Peptide, inhibitor, screening method and application

Abstract

The invention belongs to the technical field of preparation of inhibitory peptides, and particularly relates to a peptide, an inhibitor, a screening method and application. The invention provides an inhibitory peptide or umami peptide. The inhibitory peptide comprises a first polypeptide and / or a second polypeptide; the amino acid sequence of the first polypeptide is as shown in SEQ ID NO. 1; the amino acid sequence of the second polypeptide is as shown in SEQ ID NO. 2. The inhibitory peptide provided by the invention is extracted from shiitake mushrooms, and has inhibitory activity on ACE and DPP-IV.

Description

Technical Field

[0001] The present invention belongs to the technical field of peptide preparation, and in particular relates to a peptide, an inhibitor, a screening method and an application. Background Art

[0002] Bioactive peptides generally contain 2 to 20 amino acids, with a molecular weight of less than 6000Da and good biological activity. At present, the research on bioactive peptides in edible fungi mainly focuses on antioxidant, antihypertensive, antibacterial and immunomodulatory aspects. Antioxidants are essential for human aging and immune system. Antioxidant peptides can activate the Keap1-Nrf2 / ARE signaling pathway, thereby triggering the expression of proteins with antioxidant and cytoprotective properties. Peptides help protect the body from oxidative stress and related damage caused by free radicals such as 1,1-diphenyl-2-pyridylhydrazide (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazole-6-sulfonate) (ABTS+). At the same time, peptides also have certain food flavor characteristics, such as umami and salty taste. Flavor peptides with a molecular weight of less than 3000Da extracted from food proteins or synthesized from amino acids have attracted widespread attention because of their unique flavor in natural and processed foods. At present, there are many studies on the preparation of flavor peptides from various edible fungi such as Lentinula edodes, Stropharia rugosoannulata, and Hypsizygus marmoreus. Compared with traditional seasonings, flavor peptides with biological activity have better development prospects and additional application value. Lentinula edodes is rich in active ingredients such as polysaccharides, proteins, and amino acids, and has certain therapeutic and pharmacological effects. It is a high-quality raw material for the development of bioactive peptides. At present, there are no reports on peptides extracted from Lentinula edodes with umami and salty taste and potential inhibitory activity of angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV). Summary of the invention

[0003] The purpose of the present invention is to provide a peptide, an inhibitor, a screening method and an application. The provided peptide has umami taste and salty taste and has ACE and DPP-IV inhibitory activity.

[0004] In order to solve the above technical problems, the following technical solutions are proposed:

[0005] The present invention provides a peptide, comprising a first polypeptide and / or a second polypeptide;

[0006] The amino acid sequence of the first polypeptide is shown in SEQ ID NO.1;

[0007] The amino acid sequence of the second polypeptide is shown in SEQ ID NO.2.

[0008] The present invention provides the use of the peptide described in the above technical solution in the preparation of angiotensin converting enzyme and / or dipeptidyl peptidase IV inhibitors.

[0009] Preferably, the active ingredient of the inhibitor includes the peptide described in the above technical solution; or the only active ingredient of the inhibitor is the peptide described in the above technical solution.

[0010] The present invention provides the use of the peptide described in the above technical solution in preparing at least one of the following products 1) and 2);

[0011] 1) Products for the prevention and / or treatment of cardiovascular diseases;

[0012] 2) Products for the prevention and / or treatment of metabolic diseases.

[0013] Preferably, the product types of the inhibitor include medicines and / or health products.

[0014] The present invention provides an angiotensin converting enzyme inhibitor and / or a dipeptidyl peptidase IV inhibitor, wherein the active ingredient of the inhibitor comprises the peptide described in the above technical solution; or the only active ingredient of the inhibitor is the peptide described in the above technical solution.

[0015] The present invention provides a product, wherein the active ingredient of the product includes the peptide described in the above technical solution; or the only active ingredient of the product is the peptide described in the above technical solution;

[0016] The product is at least one of the following Ⅰ) and Ⅱ);

[0017] Ⅰ) Products for the prevention and / or treatment of cardiovascular diseases;

[0018] II) Products for the prevention and / or treatment of metabolic diseases.

[0019] The present invention provides application of the peptide described in the above technical solution in preparing fresh products.

[0020] The present invention provides a fresh product, wherein the active ingredient of the fresh product comprises the peptide described in the above technical solution; or the only active ingredient of the fresh product is the peptide described in the above technical solution.

