Sweet almond ACE (angiotensin converting enzyme) inhibitory peptide as well as composition, preparation method and application thereof

Abstract

The invention relates to the technical field of functional food processing, in particular to a sweet almond ACE (angiotensin converting enzyme) inhibitory peptide as well as a composition, a preparation method and application thereof. The invention provides a sweet almond ACE inhibitory peptide. The amino acid sequence of the sweet almond ACE inhibitory peptide is shown as SEQ ID NO: 1. The sweet almond ACE inhibitory peptide composition with high yield and strong activity is prepared by taking cold-pressed sweet almond meal as a test material, adopting immobilized enzyme and optimizing an enzymolysis process. Amino acid sequence analysis is carried out on sweet almond ACE inhibitory peptide components of 1-3kDa components by adopting an LC-TOFMS (Liquid Chromatography-Time of Flight Mass Spectrometry) method, most hydrogen bonds are formed between screened polypeptide with an amino acid sequence shown as SEQ ID NO: 1 and key active sites of ACE, and a better ACE inhibitory effect is shown.

Description

Technical Field

[0001] The present invention relates to the technical field of functional food processing, and particularly relates to a sweet almond ACE inhibitory peptide, its composition, preparation method and application. Background Art

[0002] Sweet almond is a high-nutrition and high-quality food with the characteristics of both medicine and food. However, the almond meal produced after almond oil extraction is usually discarded or only used as feed, resulting in a great waste of resources. Almond protein is an important component of almonds and is also a good plant protein with both medicine and food uses. It has a complete variety of amino acids, including 18 kinds of amino acids required by the human body, and can be used as a protein nutritional fortifier and additive.

[0003] Extracting the protein from sweet almond meal and carrying out intensive processing and utilization can turn sweet almond meal from "waste" into "treasure". Research has found that almond protein peptides can have a variety of functional activities, such as antioxidant, lipid-lowering, hypoglycemic and blood pressure-lowering functions or activities. In recent years, scientific researchers have successfully isolated bioactive peptides with specific effects, such as antioxidant peptides, blood pressure-lowering peptides, and hypoglycemic peptides, from almond proteins by using different extraction technologies, greatly increasing the added value of almonds. Since there are significant differences in the contents of components such as amygdalin and protein between bitter almonds and sweet almonds, and there is currently little research on the active ingredients and efficacy of sweet almonds, the existing research results of bitter almond polypeptides cannot be directly copied, and targeted research needs to be carried out again. Therefore, how to turn sweet almond meal from "waste" into "treasure", improve the utilization rate of sweet almond protein, and increase the economic value has become an important research topic.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a sweet almond ACE inhibitory peptide, its composition, preparation method and application.

[0006] The present invention provides a composition of sweet almond ACE inhibitory peptide, and the composition of sweet almond ACE inhibitory peptide is prepared by the following method:

[0007] S1. Extract almond protein from cold-pressed sweet almond meal by salting-out method;

[0008] S2. Use genipin as a cross-linking agent, take eggshell membrane as a carrier, and prepare an immobilized enzyme by covalently cross-linking protease with eggshell membrane;

[0009] S3. Use the immobilized enzyme to enzymatically hydrolyze almond protein, and the enzymatic hydrolysis product is separated by centrifugation and ultrafiltration to obtain a fraction of 1-3 kDa, and the sweet almond ACE inhibitory peptide composition is obtained after desalting treatment.

[0010] The present invention provides a sweet almond ACE inhibitory peptide, the amino acid sequence of which is shown as SEQ ID NO:1.

[0011] The present invention also provides the application of a sweet almond ACE inhibitory peptide composition or a sweet almond ACE inhibitory peptide in the preparation of a drug for preventing and treating hypertension.

[0012] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0013] The present invention uses cold-pressed sweet almond meal as the test material, optimizes the enzymatic hydrolysis process, and prepares a sweet almond ACE inhibitory peptide composition with high yield and strong activity. The present invention uses immobilized enzyme ESM-BAP, which shows higher tolerance in terms of temperature and pH compared with free enzyme. ESM-BAP still has 75.39% enzyme activity after being reused 6 times, showing good reusability stability. The present invention continues to use LC-TOFMS method to analyze the amino acid sequence of the sweet almond ACE inhibitory peptide component in the 1-3 kDa fraction, and then uses molecular docking simulation method to analyze the structure-activity relationship of this ACE inhibitory peptide component. 68 amino acid sequences are detected by LC-TOFMS, and 5 amino acid sequences with good water solubility are selected for molecular docking simulation. The results show that these amino acid sequences are rich in hydrophobic amino acids and aromatic amino acids, and the polypeptide with the amino acid sequence shown as SEQ ID NO:1 forms the most hydrogen bonds with the key active sites of ACE, showing better ACE inhibitory effect. Description of the Drawings

[0014] Figure 1 Diagrams of freeze-dried samples of ESM and ESM-BAP: A: ESM, B: ESM-BAP;

[0015] Figure 2 SEM diagrams of both sides of ESM and ESM-BAP: A: outer surface of ESM; B: outer surface of ESM-BAP; C: inner surface of ESM; D: inner surface of ESM-BAP; Observation size unit of scanning electron microscope (SEM): 10 μm;

[0016] Figure 3 Diagram of the change in degree of hydrolysis of sweet almond protein powder enzymatically hydrolyzed by five proteases;

[0017] Figure 4 Comparison diagram of ACE inhibitory activities of five sweet almond polypeptides;

[0018] Figure 5 Diagram of the effect of genipin concentration on enzyme activity recovery rate, enzyme loading amount and enzyme activity loading amount;

[0019] Figure 6Figure showing the effects of enzyme concentration on enzyme activity recovery rate, enzyme loading, and enzyme activity loading;

[0020] Figure 7 Figure showing the effects of crosslinking temperature on enzyme activity recovery rate, enzyme loading, and enzyme activity loading;

[0021] Figure 8 Figure showing the effects of crosslinking time on enzyme activity recovery rate, enzyme loading, and enzyme activity loading;

[0022] Figure 9 Figure showing the effects of enzymolysis time on degree of hydrolysis and ACE inhibition rate;

[0023] Figure 10 Figure showing the effects of temperature on the activities of free enzyme and ESM - BAP;

[0024] Figure 11 Figure showing the effects of enzyme dosage on degree of hydrolysis and ACE inhibition rate;

[0025] Figure 12 Figure showing the effects of substrate concentration on degree of hydrolysis and ACE inhibition rate;

[0026] Figure 13 Figure showing the effects of pH on degree of hydrolysis and ACE inhibition rate;

[0027] Figure 14 Figure showing the effects of pH on the activities of free enzyme and ESM - BAP;

[0028] Figure 15 Double - reciprocal Km curve diagram of free enzyme and immobilized enzyme;

[0029] Figure 16 Reusability stability of ESM - BAP. Different lowercase letters indicate significant differences in the same experiment (P < 0.05);

[0030] Figure 17 shows the diagram of the effects of interaction of different factor variables on the ACE - inhibiting effect of sweet almond protein;

