ACE inhibitory peptide, preparation method and application

Abstract

The invention relates to the technical field of biology, in particular to an ACE inhibitory peptide, a preparation method and application, the ACE inhibitory peptide has the amino acid sequence of Trp-Pro-Phe (WPF) or Phe-Pro-Trp (FPW) or Phe-Gly-Trp (FGW) or Leu-Pro-Val-Pro-Ala-Phe-Asn (LPVPAFN) or Leu-Trp-Trp-Leu (LWWL), the ACE inhibitory peptide WPF, FPW, FGW, LPVPAFN and LWWL have outstanding antihypertensive activity, the IC50 values of the WPF, FPW, FGW, LPVPAFN and LWWL are 0.264 mg / mL, 0.800 mg / mL, 0.106 mg A variety of aquatic product peptide powder sources are separated and purified by an affinity ultrafiltration screening method, an ultrafiltrate component is selected for amino acid sequence identification, and a new amino acid sequence with ACE inhibitory activity is screened out; the ACE inhibitory peptides WFP, FPW, FGW, LPVPAFN and LWWL prepared by the invention have outstanding antihypertensive activity.

Description

Technical Field

[0001] The present application relates to the technical field of bioactive peptides, and mainly to ACE inhibitory peptides, preparation methods and applications. Background Art

[0002] Hypertension is a common chronic disease and one of the major causes of premature death in humans. ACE is an important enzyme in the human body that is mainly involved in the conversion of angiotensin I to angiotensin II. Angiotensin II is a potent vasoconstrictor that can cause vasoconstriction and thus increase blood pressure. Therefore, inhibiting the activity of ACE can reduce the production of angiotensin II, thereby helping to lower blood pressure.

[0003] However, although the synthetic drugs currently used to treat hypertension can effectively regulate blood pressure, they all have certain side effects that affect human health. Safe and effective treatment of hypertension is still an urgent problem to be solved. Food-borne ACE inhibitory peptides (ACEIPs) have the characteristics of safety, mild action, and easy absorption, making them a research hotspot for the prevention and treatment of hypertension. About 71% of the earth's area is ocean, and marine biological resources are rich, accounting for more than half of the global biodiversity. Marine biological resources are considered to be the most promising source of new bioactive substances with therapeutic and nutritional health functions. Marine organisms include marine fish, marine mollusks, marine algae, marine fungi and marine bacteria. These organisms have extremely high edible and medicinal value. Their rich protein content makes them one of the high-quality sources of bioactive peptides. Studying the activity and mechanism of action of ACE inhibitory peptides in aquatic products and finding new ACE inhibitory peptides are of great significance to the development and development of peptide antihypertensive drugs.

[0004] The purpose of the present application is to provide ACE inhibitory peptides derived from various aquatic product peptide powders, preparation methods and applications. Summary of the invention

[0005] To achieve the above objectives, this application is implemented through the following technical solutions: An ACE inhibitory peptide, wherein the amino acid sequence of the ACE inhibitory peptide is Trp-Pro-Phe (WPF) or Phe-Pro-Trp (FPW) or Phe-Gly-Trp (FGW) or Leu-Pro-Val-Pro-Ala-Phe-Asn (LPVPAFN) or Leu-Trp-Trp-Leu (LWWL).

[0006] Preferably, the source of the ACE inhibitory peptide is aquatic products.

[0007] Preferably, the source of the ACE inhibitory peptide is sea cucumber or sea cucumber flower or tuna or oyster.

[0008] In addition, the present invention also provides a method for preparing the above-mentioned ACE inhibitory peptide, comprising the following steps: S1: crush the source material and set aside; S2: adding the crushed source material into water, adjusting the pH to 6-9, adding enzymes, performing enzymolysis, and obtaining an enzymolysis solution; S3: inactivating the enzyme in the enzymatic hydrolysate, taking the supernatant, and obtaining an enzyme-inactivated enzymatic hydrolysate; S4: spray drying the enzyme-killed hydrolyzate to obtain peptide powder; S5: Add the peptide powder to water to obtain a peptide powder solution, and obtain the ACE inhibitory peptide after amino acid content determination, affinity ultrafiltration screening, high performance liquid chromatography purification, and amino acid sequence determination.

