGF peptide is: H-Glu-Trp-Glu-Gly-Phe-OH; The GLGPF peptide has the following amino acid sequence: H-Gly-Leu-Gly-Pro-Phe-OH; The LLEW peptide has the following amino acid sequence: H-Leu-Leu-Glu-Trp-OH; or The amino acid sequence of the LGKW peptide is: 1H-Leu-Gly-Lys-Trp-OH.

3. The highly active ACE inhibitory peptide containing tryptophan at the C-terminus of claim 1, comprising the following peptide segments: The GDGLKW peptide has the following amino acid sequence: H-Gly-Asp-Gly-Leu-Lys-Trp-OH; as well as, The amino acid sequence of the GKGLW peptide is: H-Gly-Lys-Gly-Leu-Trp-OH.

4. The method for preparing the highly active ACE inhibitory peptide containing tryptophan at the C-terminus according to claim 1, characterized in that: The method comprises the following steps: Step 1: Perform enzymatic separation and purification on various common food protein sources, analyze their endogenous ACE inhibitory peptides, and determine that soybeans are the raw material for obtaining the highly active ACE inhibitory peptide containing tryptophan at the C-terminus; Step 2: Using different commercial proteases, the optimal hydrolytic protease for preparing highly active ACE inhibitory peptides was selected based on the degree of hydrolysis, peptide content, and ACE inhibition rate; Step 3: Using ultrafiltration and gel chromatography technology, the soybean protein is hydrolyzed with the hydrolyzing protease to screen the peptide component with the highest ACE inhibition rate; Step 4: Based on the blood pressure reduction experiment, the highly active ACE inhibitory peptide containing tryptophan at the C-terminus is screened out.

5. The preparation method according to claim 4, characterized in that The hydrolytic protease in step 2 includes alkaline protease and chymotrypsin.

6. The preparation method according to claim 4, characterized in that The enzymatic hydrolysis conditions in step 3 were: time 2 h, enzyme-substrate ratio 2000 U / g, and substrate concentration 5 mg / mL.

7. The method for using the highly active ACE inhibitory peptide containing tryptophan at the C-terminus according to any one of claims 1 to 6, characterized in that: The highly active ACE inhibitory peptide containing tryptophan at the C-terminus is used for preparing functional blood pressure-lowering protein food.

8. A blood pressure lowering soy milk, which is prepared by adding the C-terminal tryptophan-containing high-activity ACE inhibitory peptide according to any one of claims 1 to 6 to soy milk and enzymatically hydrolyzing it for at least 2 hours.

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