[0031] Figure 18 Total ion current chromatogram of TOF MS;

[0032] Figure 19 Molecular docking diagram of DIIALPAGVAY;

[0033] Figure 20 Molecular docking diagram of DVVAIPAGVAYW;

[0034] Figure 21 Molecular docking diagram of GRGVLGAVF;

[0035] Figure 22 Molecular docking diagram of IIALPAGVAY;

[0036] Figure 23 Molecular docking diagram of VVAIPAGVAY. Specific implementation manners

[0037] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, without conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0038] Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention may also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0039] ACE (Angiotensin-I Converting Enzyme) - angiotensin converting enzyme; BAP (Alkaline protease): alkaline protease; DH (Degree of hydrolysis): degree of hydrolysis; ESM (Egg shell film): eggshell membrane; FAPGG (N-[3-(2-furylacryloyl)]-L-phenyalanyl-glycyl-glycine): N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine; LC-TOF MS (Ultrahigh performance liquid chromatography tandem time of flight mass spectrometry): ultra-high performance liquid chromatography tandem time of flight mass spectrometry; SEM (Scanning electron microscope): scanning electron microscope.

[0040] Angiotensin converting enzyme (angiotensin-I converting enzyme, ACE) is a zinc-containing bioactive polypeptide and belongs to glycoprotein. It is widely distributed on the inner surface of human blood vessels and in plasma, and plays a crucial role in the regulation of the human blood regulation system. Hypertension is a chronic cardiovascular disease caused by the long-term high-pressure state of blood vessels in the human body. In today's society, continuous stress, intense social competition and unhealthy eating habits have all increased the incidence of hypertension. If not detected in time and effective management measures are not taken, it may induce a series of complications including coronary heart disease, cerebral hemorrhage, heart failure, etc., causing irreversible damage to the body.

[0041] An embodiment of the present invention provides a sweet almond ACE inhibitory peptide composition, which is obtained by extracting from cold-pressed sweet almond meal. The specific preparation method is as follows:

[0042] S1. Extract almond protein from cold-pressed sweet almond meal by salting-out method;

[0043] S2. Use genipin as a cross-linking agent, take eggshell membrane as a carrier, and prepare an immobilized enzyme by covalently cross-linking protease with eggshell membrane;

[0044] S3. Use the immobilized enzyme to enzymatically hydrolyze almond protein. The enzymatic hydrolysis product is separated by centrifugation and ultrafiltration to obtain a fraction of 1-3 kDa, and then desalted to obtain the sweet almond ACE inhibitory peptide composition.

[0045] As a preferred technical solution of the embodiment of the present invention, S1 includes:

[0046] S11. Prepare an almond solution with cold-pressed sweet almond meal;

[0047] S12. Crush the almond solution and centrifuge to obtain the supernatant;

[0048] S13. Add solid ammonium sulfate to the supernatant for salting-out. The obtained precipitate is dissolved and then dialyzed to obtain almond protein. Optionally, the salting-out is stepwise salting-out. First, add solid ammonium sulfate to a saturation of 20%-25% and dissolve it fully, then centrifuge to obtain the supernatant and add solid ammonium sulfate to a concentration of 40%-50%, and centrifuge to obtain the precipitate for dialysis. The dialysis conditions can be: add it to a dialysis bag with MW3500 treated with a mixed solution of sodium bicarbonate and EDTA, and dialyze with PBS solution for 8-16 hours. To ensure the activity of the protein, the temperature of crushing and centrifugation is operated at 0-10°C, such as 2-8°C, or 4°C, 5°C.

[0049] As a preferred technical solution of the embodiment of the present invention, the protease is selected from one or more of papain, alkaline protease, neutral protease, trypsin, and bromelain, and preferably alkaline protease is used.

[0050] As a preferred technical solution of an embodiment of the present invention, S2 includes: taking eggshell membrane, soaking it in genipin solution, washing it, and then adding it to protease solution for crosslinking to obtain immobilized enzyme ESM-BAP. Specifically, the temperature of the crosslinking reaction is 42°C to 48°C, for example, 45°C can be adopted. The time of the crosslinking reaction is 1 to 4 hours, for example, 2 hours can be adopted. The mass percentage concentration of the genipin solution is 0.5% to 1%, for example, 0.75% can be adopted. The concentration of the protease solution is 2 to 10 mg / mL, for example, 5 mg / mL can be adopted. Compared with free alkaline protease (BAP), ESM-BAP has higher temperature and pH tolerance, and still has 75.39% enzyme activity after being reused 6 times, thus significantly increasing the yield of the preparation of sweet almond ACE inhibitory peptide composition, improving the efficiency, and being applicable to large-scale production applications.

[0051] As a preferred technical solution of an embodiment of the present invention, in S3, the conditions for enzymatic hydrolysis using crosslinked enzyme are specifically as follows: the temperature is 50 to 60°C, preferably 55 to 56°C. The pH is 10 to 12, preferably 10.7 to 11. The time is 3 to 5 hours, preferably 4 to 4.1 hours. The enzyme addition amount is 5000 to 9000 U / g, preferably 7000 to 7200 U / g. The concentration of the substrate is 3% to 5%, preferably 4%.

[0052] As a preferred technical solution of an embodiment of the present invention, in S3, the supernatant obtained by centrifuging the enzymatic hydrolysis product is first vacuum filtered through a 0.55 μm aqueous filter membrane, and then centrifuged using a 3 kDa ultrafiltration centrifugal tube. The supernatant solution is centrifuged using a 1 kDa ultrafiltration centrifugal tube, and a fraction of 1 to 3 kDa is obtained by ultrafiltration separation. Specifically, the centrifugation conditions are at 0 to 5°C, the rotation speed is 6000 to 8000 rpm, and the time is 20 to 40 min. For example, centrifugation can be carried out at 4°C with a rotation speed of 7000 rpm for 30 min.

[0053] As a preferred technical solution of an embodiment of the present invention, the sweet almond ACE inhibitory peptide composition contains polypeptides with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:4. The amino acid sequence of SEQ ID NO:1 is: VVAIPAGVAY; the amino acid sequence of SEQ ID NO:2 is: GRGVLGAVF; the amino acid sequence of SEQ ID NO:3 is: DIIALPAGVAY; the amino acid sequence of SEQ ID NO:4 is: DVVAIPAGVAYW.

[0054] An embodiment of the present invention also provides a sweet almond ACE inhibitory peptide, and its amino acid sequence is as shown in SEQ ID NO:1. In the embodiment of the present invention, multiple amino acid sequences with good water solubility are screened for molecular docking simulation. The results show that the content of hydrophobic amino acids and aromatic amino acids in these amino acid sequences is rich. Among them, the polypeptide with the amino acid sequence as shown in SEQ ID NO:1 forms the most hydrogen bonds with the key active sites of ACE, showing better ACE inhibitory effect. Through enzyme activity experiments, this polypeptide has excellent ACE inhibition rate and higher biological activity.