[0009] Preferably, the enzyme in step S2 is a mixture of alkaline protease and neutral protease, and the mass concentration of the enzyme is 0.2-0.5%.

[0010] Preferably, step S3 is specifically as follows: heating the enzymatic hydrolysate at 80-100° C. for 10-30 min to inactivate the enzyme, then centrifuging at 3000-5000 r / min for 10-30 min, taking the supernatant, and obtaining the enzyme-inactivated enzymatic hydrolysate.

[0011] Preferably, the specific method of step S5 is: S51: Mix the peptide powder solution and ACE in a volume ratio of 1:1-5 and incubate at 30-40°C for 1-3h; S52: After the incubation, centrifuge at 3000-5000 r / min for 10-30 min using a 5-10 kDa ultrafiltration centrifuge tube to remove the peptides not bound to ACE and obtain the complex formed by the binding of ACE and the enzymatic hydrolysate; S53: The complex formed by the combination of ACE and the hydrolysate was incubated with 50-70% acetonitrile for 10-30 min, and centrifuged at 3000-5000 r / min for 10-30 min to release the peptides in the complex. The filtrate was collected as the affinity ultrafiltration hydrolysate. S54: The affinity ultrafiltration hydrolyzate is purified by high performance liquid chromatography and subjected to amino acid sequencing to obtain an ACE inhibitory peptide; In step S51, the concentration of the peptide powder solution is 3-10 mg / mL, and the concentration of ACE is 100-300 mU / mL.

[0012] Preferably, in step S53, the HPLC purification is performed by gradient elution, and the mobile phases are mobile phase A and mobile phase B, wherein mobile phase A is a formic acid aqueous solution with a volume concentration of 0.05-0.15%, and mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.05-0.15%. The specific steps are: From 0 to 50 min, the volume content of mobile phase B in the mobile phase was 4 to 50%, and the volume content of mobile phase A in the mobile phase was 96 to 50%; At 50-54 min, the volume content of mobile phase B in the mobile phase was 50-100%, and the volume content of mobile phase A in the mobile phase was 50-0%; At 54-60 min, the volume content of mobile phase B in the mobile phase was 100%.

[0013] Preferably, during the HPLC purification process, the mobile phase flow rate is 1-2 mL / min.

[0014] Finally, the present invention also provides the application of the above ACE inhibitory peptide for preparing a drug for reducing hypertension.