[0021] The present invention provides a method for screening the peptides described in the above technical solution, comprising:

[0022] Use the PeptideRanker tool to score the bioactive peptides to be screened, and obtain active peptides with scores > 0.5;

[0023] The obtained active peptides were screened by BIOPEP-UWM, and the bioactive peptides with angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities were obtained by molecular docking and molecular dynamics simulation methods;

[0024] The toxicity, sensitization, water solubility and stability of the bioactive polypeptides were screened by ToxinPred tool, AllerTOP v.2.0 tool, Innovagen tool and ExpasyProtParam tool in sequence to obtain non-toxic, non-allergenic, water-soluble and stable peptides.

[0025] Technical effect of the present invention: The present invention provides a peptide, the peptide comprising a first polypeptide and / or a second polypeptide;

[0026] The amino acid sequence of the first polypeptide is shown in SEQ ID NO.1;

[0027] The amino acid sequence of the second polypeptide is shown in SEQ ID NO.2.

[0028] The first polypeptide and the second polypeptide provided by the present invention are both prepared from shiitake mushrooms, have umami and salty tastes, and have ACE and DPP-IV inhibitory activities.

[0029] The results of the examples show that the peptide provided by the present invention has inhibitory activity on angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), and has a freshening effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The diagrams are the structures of bioactive peptides and ACE and DPP-IV receptors, where A and B are the three-dimensional surface structures of ACE and DPP-IV receptors; C and D are the docking and overlap results of 15 potential bioactive peptides with ACE and DPP-IV receptors; Figure 1 The "peptide color" from top to bottom are LPFDFPK, DPPHLPPLPR, APPPDPNRP, PPPDPNRP, DPTPPPPR, ADHDLPF, APPPDPNRPS, DAPLPHPNR, GSEDPLPGAK, LPDWGLPHE, LPPPEEYF, DIGIPDPTGR, TPGPDRP, NPVDAYPPGSPAR, HPDLPPHQ;

[0031] Figure 2 The three-dimensional and two-dimensional ligand interaction diagrams of the molecular docking of the peptide DAPLPHPNR and the ACE protein, wherein the left side is the three-dimensional interaction diagram and the right side is the two-dimensional interaction diagram;

[0032] Figure 3 The three-dimensional and two-dimensional ligand interaction diagrams of the molecular docking of the peptide GSEDPLPGAK and the ACE protein, wherein the left side is the three-dimensional interaction diagram and the right side is the two-dimensional interaction diagram;

[0033] Figure 4 The three-dimensional and two-dimensional ligand interaction diagrams of the molecular docking of the peptide DAPLPHPNR and the DPP-IV protein, wherein the left side is the three-dimensional interaction diagram and the right side is the two-dimensional interaction diagram;

[0034] Figure 5 The three-dimensional and two-dimensional ligand interaction diagrams of the molecular docking of the peptide GSEDPLPGAK and the DPP-IV protein, wherein the left side is the three-dimensional interaction diagram and the right side is the two-dimensional interaction diagram;

[0035] Figure 6 The molecular dynamics interactions of two potential bioactive peptides with ACE and DPP-IV receptors within 50 ns; A represents the root mean square deviation (RMSD) of system stability, B represents the root mean square fluctuation (RMSF), C represents the Rg curve, and D represents the number of hydrogen bonds of the peptide receptor complex;

[0036] Figure 7 These are sensory evaluation diagrams of synthetic peptides, where A is the sensory characterization result and B is the electronic tongue analysis diagram. DETAILED DESCRIPTION

[0037] The present invention provides a peptide, comprising a first polypeptide and / or a second polypeptide;

[0038] The amino acid sequence of the first polypeptide is shown in SEQ ID NO.1;

[0039] The amino acid sequence of the second polypeptide is shown in SEQ ID NO.2.

[0040] In the present invention, the expression of the first polypeptide and the second polypeptide is only for distinguishing different polypeptides and has no special designated meaning. In the present invention, the first polypeptide and the second polypeptide are preferably extracted from Lentinus edodes as raw materials, and can also be artificially synthesized according to the amino acid sequence of the polypeptide.

[0041] The first polypeptide and the second polypeptide of the present invention both have an inhibitory effect on angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (Dipeptidylpeptidase IV, DPP-IV). The first polypeptide and the second polypeptide of the present invention still have an inhibitory effect on ACE and DPP-IV after being mixed. The first polypeptide and the second polypeptide can both be polypeptides extracted from shiitake mushrooms, which are safer and more reliable. As natural inhibitors, they can inhibit the activity of ACE and DPP-IV, reduce adverse reactions in the body, and may have fewer side effects. The first polypeptide and the second polypeptide of the present invention have similar taste characteristics, have stronger umami and salty taste, weaker sour and bitter taste, and show more pleasant taste characteristics, and the umami and salty taste are more prominent.