[0055] An embodiment of the present invention also provides the application of the above sweet almond ACE inhibitory peptide composition or the above sweet almond ACE inhibitory peptide in the preparation of drugs for preventing and treating hypertension. The sweet almond ACE inhibitory peptide composition proposed in the embodiment of the present invention has high biological activity, can be used in the preparation of drugs for preventing and treating hypertension, and has broad application prospects. The screened active polypeptide shows better ACE inhibitory effect, and can be prepared in large quantities by chemical methods or biological fermentation methods in the future, and has even broader application prospects.

[0056] Example 1

[0057] This example is used to illustrate the conditions for extracting sweet almond protein by salting-out method:

[0058] 1. Take a certain mass of cold-pressed sweet almond meal, add PBS solution with a concentration of 0.1mol / L and pH = 7.2, stir and dissolve it. The ratio of sweet almond meal to PBS is 1:12. After preparing the almond solution, let it stand at room temperature for 0.5h.

[0059] 2. Crush the almond solution at an ultrasonic power of 100W for 30min, and control the water temperature below 10℃ to prevent protein denaturation.

[0060] 3. Under the conditions of 4℃ and 7000rpm, freeze-centrifuge the almond solution for 15min, and then take the supernatant.

[0061] 4. Add ammonium sulfate solid to the supernatant obtained after centrifugation until the saturation is 25%, that is, the addition amount is 144g / L. Stir for 30min to fully dissolve it, and then freeze-centrifuge it at 4℃ and 9000rpm for 20min to take the supernatant.

[0062] 5. Add ammonium sulfate solid to the supernatant again until the concentration is 50%, that is, the addition amount is 158g / L. Stir for 30min to fully precipitate the sweet almond protein, and then freeze-centrifuge it at 4℃ and 9000rpm for 20min to take the precipitate.

[0063] 6. Add an appropriate amount of 0.01 mol / L PBS solution to the precipitate to dissolve it, place it in a dialysis bag of MW3500 treated with a mixed solution of 2% sodium bicarbonate and 1 mmol / L EDTA, and dialyze it with 0.01 mol / L PBS solution for 12 h until no precipitate appears when 1% barium chloride solution is added to the dialysis solution.

[0064] 7. Take it out and pour it into a petri dish, cover it with plastic wrap, make holes, fix it with a rubber band, pre-freeze it, and vacuum freeze-dry it for 48 h.

[0065] 8. Measure the extraction rate of the finished product, and then store it frozen and sealed in a low-temperature refrigerator.

[0066] 18.5 g of sweet almond protein powder was obtained by salting out method from every 100 g of cold-pressed sweet almond meal. The protein content of cold-pressed sweet almond meal was measured to be 60.54% by using an automatic Kjeldahl nitrogen analyzer, and the protein content of almond protein powder was 56.28%. Based on this, the extraction rate of sweet almond protein was calculated to be 17.19%.

[0067] Example 2

[0068] This example is used to illustrate the preparation method and structural characterization of the ESM-BAP sweet almond ACE inhibitory peptide composition:

[0069] 1. Preparation of ESM

[0070] Discard the egg yolks from commercially available eggs and take the eggshells. Then soak the eggshells in a 20% acetic acid solution for 12 - 18 h, separate the eggshells and eggshell membranes (ESM), rinse the eggshell membranes with ultrapure water until neutral, and then place them in a phosphate buffer solution with pH = 7.4 and store them at 4°C.

[0071] 2. Preparation of ESM-BAP

[0072] Take an appropriate amount of ESM, soak it in a cross-linking agent with a certain concentration in a constant-temperature water bath at 25°C and 80 RPM for 3.5 h, wash away the excess cross-linking agent on the ESM with ultrapure water, and then slowly add a pre-adjusted enzyme solution of alkaline protease with a certain concentration. Then carry out constant-temperature oscillation cross-linking at 45°C and 80 rpm. After cross-linking for a certain time, wash it with phosphate buffered saline solution to finally obtain ESM-BAP.

[0073] 3. Characterization results of ESM and ESM-BAP

[0074] The freeze-dried physical diagram is as Figure 1 shown, and the scanning electron microscope analysis is as Figure 2 shown.

[0075] According to Figure 1It can be seen that ESM presents off-white after freeze-drying, while ESM-BAP presents dark blue after freeze-drying. The dark blue is the characteristic color reaction of the successful cross-linking of enzyme molecules on ESM through the cross-linking agent genipin. This indicates that the enzyme molecules are successfully immobilized on ESM to form ESM-BAP. According to Figure 2 Observation shows that the outer surface of ESM presents a reticular structure composed of fibers. The fibers are densely intertwined, and various-shaped and -sized particles are distributed on it, making the surface appear rough. The keratin fiber structure on the inner surface of ESM is dense and uneven; the outer surface of ESM-BAP becomes smoother after being wrapped by enzyme molecules but still presents a reticular structure, and the inner surface of ESM-BAP becomes denser and smoother, indicating that the enzyme molecules are successfully immobilized on the surface of ESM.

[0076] Example 3

[0077] This example is used to illustrate the preparation method of the sweet almond ACE inhibitory peptide composition:

[0078] Take the sweet almond protein powder prepared in Example 1 to prepare a 4% substrate enzymatic hydrolysis solution, add ESM-BAP prepared in Example 2 at 7000 U / g, the pH of enzymatic hydrolysis is 11, the temperature of enzymatic hydrolysis is 55 °C, and enzymatic hydrolysis is carried out for 4 hours. After enzymatic hydrolysis, inactivate the enzyme in a water bath at 85 °C, and adjust the pH to 4 with hydrochloric acid to obtain an almond protein hydrolysate. The almond protein hydrolysate is centrifuged at 4 °C and 7000 rpm for 15 min, and the supernatant is taken. The supernatant is first vacuum filtered through a 0.55 μm aqueous filter membrane, and then a 3 kDa ultrafiltration centrifugal tube is used to centrifuge at 4 °C and 7000 rpm for 30 min in a high-speed refrigerated centrifuge to separate large molecular polypeptide chains. The remaining solution is then centrifuged at 4 °C and 7000 rpm for 30 min using a 1 kDa ultrafiltration centrifugal tube in a high-speed refrigerated centrifuge, and the 1 - 3 kDa fraction is ultrafiltered and separated for subsequent desalting treatment. The ACE inhibitory peptide solution of the optimal component obtained through ultrafiltration separation and determination of the results is put into an MW100 dialysis bag, the temperature is controlled at 10 °C, and dialysis is carried out under the action of a magnetic stirrer until there is no BacL 2 precipitate.

[0079] Example 4

[0080] This example is used to illustrate the screening process of the optimal protease:

[0081] Take an appropriate amount of freeze-dried sweet almond protein powder to prepare a 4% substrate enzymatic hydrolysis solution, add protease at 7000 U / g, and hydrolyze under the optimal temperature and pH conditions of each enzyme. The optimal enzymatic hydrolysis conditions are shown in Table 1 in detail. During the hydrolysis process, the degree of hydrolysis is measured, and after reacting for 6 h, the enzyme is inactivated in a water bath at 85 °C, and the pH is adjusted to 4 with hydrochloric acid. After centrifuging at 8000 rpm for 15 min, the supernatant is taken, and its ACE inhibitory rate is measured.