[0015] Compared with the known public technologies, the technical solution provided by this application has the following beneficial effects: (1) The ACE inhibitory peptides WPF, FPW, FGW, LPVPAFN, and LWWL of the present application have outstanding antihypertensive activity, and their IC 50 The values ​​were 0.264 mg / mL, 0.800 mg / mL, 0.106 mg / mL, 0.568 mg / mL, and 0.109 mg / mL, respectively, which are more active than most existing ACE inhibitory peptides; (2) The present application uses affinity ultrafiltration screening method to separate and purify a variety of aquatic product peptide powder sources, selects ultrafiltrate components for amino acid sequence identification, and screens out new amino acid sequences with ACE inhibitory activity; the ACE inhibitory peptides WFP, FPW, FGW, LPVPAFN, and LWWL prepared by the present invention have outstanding antihypertensive activity, and their structure-activity relationship is preliminarily evaluated by modern means such as molecular docking, which further improves the application value of the ACE inhibitory peptide in the field of chronic disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 a is the total ion current diagram of the sea cucumber affinity ultrafiltration control group in the present invention; Figure 1 b is the total ion current diagram of the sea cucumber affinity ultrafiltration group in the present invention; Figure 2 a is the total ion current diagram of the sea cucumber flower affinity ultrafiltration control group in the present invention; Figure 2 b is the total ion current diagram of the sea cucumber flower affinity ultrafiltration group in the present invention; Figure 3 a is the total ion current diagram of the tuna affinity ultrafiltration control group in the present invention; Figure 3 b is the total ion current diagram of the tuna affinity ultrafiltration group in the present invention; Figure 4 a is the total ion current diagram of the oyster affinity ultrafiltration control group in the present invention; Figure 4 b is the total ion current diagram of the oyster affinity ultrafiltration group in the present invention; Figure 5 a is the total ion current diagram of the sturgeon cartilage affinity ultrafiltration control group in the present invention; Figure 5 b is the total ion current diagram of the affinity ultrafiltration group of sturgeon cartilage in the present invention; Figure 6 a is the total ion current diagram of the cod affinity ultrafiltration control group in the present invention; Figure 6 b is the total ion current diagram of the cod affinity ultrafiltration group in the present invention; Figure 7 a is the primary mass spectrum of WFP in the present invention; Figure 7 b is the secondary mass spectrum of WFP in the present invention; Figure 8 a is the primary mass spectrum of FGW in the present invention; Figure 8 b is the secondary mass spectrum of FGW in the present invention; Fig. 9 a is the primary mass spectrum of FPW in the present invention; Fig. 9 b is the secondary mass spectrum of FPW in the present invention; Fig.10 a is the primary mass spectrum of LPVPAFN in the present invention; Fig.10 b is the secondary mass spectrum of LPVPAFN in the present invention; Fig.11 a is the primary mass spectrum of LWWL in the present invention; Fig.11 b is the secondary mass spectrum of LWWL in the present invention; Fig.12 a is the molecular docking diagram of ACE and ALLL in the present invention; Fig.12 b is a diagram for analyzing the interaction between ACE and ALLL in the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0019] The amino acid sequence of the ACE inhibitory peptide of the present invention is Trp-Pro-Phe (abbreviated as WFP, with a theoretical molecular weight of 448.21 and a structural formula of C 25 H 28 N4O4), Phe-Pro-Trp (abbreviated as FPW, with a theoretical molecular weight of 448.21 and a structural formula of C 25 H 28 N4O4), Phe-Gly-Trp (abbreviated as FGW, with a theoretical molecular weight of 408.18 and a structural formula of C 22 H 24 N4O4), Leu-Pro-Val-Pro-Ala-Phe-Asn (LPVPAFN for short, with a theoretical molecular weight of 756.42 and a structural formula of C 37 H 56 N8O9), Leu-Trp-Trp-Leu (abbreviated as LWWL, with a theoretical molecular weight of 616.34 and a structural formula of C 34 H 44 N6O5).

[0020] Specifically, glycine is Gly (G), alanine is Ala (A), valine is Val (V), leucine is Leu (L), isoleucine is Ile (I), methionine is Met (M), proline is Pro (P), alanine is Phe (F), tyrosine is Tyr (Y), tryptophan is Trp (W), arginine is Arg (R), lysine is Lys (K), histidine is His (H), aspartic acid is Asp (D), glutamic acid is Glu (E), cysteine ​​is Cys (C), serine is Ser (S), threonine is Thr (T), asparagine is Asn (N), and glutamine is Gln (Q).

[0021] Specifically, the structural formula of WFP is as follows: .

[0022] The structural formula of FPW is shown below: .

[0023] The structural formula of FGW is as follows: .

[0024] The structural formula of LPVPAFN is as follows: .

[0025] Specifically, the structural formula of LWWL is as follows: .

[0026] The ACE inhibitory peptide was prepared according to the method of the following example.