[0042] The present invention provides the use of the peptide described in the above technical solution in the preparation of angiotensin converting enzyme and / or dipeptidyl peptidase IV inhibitors. As an practicable manner, the active ingredient of the inhibitor of the present invention includes the peptide described in the above technical solution; or the only active ingredient of the inhibitor is the peptide.

[0043] The present invention provides the use of the peptide described in the above technical solution in the preparation of products for preventing and / or treating cardiovascular diseases. As an practicable manner, the product types described in the present invention preferably include medicines and / or health products.

[0044] The cardiovascular disease of the present invention may be hypertension, and the ACE is a Zn-containing 2+ ACE is a target for the treatment of hypertension. It inactivates bradykinin with vasodilatory effect by catalyzing the conversion of non-hypertensive angiotensin I to hypertensive angiotensin II, leading to increased blood pressure. Inhibiting ACE activity can lower blood pressure. Both the first polypeptide and the second polypeptide of the present invention have an inhibitory effect on angiotensin converting enzyme (ACE), and have potential value in preventing and / or treating cardiovascular diseases.

[0045] The present invention provides the use of the peptide described in the above technical solution in the preparation of products for preventing and / or treating metabolic diseases. As an practicable manner, the product types described in the present invention preferably include medicines and / or health products.

[0046] The metabolic disease described in the present invention may be hyperglycemia and / or diabetes. Dipeptidylpeptidase IV (DPP-IV) is a serine peptidase located on the cell surface and belongs to a membrane protein. DPP-IV is a target for the treatment of diabetes. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin-releasing peptide (GIP) can induce β-cell differentiation and stimulate the production and release of insulin, but their half-lives are extremely short and are rapidly degraded by DPP-IV in the body. Therefore, by inhibiting DPP-IV to extend the half-life of GLP-1 and GIP in the body, blood sugar stability can be maintained. The first polypeptide and the second polypeptide of the present invention both have an inhibitory effect on ACE and DPP-IV, and have potential value in preventing and / or treating metabolic diseases.

[0047] The present invention provides an angiotensin converting enzyme inhibitor, the active ingredient of the inhibitor includes the peptide described in the above technical solution; or the only active ingredient of the inhibitor is the peptide; the inhibitor preferably also includes an excipient. As an implementable method, the present invention can use ingredients that have preventive and / or therapeutic effects on cardiovascular diseases as other effective ingredients in combination with the peptide. The first polypeptide and the second polypeptide of the present invention can be used alone or in combination. The present invention does not specifically limit the types of excipients, and conventional excipient ingredients can be used. The present invention does not specifically limit the dosage form of the inhibitor, and conventional dosage forms can be used.

[0048] The present invention provides a dipeptidyl peptidase IV inhibitor, the active ingredient of the inhibitor includes the peptide described in the above technical solution or the only active ingredient is the peptide; the inhibitor preferably also includes an excipient. As an implementable method, the present invention can use ingredients that have preventive and / or therapeutic effects on metabolic diseases as other effective ingredients in combination with the peptide. The first polypeptide and the second polypeptide of the present invention can be used alone or in combination, and can also be used in combination with other ingredients that have preventive and / or therapeutic effects on metabolic diseases. The present invention does not specifically limit the types of excipients, and conventional excipient ingredients can be used. The present invention does not specifically limit the dosage form of the inhibitor, and conventional dosage forms can be used.

[0049] The present invention provides a product, wherein the active ingredient of the product includes the peptide described in the above technical solution; or the only active ingredient of the product is the peptide described in the above technical solution;

[0050] The product is at least one of the following Ⅰ) and Ⅱ);

[0051] Ⅰ) Products for the prevention and / or treatment of cardiovascular diseases;

[0052] II) Products for the prevention and / or treatment of metabolic diseases.

[0053] As an practicable manner, the first polypeptide and the second polypeptide of the present invention can be used alone or in combination, or can be used in combination with other components that have preventive and / or therapeutic effects on metabolic diseases or cardiovascular diseases.

[0054] As an practicable manner, the product of the present invention preferably further comprises excipients. The present invention has no special limitation on the type of excipients, and conventional excipient components can be used. The present invention has no special limitation on the dosage form of the product, and conventional dosage forms can be used.

[0055] The present invention provides the use of the peptides described in the above technical solution in the preparation of fresh products. The first polypeptide and the second polypeptide of the present invention have similar taste characteristics, have stronger umami and salty taste, weaker sour and bitter taste, and show more pleasant taste characteristics, with more prominent umami and salty taste.

[0056] The present invention provides a fresh product, wherein the active ingredient of the fresh product includes the umami peptide described in the above technical solution; or the only active ingredient of the fresh product is the umami peptide described in the above technical solution. As an practicable manner, the present invention can use ingredients with fresh effects as other active ingredients in combination with the umami peptide.