[0082] Table 1: Optimal enzymatic hydrolysis conditions of five proteases

[0083] Enzyme type Papain Neutral protease Bromelain Trypsin Alkaline protease Temperature (°C) 50 45 55 37 55 pH 7.2 7 7 8 10.5

[0084] Method for determining the enzyme activity of five proteases: Folin-Ciocalteu method in the national standard SBT10317-1999

[0085] Table 2: Enzyme activities of 5 proteases

[0086] Enzyme type Trypsin Papain Bromelain Neutral protease Alkaline protease Enzyme activity 171492±996 357026±919 304813±517 64324±276 11751±780

[0087] 1. The method for determining the degree of hydrolysis (DH) adopts the pH-stat method. The change diagram of the degree of hydrolysis of five proteases hydrolyzing sweet almond protein powder is as Figure 3 shown. 2. The determination of the ACE inhibitory rate of sweet almond polypeptides adopts the 96-well plate assay method of Luo Lin (Luo Lin, Ding Qingzhi, Ma Haile; The 96-well plate method is used for the in vitro detection of high-throughput angiotensin-converting enzyme inhibitors [J], Chinese Journal of Analytical Chemistry, 2012, 40(01):129-134.).

[0088] The comparison of the ACE inhibitory activities of five sweet almond polypeptides is as Figure 4 shown. Considering the results of the two indexes of the degree of hydrolysis and the ACE inhibitory rate comprehensively, alkaline protease is selected as the optimal enzyme.

[0089] Example 5

[0090] This example is used to illustrate the influence of genipin concentration on the enzymatic activity characteristics of immobilized enzyme

[0091] Take the same weight of ESM, and soak it in genipin solutions with concentrations of 0.10%, 0.25%, 0.50%, 0.75% and 1.00% respectively at 25°C and 80 rpm for 3.5 h. Wash the excess cross-linking agent on the ESM with ultrapure water, add the ESM into the enzyme solution with a concentration of 5 mg / mL, and under the condition of a constant temperature water bath oscillation at 45°C and 80 rpm, after cross-linking for 2 h, wash it with PBS to obtain ESM-BAP. While collecting the washing solution to measure the protein content, measure the enzyme activity of ESM-BAP, calculate the enzyme activity recovery rate of ESM-BAP, as well as the enzyme loading amount and enzyme activity loading amount of ESM. Method for determining the enzymatic activity index: Determine the protein concentration according to the Bradford method using bovine serum albumin as the standard. The experimental results are as Figure 5 shown. When the genipin concentration is 0.75%, it reaches the peak value of 61.87%. Therefore, considering the cost and effect comprehensively, a genipin concentration of 0.75% is selected.

[0092] Example 6

[0093] This example is used to illustrate the influence of enzyme concentration on the enzymatic activity characteristics of immobilized enzyme

[0094] Take the same weight of ESM and soak it in 0.75% genipin solution at 25 °C and 80 rpm for 3.5 h. Wash off the excess cross-linking agent on the ESM with ultrapure water. Add the ESM into enzyme solutions with concentrations of 1 mg / mL, 2 mg / mL, 5 mg / mL, 8 mg / mL, and 10 mg / mL respectively, and under the condition of constant temperature water bath oscillation at 45 °C and 80 rpm, after cross-linking for 2 h, wash it with PBS to obtain ESM-BAP. While collecting the washing solution to measure the protein content, measure the enzyme activity of ESM-BAP, calculate the enzyme activity recovery rate of ESM-BAP, as well as the enzyme loading and enzyme activity loading of ESM. The experimental results are as Figure 6 shown. Considering both cost and effect, select the enzyme concentration added to be 5 mg / mL.

[0095] Example 7

[0096] This example is used to illustrate the influence of cross-linking temperature on the enzymatic activity characteristics of the immobilized enzyme.

[0097] Take the same weight of ESM and soak it in 0.75% genipin solution at 25 °C and 80 rpm for 3.5 h. Wash off the excess cross-linking agent on the ESM with ultrapure water. Add the ESM into 5 mg / mL enzyme solutions at 25 °C, 35 °C, 45 °C, 55 °C, and 65 °C respectively, and perform water bath oscillation cross-linking at 80 rpm. After cross-linking for 2 h, wash it with PBS to obtain ESM-BAP. While collecting the washing solution to measure the protein content, measure the enzyme activity of the immobilized enzyme, calculate the enzyme activity recovery rate of the immobilized enzyme, as well as the enzyme loading and enzyme activity loading of ESM. The experimental results are as Figure 7 shown. Considering the effect comprehensively, select the cross-linking temperature to be 45 °C.

[0098] Example 8

[0099] This example is used to illustrate the influence of cross-linking time on the enzymatic activity characteristics of the immobilized enzyme.

[0100] Take the same weight of ESM and soak it in 0.75% genipin solution at 25 °C and 80 rpm for 3.5 h. After washing off the excess cross-linking agent on the ESM with ultrapure water, add the ESM into 5 mg / mL enzyme solution, and under the condition of constant temperature water bath oscillation at 45 °C and 80 rpm, after cross-linking for 1 h, 2 h, 4 h, 6 h, and 8 h respectively, wash it with PBS to obtain ESM-BAP. While collecting the washing solution to measure the protein content, measure the enzyme activity of ESM-BAP, calculate the enzyme activity recovery rate of the immobilized enzyme, as well as the enzyme loading and enzyme activity loading of ESM. The experimental results are as Figure 8 shown. Considering both time and effect, select the enzymatic hydrolysis time to be 2 h.

[0101] Example 9

[0102] This example is used to illustrate the effect of the enzymatic hydrolysis time of the immobilized enzyme on the hydrolysis effect:

[0103] The freeze-dried sweet almond protein powder was made into a protein solution with a concentration of 4%, the pH value was adjusted to 11, and 7000 U / g of immobilized enzyme was added at 55 °C. The enzymatic hydrolysis was carried out for 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h respectively. The degree of hydrolysis (DH) was measured during the enzymatic hydrolysis process. Subsequently, the enzyme was inactivated in a water bath at 85 °C for 15 min, hydrochloric acid was added to adjust the pH value of the enzymatic hydrolysis solution to 4.5, and centrifuged at 8500 rpm for 15 min. The ACE inhibition rate of the supernatant was compared to obtain the appropriate enzymatic hydrolysis time. The experimental results are as Figure 9 shown. According to Figure 9 observations, an enzymatic hydrolysis time of 4 h was selected.