[0027] Example 1 A method for preparing an ACE inhibitory peptide having an amino acid sequence of Trp-Pro-Phe (WPF) and Phe-Pro-Trp (FPW), comprising: S1: Crush the sea cucumber and set aside; S2: Add the crushed sea cucumber into water (solid-liquid ratio 1:10, i.e. 1 g of sea cucumber into 10 mL of water), adjust the pH to 7 with NaOH, add alkaline protease and neutral protease in a ratio of 1:1, and the enzyme mass concentration is 0.2%, and react at 53°C for 5 h to obtain an enzymatic solution; S3: heating the enzymatic hydrolysate at 90°C for 20 min to inactivate the enzyme, and then centrifuging at 3000 r / min for 30 min, taking the supernatant to obtain the inactivated enzymatic hydrolysate; S4: spray drying the enzyme-killed hydrolyzate to obtain sea cucumber peptide powder; S51: Add sea cucumber peptide powder to water to obtain a sea cucumber peptide powder solution with a concentration of 8 mg / mL. Mix the sea cucumber peptide powder solution and 300 mU / mL ACE at a volume ratio of 1:1, and incubate at 30°C for 3 h. Another ACE inactivation (100°C water bath for 10 min) was used as a blank group. S52: After the incubation, the mixture was centrifuged at 3000 r / min for 30 min using a 5 kDa ultrafiltration centrifuge tube to remove the peptides not bound to ACE and obtain the complex formed by the binding of ACE and the enzymatic hydrolysate; S53: The complex formed by the combination of ACE and the hydrolysate was incubated with 50% acetonitrile for 10 min, and centrifuged at 3000 r / min for 30 min to release the peptides in the complex. This was repeated three times, and the filtrate was collected as the affinity ultrafiltration hydrolysate. S54: The affinity ultrafiltration hydrolyzate is added with water to prepare a solution with a concentration of 50 mg / mL, and after high performance liquid chromatography purification and amino acid sequencing, an ACE inhibitory peptide is obtained. The high performance liquid chromatography purification is separated and purified by gradient elution, and the mobile phases used are mobile phase A and mobile phase B. Mobile phase A is a formic acid aqueous solution with a volume concentration of 0.05%, and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.05%. The specific steps are: From 0 to 50 min, the volume content of mobile phase B in the mobile phase was 4%, and the volume content of mobile phase A in the mobile phase was 96%; At 50-54 min, the volume content of mobile phase B in the mobile phase was 50%, and the volume content of mobile phase A in the mobile phase was 50%; At 54-60 min, the volume content of mobile phase B in the mobile phase was 100%.

[0028] The liquid chromatography column was an Agilent ZORBAXSB-C18 column (Agilent Technologies, Wilmington, DE), the mobile phase flow rate was 1 mL / min, and the sample volume was 10 μL.

[0029] Example 2 A method for preparing an ACE inhibitory peptide having an amino acid sequence of Phe-Pro-Trp (FPW), comprising: S1: Crush the sea cucumber flowers and set aside; S2: Add the crushed sea cucumber flower into water (solid-liquid ratio 1:10, i.e., 1 g of sea cucumber flower is added into 10 mL of water), adjust the pH to 9 with NaOH, add alkaline protease and neutral protease in a ratio of 1:1, and the enzyme mass concentration is 0.4%, and react at 53°C for 5 h to obtain an enzymatic solution; S3: heating the enzymatic hydrolysate at 80°C for 30 min to inactivate the enzyme, and then centrifuging at 5000 r / min for 10 min to obtain the supernatant to obtain the inactivated enzymatic hydrolysate; S4: spray drying the enzyme-killed hydrolyzate to obtain sea cucumber flower peptide powder; S51: Add sea cucumber flower peptide powder to water to obtain a 10 mg / mL sea cucumber flower peptide powder solution. Mix the sea cucumber flower peptide powder solution and 300 mU / mL ACE at a volume ratio of 1:2, and incubate at 40°C for 1 hour. Another ACE inactivation (100°C water bath for 10 minutes) is used as a blank group; S52: After the incubation, centrifuge at 5000 r / min for 10 min using a 5 kDa ultrafiltration centrifuge tube to remove the peptides not bound to ACE and obtain the complex formed by the binding of ACE and the enzymatic hydrolysate; S53: The complex formed by the combination of ACE and the hydrolysate was incubated with 50% acetonitrile for 10 min, and centrifuged at 5000 r / min for 10 min to release the peptides in the complex. This was repeated three times, and the filtrate was collected as the affinity ultrafiltration hydrolysate. S54: The affinity ultrafiltration hydrolyzate is added with water to prepare a solution with a concentration of 50 mg / mL, and after high performance liquid chromatography purification and amino acid sequencing, an ACE inhibitory peptide is obtained. The high performance liquid chromatography purification is separated and purified by gradient elution, and the mobile phases used are mobile phase A and mobile phase B. Mobile phase A is a formic acid aqueous solution with a volume concentration of 0.05%, and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.05%. The specific steps are: From 0 to 50 min, the volume content of mobile phase B in the mobile phase was 20%, and the volume content of mobile phase A in the mobile phase was 80%; At 50-54 min, the volume content of mobile phase B in the mobile phase was 60%, and the volume content of mobile phase A in the mobile phase was 40%; At 54-60 min, the volume content of mobile phase B in the mobile phase was 100%.

[0030] The liquid chromatography column was an Agilent ZORBAXSB-C18 column (Agilent Technologies, Wilmington, DE), the mobile phase flow rate was 1 mL / min, and the sample volume was 10 μL.

[0031] Example 3 A method for preparing an ACE inhibitory peptide having an amino acid sequence of Leu-Pro-Val-Pro-Ala-Phe-Asn (LPVPAFN), comprising: S1: Crush the tuna meat and set aside; S2: Add the crushed tuna meat into water (solid-liquid ratio 1:10, i.e., 1 g tuna meat is added into 10 mL water), adjust the pH to 8 with NaOH, add alkaline protease and neutral protease in a ratio of 1:1, and the enzyme mass concentration is 0.3%, and react at 53°C for 5 h to obtain an enzymatic hydrolyzate; S3: heating the enzymatic hydrolysate at 90°C for 20 min to inactivate the enzyme, and then centrifuging at 4000 r / min for 10 min, taking the supernatant to obtain the inactivated enzymatic hydrolysate; S4: spray drying the enzyme-killed hydrolyzate to obtain tuna peptide powder; S51: Tuna peptide powder was added to water to obtain a 5 mg / mL tuna peptide powder solution. The tuna peptide powder solution and 100 mU / mL ACE were mixed at a volume ratio of 1:2 and incubated at 37°C for 1 h. Another ACE inactivation (100°C water bath for 10 min) was used as a blank group. S52: After the incubation, centrifuge at 5000 r / min for 10 min using a 5 kDa ultrafiltration centrifuge tube to remove the peptides not bound to ACE and obtain the complex formed by the binding of ACE and the enzymatic hydrolysate; S53: The complex formed by the combination of ACE and the hydrolysate was incubated with 70% acetonitrile for 10 min, and centrifuged at 4000 r / min for 30 min to release the peptides in the complex. This was repeated three times, and the filtrate was collected as the affinity ultrafiltration hydrolysate. S54: The affinity ultrafiltration hydrolyzate is added with water to prepare a solution with a concentration of 50 mg / mL, and after high performance liquid chromatography purification and amino acid sequencing, an ACE inhibitory peptide is obtained. The high performance liquid chromatography purification is separated and purified by gradient elution, and the mobile phases used are mobile phase A and mobile phase B. Mobile phase A is a formic acid aqueous solution with a volume concentration of 0.1%, and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.1%. The specific steps are: From 0 to 50 min, the volume content of mobile phase B in the mobile phase was 45%, and the volume content of mobile phase A in the mobile phase was 55%; At 50-54 min, the volume content of mobile phase B in the mobile phase was 90%, and the volume content of mobile phase A in the mobile phase was 10%; At 54-60 min, the volume content of mobile phase B in the mobile phase was 100%.

[0032] The liquid chromatography column was an Agilent ZORBAXSB-C18 column (Agilent Technologies, Wilmington, DE), the mobile phase flow rate was 1 mL / min, and the sample volume was 10 μL.