[0104] Example 10

[0105] This example is used to illustrate the effect of the enzymatic hydrolysis temperature of the immobilized enzyme on the hydrolysis effect:

[0106] The freeze-dried sweet almond protein powder was made into a protein solution with a concentration of 4%, the pH value was adjusted to 11, and 7000 U / g of immobilized enzyme was added. The enzymatic hydrolysis was carried out at 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, and 70 °C for 4 h respectively. The degree of hydrolysis (DH) was measured during the enzymatic hydrolysis process. Subsequently, the enzyme was inactivated in a water bath at 85 °C for 15 min, hydrochloric acid was added to adjust the pH value of the enzymatic hydrolysis solution to 4.5, and centrifuged at 8500 rpm for 15 min. The ACE inhibition rate of the supernatant was compared to obtain the appropriate enzymatic hydrolysis temperature. The experimental results are as Figure 10 shown. According to Figure 10 observations, ESM-BAP showed the highest enzyme activity at 55 °C, exhibited strong tolerance, and was significantly different from the free enzyme (P < 0.05).

[0107] Example 11

[0108] This example is used to illustrate the effect of the amount of added immobilized enzyme on the hydrolysis effect:

[0109] The freeze-dried sweet almond protein powder was made into a protein solution with a concentration of 4%, the pH value was adjusted to 11, and 1000 U / g, 3000 U / g, 5000 U / g, 7000 U / g, 9000 U / g, and 11000 U / g of immobilized enzyme were added respectively at 55 °C. The enzymatic hydrolysis was carried out for 4 h. The degree of hydrolysis (DH) was measured during the enzymatic hydrolysis process. Subsequently, the enzyme was inactivated in a water bath at 85 °C for 15 min, hydrochloric acid was added to adjust the pH value of the enzymatic hydrolysis solution to 4.5, and centrifuged at 8500 rpm for 15 min. The ACE inhibition rate of the supernatant was compared to obtain the appropriate amount of added enzyme. The experimental results are as Figure 11 shown. According to Figure 11 , an amount of added enzyme of 7000 U / g was selected.

[0110] Example 12

[0111] This example is used to illustrate the effect of the substrate concentration of immobilized enzyme on the enzymatic hydrolysis effect:

[0112] Prepare protein solutions with concentrations of 1%, 2%, 3%, 4%, 5% and 6% from freeze-dried sweet almond protein powder respectively, adjust the pH value to 11, add immobilized enzyme at 7000 U / g under the condition of 55 °C, and carry out enzymatic hydrolysis for 4 h. During the enzymatic hydrolysis process, measure the degree of hydrolysis (DH). Subsequently, inactivate the enzyme in a water bath at 85 °C for 15 min, adjust the pH value of the enzymatic hydrolysate to 4.5 with hydrochloric acid, and centrifuge at 8500 rpm for 15 min. Compare the ACE inhibition rate of the supernatant to obtain the appropriate substrate concentration addition amount. The experimental results are as Figure 12 shown. According to Figure 12 , select a substrate concentration addition amount of 4%.

[0113] Example 13

[0114] This example is used to illustrate the effect of the enzymatic hydrolysis pH of immobilized enzyme on the enzymatic hydrolysis effect:

[0115] Prepare a protein solution with a concentration of 4% from freeze-dried sweet almond protein powder, adjust the pH to 7, 8, 9, 10, 11, 12 respectively, add immobilized enzyme at 7000 U g-1 under the condition of 55 °C, and carry out enzymatic hydrolysis for 4 h. During the enzymatic hydrolysis process, measure the degree of hydrolysis (DH). Subsequently, inactivate the enzyme in a water bath at 85 °C for 15 min, adjust the pH value of the enzymatic hydrolysate to 4.5 with hydrochloric acid, and centrifuge at 8500 rpm for 15 min. Compare the ACE inhibition rate of the supernatant to obtain the appropriate pH condition. The experimental results are as Figure 13 shown. According to Figure 13 it can be seen that the ACE inhibition rate is the highest when pH = 11, and select pH = 11.

[0116] Example 14

[0117] This example is used to illustrate the difference in the enzymatic hydrolysis effects between immobilized enzyme and free enzyme:

[0118] 1. Optimal catalytic pH and temperature

[0119] The free enzyme and the ESM-BAP prepared under the optimal conditions were respectively placed in buffer solutions with pH values of 5.5, 7, 8.5, 9.5, and 10.5 for reaction. At the same time, the pH value of the substrate solution was also adjusted to the corresponding value to maintain the consistency of the enzyme activity measurement conditions, without changing other conditions for enzyme activity measurement, and the enzyme activities of the free enzyme and ESM-BAP were measured. Then, the enzyme activities of the free enzyme and ESM-BAP were determined. The free enzyme and the ESM-BAP prepared under the optimal conditions were respectively reacted at 25 °C, 35 °C, 45 °C, 55 °C, 65 °C, and 85 °C, without changing other conditions for enzyme activity measurement, and the enzyme activities of the free enzyme and ESM-BAP were measured. The experimental results are as Figure 14 shown.

[0120] According to Figure 14 it can be seen that as the pH increases, the relative enzyme activity of the free enzyme first rises rapidly and then decreases. When the pH is in the range of 5.5 - 9.5, the relative enzyme activity gradually increases and shows the highest relative enzyme activity at pH 9.5. As the pH rises, the relative enzyme activity of ESM-BAP gradually increases and shows the highest relative enzyme activity at pH 10.5. Compared with the free enzyme, there is a significant difference when the pH increases from 9.5 to 10.5 (P < 0.05). Since the microenvironment of the enzyme molecule changes after immobilization, the sensitivity of the enzyme to pH decreases, enhancing its ability to resist conformational changes at high pH. In addition, ESM provides additional protection for the enzyme active site, reducing the risk of inactivation under polar conditions.

[0121] 2. Enzyme kinetic parameters

[0122] Determination of the Michaelis constant: First, casein solutions with concentrations of 1 mg / mL, 2 mg / mL, 5 mg / mL, 10 mg / mL, and 20 mg / mL were prepared. Then, the free enzyme and ESM-BAP with the same enzyme activity were respectively allowed to react in casein solutions with different concentrations, and the absorbance (OD) values were measured by the Folin-phenol method. The tyrosine content was calculated according to the casein standard curve (y = 0.0098x + 0.0081, R2 = 0.9994), and the reaction rates of the free enzyme and ESM-BAP were deduced according to the literature. Finally, the Michaelis constant values Km of the free enzyme and ESM-BAP were calculated using the Lineweaver-Burk double reciprocal method. The experimental results are as Figure 15 shown.

[0123] According to Figure 15It can be seen that the Km values of the free enzyme ESM and the immobilized alkaline protease ESM-BAP are 6.64 mol / L and 8.952 mol / L respectively. After immobilization, the affinity of ESM-BAP for the substrate and its catalytic efficiency do not decrease significantly. This is because ESM has high biocompatibility and has little impact on enzyme molecules. It shows that ESM can effectively maintain the catalytic activity and affinity of enzymes during the enzyme immobilization process.

[0124] 3. Reusability and stability of ESM-BAP

[0125] Use ESM-BAP to enzymatically hydrolyze the substrate casein six times, measure the enzyme activity, calculate the enzyme activity recovery rate, and analyze the operational stability and usage characteristics of ESM-BAP. The experimental results are as Figure 16 shown.