[0033] Example 4 A method for preparing an ACE inhibitory peptide having an amino acid sequence of Leu-Trp-Trp-Leu (LWWL), comprising: S1: Crush the oyster meat and set aside; S2: Add the crushed oyster meat into water (solid-liquid ratio 1:10, i.e., 1 g of oyster meat is added into 10 mL of water), adjust the pH to 6 with NaOH, add alkaline protease and neutral protease in a ratio of 1:1, and the enzyme mass concentration is 0.5%, and react at 53°C for 5 h to obtain an enzymatic hydrolyzate; S3: heating the enzymatic hydrolysate at 90°C for 20 min to inactivate the enzyme, and then centrifuging at 5000 r / min for 10 min, taking the supernatant to obtain the inactivated enzymatic hydrolysate; S4: spray drying the enzyme-killed hydrolyzate to obtain oyster meat peptide powder; S51: Add oyster meat peptide powder to water to obtain a 6 mg / mL tuna peptide powder solution. Mix the oyster meat peptide powder solution and 100 mU / mL ACE at a volume ratio of 1:5, and incubate at 37°C for 1 hour. Another ACE inactivation (100°C water bath for 10 minutes) was used as a blank group. S52: After the incubation, centrifuge at 5000 r / min for 10 min using a 5 kDa ultrafiltration centrifuge tube to remove the peptides not bound to ACE and obtain the complex formed by the binding of ACE and the enzymatic hydrolysate; S53: The complex formed by the combination of ACE and the hydrolysate was incubated with 70% acetonitrile for 10 min, and centrifuged at 4000 r / min for 30 min to release the peptides in the complex. This was repeated three times, and the filtrate was collected as the affinity ultrafiltration hydrolysate. S54: The affinity ultrafiltration hydrolyzate is added with water to prepare a solution with a concentration of 50 mg / mL, and after high performance liquid chromatography purification and amino acid sequencing, an ACE inhibitory peptide is obtained. The high performance liquid chromatography purification is separated and purified by gradient elution, and the mobile phases used are mobile phase A and mobile phase B. Mobile phase A is a formic acid aqueous solution with a volume concentration of 0.1%, and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.1%. The specific steps are: From 0 to 50 min, the volume content of mobile phase B in the mobile phase was 15%, and the volume content of mobile phase A in the mobile phase was 85%; At 50-54 min, the volume content of mobile phase B in the mobile phase was 70%, and the volume content of mobile phase A in the mobile phase was 30%; At 54-60 min, the volume content of mobile phase B in the mobile phase was 100%.

[0034] The liquid chromatography column was an Agilent ZORBAXSB-C18 column (Agilent Technologies, Wilmington, DE), the mobile phase flow rate was 1 mL / min, and the sample volume was 10 μL.

[0035] Comparative Example 1 The difference between this comparative example and Example 3 is that the source material is different. The source material used in this comparative example is sturgeon cartilage.

[0036] Comparative Example 2 The difference between this comparative example and Example 3 is that the source material is different. The source material used in this comparative example is cod.

[0037] Determination method Determination of amino acid content: The determination was carried out with reference to the national standard GB5009.124-2016 “National Food Safety Standard Determination of Amino Acids in Foods”. The measured values ​​are shown in Tables 1 to 6, where * indicates essential amino acids for the human body.

[0038] Table 1 Amino acid content of sea cucumber hydrolysate

[0039] Table 2 Amino acid content of sea cucumber flower hydrolysate

[0040] Table 3 Amino acid content of tuna hydrolysate

[0041] Table 4 Amino acid content of oyster hydrolysate

[0042] Table 5 Amino acid content of sturgeon cartilage hydrolysate

[0043] Table 6 Amino acid content of Alaska pollock hydrolysate

[0044] As shown in Tables 1 to 6, hydrophobic amino acids Ser, Leu, Pro, Ile, Phe and positively charged amino acids Lys and Arg have a relatively high proportion in these seven peptide powders, so these seven peptide powders have the potential to prepare ACE inhibitory peptides.

[0045] ACE inhibition rate test method: In a 1.5mL centrifuge tube, add 10μL of sample and 20μL of ACE (100mU / mL) respectively and react at 37℃ for 5min. Add 30μL of 2.5mmol / L HHL (i.e., hippurylhistidylleucine) and react at 37℃ for 1h. Finally, add 60μL of 1M HCL solution to terminate the reaction. Then, add 200μL of water to the centrifuge tube for dilution and analyze by HPLC after passing through a 0.22μm filter membrane.

[0046] HPLC chromatographic conditions: Liquid chromatography column: Agilent ZORBAXSB-C18 (5μm, 250×4.6mm); detection wavelength: 228nm; flow rate 0.8mL / min; mobile phases are mobile phase A and mobile phase C, mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase C is acetonitrile; injection volume: 5μL; elution conditions: the volume ratio of mobile phase A and mobile phase C is 82:12. Use ultrapure water for blank experiment (i.e. control group). The calculation formula of the inhibition rate of each component on ACE activity is:

[0047] Where ΔA is the peak area of ​​HA (hippuric acid) in the control group, and ΔB is the peak area of ​​HA in the eluted component.

[0048] According to the binding energy of molecular docking, the amino acid sequences of some inhibitory peptides were screened out, such as Trp-Pro-Phe (WFP for short), Phe-Pro-Trp (FPW for short), Phe-Gly-Trp (FGW for short), Leu-Pro-Val-Pro-Ala-Phe-Asn (LPVPAFN for short), Leu-Trp-Trp-Leu (LWWL for short), Ala-Leu-Leu-Leu (ALLL for short), Trp-Pro-Trp (WPW for short), Ser-Gly-Pro-Ala-Gly-Pro-Arg-Gly-Phe (SGPAGPRGF for short), Gly-Ser-Asn-Gly-Pro-Gln-Gly-Phe-Pro (GSNGPQGFP for short), Gly-Phe-Phe (GFF for short), and Ala-Phe-Phe (AFF for short). The IC values ​​of some peptide sequences were tested. 50 The results are shown in Table 7.

[0049] Table 7 Synthetic peptide IC 50 value

[0050] It can be seen from Table 7 that the five ACE inhibitory peptides, Trp-Pro-Phe (WFP for short), Phe-Pro-Trp (FPW for short), Phe-Gly-Trp (FGW for short), Leu-Pro-Val-Pro-Ala-Phe-Asn (LPVPAFN for short), and Leu-Trp-Trp-Leu (LWWL for short), have high ACE inhibition rates. 50 The values ​​were 0.264 mg / mL, 0.800 mg / mL, 0.106 mg / mL, 0.568 mg / mL, and 0.109 mg / mL respectively.

[0051] Figures 1 to 6 These are the total ion currents of affinity ultrafiltration fractions of tuna peptide, oyster peptide, sea cucumber peptide, sea cucumber flower peptide, sturgeon cartilage peptide, and Alaska pollock peptide. 58 potential ACE inhibitory peptide sequences were identified in tuna peptide affinity ultrafiltration hydrolysate, 515 in oyster peptide, 336 in sea cucumber peptide, 30 in sturgeon cartilage peptide, 42 in Alaska pollock peptide, and 455 in sea cucumber flower peptide.

[0052] The affinity ultrafiltration hydrolysate was separated by capillary high performance liquid chromatography and then analyzed by mass spectrometry using a QExactive mass spectrometer (ThermoFisher). The analysis time was 60 min. Detection method: positive ion. The mass-to-charge ratios of peptides and peptide fragments were collected according to the following method: 10 fragment spectra (MS2scan) were collected after each full scan (fullscan). Figures 7 to 11 These are the mass spectra of inhibitory peptides WPF, FPW, FGW, LPVPAFN, and LWWL. It can be seen that the molecular weight of each inhibitory peptide is relatively close to its theoretical molecular weight.

[0053] The following is a further molecular docking study on the Ala-Leu-Leu-Leu (ALLL) ACE inhibitory peptide: The three-dimensional structure of ACE (PDB code: 1O8A) was obtained from the RCSB protein database (www.pdb.org), and the protein and ligand were prepared using the AutoDockTools program. The protein structure in the protein database is saved in PDB format. After removing water molecules, adding polar hydrogen, calculating the Coleman charge, and merging non-polar hydrogen, it was saved as a pdbqt format file. The 3D structure was processed using AutoDockTools and saved as a pdbqt format file. The center of the grid is located at X=40.607, Y=32.451, Z=43.579, the size of the grid box is 60Å×70Å×52Å, and the grid spacing is 0.375Å. The docking program was executed by the AutoDock4.2.6 program, and a total of 100 docking operations were performed to calculate the binding energy. The screened ACE inhibitory active peptide ALLL was selected as an example to explain in detail the mechanism of action of the peptide inhibiting ACE. The molecular docking conformation is shown in the figure. Fig.12 As shown. Hydrogen bonds, electrostatic interactions and hydrophobic interactions are the main forces between peptide ALLL and ACE, and two hydrogen bonds are formed between ACE and ALLL. This shows that peptide ALLL can bind to the ACE enzyme through multiple forces to exert antihypertensive activity.

[0054] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of the present application.