[0126] According to Figure 16 it can be seen that after the immobilized enzyme is reused 6 times, the remaining enzyme activity is still 75.39%. The decrease in enzyme activity may be due to the detachment of enzyme molecules from the carrier or their slow inactivation during repeated operations. The immobilized enzyme has higher operational stability because the carrier material can provide support and protection for enzyme molecules, reduce the loss and inactivation of enzyme molecules, thereby extending the service life of the enzyme and providing favorable conditions for industrial applications.

[0127] Example 15

[0128] Response surface optimization test of the process parameters for the enzymatic hydrolysis of the immobilized enzyme

[0129] Based on the experimental results of the above examples, the Box-Behnken method is used for experimental design. The substrate concentration is controlled at 4%, and the enzyme dosage (A), temperature (B), pH (C), and hydrolysis time (D) are selected as independent variables, with the ACE inhibition rate as the response value (Y). A 4-factor 3-level experiment is designed to determine the optimal process conditions for the enzymatic hydrolysis of sweet almond protein by the immobilized enzyme. The factors and levels are shown in Table 3:

[0130] Table 3: Factor and level table for the response surface experiment

[0131]

[0132] 1. Establishment of the response surface model

[0133] The ACE inhibitory activity results of sweet almond protein hydrolysate were analyzed using the response surface Box-Behnken method, and the regression mathematical model of enzyme dosage (A), time (B), pH (C), and temperature (D) was obtained as Y = 65.07 + 0.89A + 1.43B - 1.74C + 0.73D - 0.92AB + 0.07AC - 0.11AD - 1.49BC - 0.28BD + 0.83CD - 2.64A 2 - 4.09B 2 - 3.41C 2 - 2.04D 2

[0134] Table 4: Response surface analysis scheme and experimental results

[0135] Test serial number A B C D ACE inhibition rate % 1 -1 0 0 -1 57.84 2 1 0 1 0 58.13 3 0 0 -1 1 60.94 4 -1 1 0 0 59.88 5 0 1 -1 0 62.03 6 0 -1 0 -1 56.69 7 1 0 0 -1 59.48 8 0 0 0 0 64.65 9 0 1 0 -1 60.74 10 0 0 1 -1 57.41 11 -1 0 0 1 60.65 12 0 0 0 0 65.71 13 1 0 -1 0 61.86 14 -1 0 1 0 56.40 15 0 0 0 0 65.94 16 1 0 0 1 61.86 17 0 1 1 0 54.54 18 0 0 -1 -1 61.40 19 -1 -1 0 0 55.26 20 0 1 0 1 60.74 21 0 0 1 1 60.28 22 0 0 0 0 65.11 23 0 -1 1 0 55.23 24 -1 0 -1 0 60.17 25 1 1 0 0 60.37 26 0 -1 0 1 57.81 27 0 -1 -1 0 56.75 28 1 -1 0 0 59.42 29 0 0 0 0 63.96

[0136] Table 5: ANOVA of ACE inhibition rate and analysis of regression model coefficients

[0137]

[0138] Note: * indicates significant difference (P < 0.05), ** indicates extremely significant difference (P < 0.01).

[0139] Analysis of Table 4 and Table 5 shows that the binomial model of ACE inhibition rate has highly statistical significance, with an F value of 23.93 and a P value much less than 0.0001, indicating that the model is extremely significant. The P value of the non-linear term in the model is 0.425, indicating insignificance, which shows that the model has a high fitting quality and small experimental error. The determination coefficient R 2 is 0.9599, showing a high degree of fit between the experimental data and the predicted values and an ideal experimental effect. The enzymatic hydrolysis process of sweet almond protein can be used for model analysis, and the F value can judge the significance of the influence of the four factors. A, B, C, D, BC, A2, B2, C2, D2 are significant terms (P < 0.05), indicating that the enzyme dosage, temperature, pH, enzymatic hydrolysis time, and the interaction term of temperature and pH have a significant impact on the ACE inhibition rate.

[0140] 2. Analysis of ACE inhibition rate

[0141] By analyzing the response surface and contour plots of the interaction, the influence degree of different variables on the ACE inhibition rate can be evaluated. The results are shown in Figure 17.

[0142] Figure 17 shows the influence of the interaction of different factor variables on the ACE inhibitory rate of sweet almond protein. Ranking by F value: pH > temperature > enzyme dosage > enzymolysis time. This indicates that among the factors affecting the ACE inhibitory rate, pH has the most significant influence, followed by temperature, enzyme dosage ranks third, and enzymolysis time has the least influence. The steeper the slope of the response surface, the greater the influence of the factor and its interaction on the ACE inhibitory rate. A smaller or flatter slope indicates a smaller or insignificant influence on the ACE inhibitory rate. Among them, the slopes of temperature and pH are steeper than those of other factors, which also proves that the interaction between BC is stronger.

[0143] 3. Determination of optimized process parameters for enzymolysis

[0144] In the experimental condition range of enzyme dosage 5000 - 9000 U / g, temperature 50 - 60 °C, pH = 10 - 12, and enzymolysis time 3 - 5 h, the optimal experimental conditions obtained by Design-expert11 for the ACE inhibitory rate equation are: enzyme dosage 726835 U / g, temperature 56.0604 °C, pH = 10.7115, and enzymolysis time 4.10127 h, with an ACE inhibitory rate of 65.5658%. Considering practical operability, the optimized conditions are adjusted to: enzyme dosage 7200 U / g, temperature 56.1 °C, pH adjusted to 10.7, and enzymolysis time 4.1 h.

[0145] To verify the reliability and effectiveness of the experiment, according to the set process flow, the experiment was repeated 3 times, and the average value of the ACE inhibitory rate obtained was 65.13%. The error between the verification result and the model prediction value was only 0.43%, indicating that the model fitting effect is good.

[0146] Example 16

[0147] This example is used to illustrate the separation and purification of sweet almond protease hydrolysate.

[0148] 1. Ultrafiltration separation

[0149] The prepared almond protease hydrolysate was centrifuged at 4 °C and 7000 rpm for 15 min, and the supernatant was taken. The supernatant was first vacuum filtered through a 0.55 μm aqueous filter membrane, and then a 3 kDa ultrafiltration centrifugal tube was used to centrifuge at 4 °C and 7000 rpm for 30 min in a high-speed refrigerated centrifuge to separate large molecular polypeptide chains. The remaining solution was then centrifuged at 4 °C and 7000 rpm for 30 min using a 1 kDa ultrafiltration centrifugal tube. Finally, three component enzyme hydrolysates with >3 kDa, 1 - 3 kDa, and <1 kDa were obtained by ultrafiltration separation. The volume ratios of the three component enzyme hydrolysates of >3 kDa, 1 - 3 kDa, and <1 kDa are 55%, 33%, and 12% respectively. The ACE inhibitory rate of each group was measured to screen out the component with the best ACE inhibitory effect for subsequent dialysis desalting preparation.

[0150] Table 6: Comparative analysis of ACE inhibition rates of almond protease hydrolysates with different molecular weight cut-offs

[0151] Name ACE inhibition rate (%) Almond enzymolysis solution under optimal conditions 65.14±0.56b > 3 kDa 60.68±1.12c 1 - 3 kDa 68.32±1.92a < 1 kDa 64.13±1.48b

[0152] According to the results, three enzyme hydrolysate components with different molecular cut-offs (1 - 3 kDa, 1 kDa, 3 kDa) were obtained by ultrafiltration centrifugal tubes. The measured ACE inhibition rates were 1 - 3 kDa > 1 kDa > 3 kDa from high to low, and there were significant differences (P < 0.05). Among them, the ACE inhibition rate of the 1 - 3 kDa component was the highest (68.32%). The ACE inhibition effect of 1 - 3 kDa was better than that below 1 kDa, indicating that the ACE inhibitory peptides in almond protein were mainly distributed in the range of 1 - 3 kDa. Therefore, a molecular cut-off of 1 - 3 kDa was selected for subsequent desalting treatment.

[0153] 2. Dialysis desalting

[0154] The ACE inhibitory peptide solution of the best component obtained by ultrafiltration separation and measurement was put into a MW100 dialysis bag, the temperature was controlled at 10 °C, and dialysis was carried out under the action of a magnetic stirrer until there was no BacL 2 precipitate. After dialysis, the desalting rate and ACE inhibition rate were measured.

[0155] Table 7: Desalting results of ACE inhibitory peptides

[0156] Desalination rate (%) ACE inhibition rate (%) ACE inhibitory peptide 82.25 67.03

[0157] According to Tables 3 - 6, the desalting rate of the 1 - 3 kDa component was 82.25%, and the desalting effect was excellent.

[0158] 3. Identification and analysis of the amino acid sequence of the sweet almond ACE inhibitory peptide composition

[0159] Elution conditions for ultra-high performance liquid chromatography: Chromatographic column: ACQUITY C18 BEH column (2.1x100 mm, 1.7 mm particle size); Mobile phase A: water (containing 0.1% formic acid); Mobile phase B: acetonitrile (containing 0.1% formic acid); Column temperature was set at 45 °C, injection volume was 3 μL, and flow rate was set at 300 nL / min.

[0160] Mass spectrometry detection conditions: Ion spray (IS) voltage 5.0 kV; Turbomolecular gun temperature: 500 °C; Curtain gas (CUR): 35 psi; m / z of MS1 was set at 100 - 1500, and m / z of MS2 was set at 50 - 1500.

[0161] The obtained amino acid sequences were used to determine the molecular weight, toxicity, isoelectric point (pI), and hydrophobicity of each amino acid sequence through online databases including pepdraw (http: / / www.tulane.edu / ~biochem / WW / PepDraw), PeptideRanker (http: / / bioware.ucd.ie / ~compass / biowareweb / ), and Expasy-Compute (https: / / web.expasy.org / compute). Then, the optimal ACE inhibitory polypeptide small molecules were screened out. The desalted enzymatic hydrolysate was analyzed for amino acid sequences using a 1920 ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer (LC-TOFMS). The total ion chromatogram and multiple amino acid sequences obtained are shown in Figure 18 ; 68 amino acid sequences were identified, and the obtained amino acid sequences were statistically analyzed, as shown in Tables 8 and 9.

[0162] Table 8: Multiple amino acid sequences of the enzymatic hydrolysate of the 1 - 3 kDa fraction

[0163]

[0164]

[0165] Table 9: Statistical results of various amino acids

[0166]

[0167]

[0168] 3. Analysis of the structure-activity relationship of ACE inhibitory peptides by molecular docking simulation method

[0169] The crystal structure of the ACE inhibitory peptide (PDB ID: 1O8A) was downloaded from the PDB database and preprocessed using AutoDock for ligand removal, water removal, and hydrogen addition. The obtained ACE polypeptide small molecules were also subjected to water removal and hydrogen addition. The ACE protein was set as the receptor, and the ACE polypeptide small molecules were set as the ligands. The docking box was set, semi-flexible docking was selected, and 50 docking operations were performed to analyze the docking binding energy and select the best conformation. Finally, further analysis was carried out using Pymol software. The single-factor experimental data were processed using OriginPro2021 software and presented graphically, and then the Box-Behnken design in Design-Expert 11 software was used for the experimental design and optimization of response surface analysis. Finally, statistical significance analysis was carried out using SPSS25.0 software. Each experimental sample was processed in triplicate.

[0170] According to the solubility and the above structure-activity analysis of amino acid sequences, five amino acid sequences, namely GRGVLGAVF (SEQ ID NO:2), IIALPAGVAY (SEQ ID NO:5), VVAIPAGVAY (SEQ ID NO:1), DIIALPAGVAY (SEQ ID NO:3), and DVVAIPAGVAYW (SEQ ID NO:4), were screened out from 68 amino acid sequences. These five amino acid sequences were respectively subjected to molecular docking simulation with the receptor ACE (1O8A). According to the Autodock docking results shown in Table 10, the specific molecular docking diagram is as shown in Figures 19 - 23 shown.

[0171] Table 10: ACE inhibitory peptide sequences of sweet almonds

[0172]

[0173] From the above results, it can be seen that among the five amino acid sequences, DVVAIPAGVAYW has the lowest binding energy value and the most stable binding to ACE.

[0174] Twelve hydrogen bonds were formed between DIIALPAGVAY and ACE, among which hydrogen bond forces were formed on the key amino acid residue Ser355; ten hydrogen bonds were formed between DVVAIPAGVAYW and ACE, among which hydrogen bond forces were formed on the key amino acid residue Ala356; eight hydrogen bonds were formed between GRGVLGAVF and ACE, among which hydrogen bond forces were formed with the key amino acid residue Ala356; seven hydrogen bonds were formed between IIALPAGVAY and ACE, but no hydrogen bonds were formed with any key amino acid residues; nine hydrogen bonds were formed between VVAIPAGVAY and ACE, among which three hydrogen bonds were formed with the key amino acid residues Ser355 and Ala356. Hydrogen bonds were generated at both the C-terminus and N-terminus of the five amino acid sequences, proving that the high hydrophobicity of the N-terminus and C-terminus is positively correlated with the ACE inhibitory effect. IIALPAGVAY did not bind to any key active sites, indicating its weak ACE inhibitory effect. The remaining four peptides were all able to bind to the key residues of ACE, indicating that these peptides have good ACE inhibitory effects. Among them, VVAIPAGVAY (SEQ ID NO:1) formed three hydrogen bonds with the key active sites of ACE, showing better ACE inhibitory effect and stronger binding stability compared to the other four peptides.