Claims

1. An ACE inhibitory peptide, characterized in that The amino acid sequence of the ACE inhibitory peptide is Trp-Pro-Phe (WPF) or Phe-Pro-Trp (FPW) or Phe-Gly-Trp (FGW) or Leu-Pro-Val-Pro-Ala-Phe-Asn (LPVPAFN) or Leu-Trp-Trp-Leu (LWWL).

2. The ACE inhibitory peptide according to claim 1, characterized in that The source of the ACE inhibitory peptide is aquatic products.

3. The ACE inhibitory peptide according to claim 2, characterized in that The source of the ACE inhibitory peptide is sea cucumber or sea cucumber flower or tuna or oyster.

4. A method for preparing an ACE inhibitory peptide, characterized in that: For preparing the ACE inhibitory peptide according to any one of claims 1 to 3, comprising the following steps: S1: crush the source material and set aside; S2: adding the crushed source material into water, adjusting the pH to 6-9, adding enzymes, performing enzymolysis, and obtaining an enzymolysis solution; S3: inactivating the enzyme in the enzymatic hydrolysate, taking the supernatant, and obtaining an enzyme-inactivated enzymatic hydrolysate; S4: spray drying the enzyme-killed hydrolyzate to obtain peptide powder; S5: Add the peptide powder to water to obtain a peptide powder solution, and obtain the ACE inhibitory peptide after amino acid content determination, affinity ultrafiltration screening, high performance liquid chromatography purification, and amino acid sequence determination.

5. The method for preparing an ACE inhibitory peptide according to claim 4, characterized in that: The enzyme in step S2 is a mixture of alkaline protease and neutral protease, and the mass concentration of the enzyme is 0.2-0.5%.

6. The method for preparing an ACE inhibitory peptide according to claim 5, characterized in that: Step S3 is specifically as follows: heating the enzymatic hydrolysate at 80-100° C. for 10-30 min to inactivate the enzyme, then centrifuging at 3000-5000 r / min for 10-30 min, taking the supernatant, and obtaining the inactivated enzymatic hydrolysate.

7. The method for preparing an ACE inhibitory peptide according to claim 4, characterized in that: The specific method of step S5 is: S51: Mix the peptide powder solution and ACE in a volume ratio of 1:1-5 and incubate at 30-40°C for 1-3h; S52: After the incubation, centrifuge at 3000-5000 r / min for 10-30 min using a 5-10 kDa ultrafiltration centrifuge tube to remove the peptides not bound to ACE and obtain the complex formed by the binding of ACE and the enzymatic hydrolysate; S53: The complex formed by the combination of ACE and the hydrolysate was incubated with 50-70% acetonitrile for 10-30 min, and centrifuged at 3000-5000 r / min for 10-30 min to release the peptides in the complex. The filtrate was collected as the affinity ultrafiltration hydrolysate. S54: The affinity ultrafiltration hydrolyzate is purified by high performance liquid chromatography and subjected to amino acid sequencing to obtain an ACE inhibitory peptide; In step S51, the concentration of the peptide powder solution is 3-10 mg / mL, and the concentration of ACE is 100-300 mU / mL.

8. The method for preparing an ACE inhibitory peptide according to claim 7, characterized in that: In step S54, the high performance liquid chromatography purification adopts a gradient elution method for separation and purification, and the mobile phases are mobile phase A and mobile phase B, the mobile phase A is a formic acid aqueous solution with a volume concentration of 0.05-0.15%, and the mobile phase B is a formic acid acetonitrile solution with a volume concentration of 0.05-0.15%. The specific steps are: From 0 to 50 min, the volume content of mobile phase B in the mobile phase was 4 to 50%, and the volume content of mobile phase A in the mobile phase was 96 to 50%; At 50-54 min, the volume content of mobile phase B in the mobile phase was 50-100%, and the volume content of mobile phase A in the mobile phase was 50-0%; At 54-60 min, the volume content of mobile phase B in the mobile phase was 100%.

9. The method for preparing an ACE inhibitory peptide according to claim 8, characterized in that: During the HPLC purification process, the mobile phase flow rate was 1~2mL / min.

10. The use of ACE inhibitory peptide, characterized in that: The ACE inhibitory peptide according to any one of claims 1 to 3 is used in the preparation of a drug for lowering hypertension.

Images