[0175] Example 17

[0176] The active center of ACE is composed of S1 (Ala354, Glu384, and Tyr523), S2 (Gln281, His353, Lys511, His513, and Tyr520), and S' (Glu162) active pockets. Usually, when the active peptide forms hydrogen bonds with the residues Trp279, Gln281, His353, Ala354, Ser355, Ala356, His383, Glu384, Glu411, Asp415, Lys454, Phe457, Lys511, His513, Tyr520, and Tyr523 in the ACE active center, high ACE inhibitory activity can be generated. The ACE inhibitory peptide sequences reported in the prior art (Zhong Yuwang, Xu Wanli, Fan Yaozhu, et al. Isolation, purification, structure identification and in vitro activity evaluation of ACE inhibitory peptides from Moringa oleifera seeds [J]. Food Science, 2023, 44(24): 118 - 126.) were docked using the same molecular docking method as in Example 16, and the experimental results are shown in Table 11:

[0177] Table 11: Reported ACE inhibitory peptide sequences

[0178]

[0179] The highest binding energy of the reported peptide sequence QGPRPQ is -6.01 Kcal / mol. At this time, 5 hydrogen bonds are formed with ACE, and the binding sites are Glu403, Arg522, Glu123, and Tyr360, and no key binding sites are generated. Key binding sites Ala 356 and Glu411 are generated at -5.64 Kcal / mol and -2.83 Kcal / mol.

[0180] The highest binding energy of the reported peptide sequence DPNNFT is -1.68 Kcal / mol. At this time, 2 hydrogen bonds are formed with ACE, and the binding sites are Lys368 and Arg522, and no key binding sites are generated. The key binding site Ala 356 is generated at -1.18 Kcal / mol.

[0181] The highest binding energy of the reported peptide sequence ARVYIHFL is -5.34 Kcal / mol. At this time, 5 hydrogen bonds are formed with ACE, and the binding sites are Ser516, ARG124, Glu123, and Asn66, and no key binding sites are generated. Key binding sites Ala 356 and Glu411 are generated at -5.32 Kcal / mol and -1.27 Kcal / mol, respectively.

[0182] According to the above experimental data, by comparing the molecular docking results of the reported peptide sequences with those of the sweet almond ACE inhibitory peptides, it is found that the docking binding energy of the sweet almond ACE inhibitory peptides is similar to that of the reported ACE inhibitory peptides, and even the binding effects of some peptide sequences with ACE are better. By comparing the binding sites, it is found that there are two peptide sequences (DIIALPAGVAY, VVAIPAGVAY) at the optimal binding energy of the almond ACE inhibitory peptides that have key binding sites. Among them, VVAIPAGVAY forms hydrogen bonds with the key binding sites Ser355 and Ala356, and the effect is better than that of the reported peptide sequences QGPRPQ, DPNNFT, and ARVYIHFL, proving that the almond ACE inhibitory peptides of the present invention have excellent ACE inhibitory activity.

[0183] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments described herein, but rather will conform to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A sweet almond ACE inhibitory peptide composition, characterized in that: The sweet almond ACE inhibitory peptide composition is prepared by the following method: S1. Extracting almond protein from cold-pressed sweet almond meal by salting-out method; S2, using genipin as a cross-linking agent and chicken egg shell membrane as a carrier, preparing an immobilized enzyme by covalently cross-linking the protease and the chicken egg shell membrane; S3. Using the immobilized enzyme, enzymolyze the almond protein, separate the enzymolysis product by centrifugation and ultrafiltration to obtain a component of 1 to 3 kDa, and obtain the sweet almond ACE inhibitory peptide composition by desalting.

2. The sweet almond ACE inhibitory peptide composition according to claim 1, characterized in that S1 includes: S11, preparing almond solution by using cold pressed sweet almond meal; S12, crushing the almond solution, centrifuging and obtaining a supernatant; S13, adding solid ammonium sulfate to the supernatant for salting out, dissolving the obtained precipitate and dialyzing it to obtain the almond protein.

3. The sweet almond ACE inhibitory peptide composition according to claim 2, characterized in that The salting out is a step-by-step salting out, firstly adding ammonium sulfate solid to a saturation of 20% to 25% to fully dissolve, then centrifuging to obtain the supernatant, then adding ammonium sulfate solid to a concentration of 40% to 50%, and then centrifuging to obtain the precipitate for dialysis; The dialysis conditions are preferably as follows: adding the mixture to a MW3500 dialysis bag treated with a mixed solution of sodium bicarbonate and EDTA, and dialysis with a PBS solution for 8 to 16 hours; Preferably, the temperature for crushing and centrifuging is 0-10°C.

4. The sweet almond ACE inhibitory peptide composition according to claim 1, characterized in that In S2, the protease is selected from one or more of papain, alkaline protease, neutral protease, trypsin, and bromelain, preferably alkaline protease.

5. The sweet almond ACE inhibitory peptide composition according to claim 1, characterized in that S2 comprises: soaking the egg shell membrane in a genipin solution, washing it, adding it into a protease solution for cross-linking, and obtaining the immobilized enzyme; Preferably, the temperature of the cross-linking reaction is 42°C to 48°C, more preferably 45°C; Preferably, the cross-linking reaction time is 1 to 4 hours, more preferably 2 hours; Preferably, the mass percentage concentration of the genipin solution is 0.5% to 1%, preferably 0.75%; Preferably, the concentration of the protease solution is 2-10 mg / mL, preferably 5 mg / mL.

6. The sweet almond ACE inhibitory peptide composition according to claim 1, characterized in that: In S3, the enzymatic hydrolysis temperature is 50-60°C, preferably 55-56°C; Preferably, the pH of the enzymatic hydrolysis is 10 to 12, preferably 10.7 to 11; Preferably, the enzymatic hydrolysis time is 3 to 5 hours, preferably 4 to 4.1 hours; Preferably, the amount of enzyme added is 5000-9000U / g, preferably 7000-7200U / g; Preferably, the concentration of the substrate is 3% to 5%, preferably 4%.

7. The sweet almond ACE inhibitory peptide composition according to claim 1, characterized in that: In S3, the supernatant obtained by centrifugation of the enzymatic hydrolysis product is first vacuum filtered through a 0.55 μm water filter membrane, and then centrifuged using a 3 kDa ultrafiltration centrifuge tube. The supernatant solution is centrifuged again using a 1 kDa ultrafiltration centrifuge tube, and ultrafiltration separation is performed to obtain 1-3 kDa components; The centrifugation conditions are 0-5°C, a rotation speed of 6000-8000 rpm, and a time of 20-40 min; preferably, at 4°C, a rotation speed of 7000 rpm for 30 min.

8. The sweet almond ACE inhibitory peptide composition according to any one of claims 1 to 7, characterized in that: The sweet almond ACE inhibitory peptide composition contains polypeptides with amino acid sequences as shown in SEQ ID NO: 1 to SEQ ID NO:

4.

9. A sweet almond ACE inhibitory peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

10. Use of the sweet almond ACE inhibitory peptide according to any one of claims 1 to 8 or the sweet almond ACE inhibitory peptide according to claim 9 in the preparation of a drug for preventing and treating hypertension.

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