[0057] The present invention provides a method for screening the peptides described in the above technical solution, comprising:

[0058] Use the PeptideRanker tool to score the bioactive peptides to be screened, and obtain the first active peptide with a score > 0.5;

[0059] The obtained active peptides were screened by BIOPEP-UWM, and the bioactive peptides with angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities were obtained by molecular docking and molecular dynamics simulation methods;

[0060] The toxicity, sensitization, water solubility and stability of the bioactive polypeptides were screened in turn by the ToxinPred tool, the AllerTOP v.2.0 tool, the Innovagen tool and the ExpasyProtParam tool to obtain non-toxic, non-allergenic, water-soluble and stable inhibitory peptides.

[0061] Based on the constructed polypeptide database to be screened, the present invention uses computer virtual screening technology to search for polypeptides with umami, salty taste and potential inhibitory activity of ACE and DPP-IV from shiitake enzymatic extracts. First, the PeptideRanker tool is used to score the bioactive peptides to be screened, and active peptides with a score of >0.5 are obtained. The PeptideRanker tool used in the present invention does not need to be set up, and the bioactive peptides are directly scored.

[0062] The bioinformatics website BIOPEP-UWM was then used to screen shiitake mushroom flavor peptides with ACE and DPP-IV inhibitory activity.

[0063] Then, the toxicity of the bioactive polypeptide was screened by the ToxinPred tool, the allergenicity of the bioactive polypeptide was screened by the AllerTOP v.2.0 tool, the water solubility of the bioactive polypeptide was screened by the Innovagen tool, and the stability of the bioactive polypeptide was screened by the ExpasyProtParam tool to obtain non-toxic, allergen-free, water-soluble and stable inhibitory peptides.

[0064] Molecular docking and molecular dynamics simulation methods were then used to further study the molecular recognition and inhibition mechanism of the inhibitory peptides on the hypertension-related target ACE and the diabetes-related target DPP-IV, and to screen out inhibitory peptides with good stability in binding to ACE and DPP-IV receptors.

[0065] Finally, a stable inhibitory peptide was synthesized by chemical synthesis. The inhibitory activity of the stable inhibitory peptide on ACE and DPP-IV was further verified by in vitro experiments. The present invention provides a new method for the rapid screening of multifunctional active flavor peptides and provides theoretical support for the multi-dimensional development and utilization of high value-added processed products of shiitake mushrooms.

[0066] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0067] Example 1 Virtual Screening of Mushroom Bioactive Peptides

[0068] The present invention provides a method for screening and preparing two mushroom flavor peptides with ACE and DPP-IV inhibitory effects, and the steps are as follows:

[0069] Online prediction and screening of ACE and DPP-IV inhibitory peptides

[0070] Based on 272 shiitake mushroom enzymatic flavor peptides, the PeptideRanker tool (https: / / distilldeep.ucd.ie / PeptideRanker / ) was used to screen potential bioactive peptides with a score > 0.5, and then BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) was used to screen potential bioactive peptides with ACE and DPP-IV inhibitory activity. Further, the ToxinPred tool (https: / / crdd.osdd.net / raghava / toxinpred / index.html), AllerTOP v.2.0 tool (https: / / www.ddg-pharmfac.net / AllerTOP / ), Innovagen tool (https: / / www.innovagen.com / proteomics-tools) and Expasy ProtParam tool (https: / / web.expasy.org / protparam / ) were used to screen potential bioactive peptides that were non-toxic, non-allergenic, water-soluble and stable. Specifically:

[0071] Peptideranker is used to predict the biological activity of peptides, and peptides with a score > 0.50 are considered to have potential biological activity. Among the 272 flavor peptides with potential salty and umami characteristics screened, 95 peptides with a score > 0.50 were found. BIO-UWM was used to further analyze the inhibitory activity of the 95 predicted peptides on ACE and DPP-IV. It was found that there were 50 peptides with an inhibitory activity score ≥ 0.5 on ACE and DPP-IV. Among them, 48 peptides were predicted to be non-toxic by the ToxinPred tool. The 48 non-toxic peptides were predicted by the AllerTOPv.2.0 tool, of which 19 were allergens and 29 were non-allergens. The water solubility and stability of 29 non-allergenic peptides were predicted by Innovagen tool and ExpasyProtParam tool, and 15 peptides with potential inhibitory activity of ACE and DPP-IV, good water solubility and stability were screened out from the 29 non-allergenic peptides, including 3 heptapeptides, 4 octapeptides, 3 nonapeptides, 4 decapeptides and 1 tripeptide.

[0072] According to the prediction results of BIO-UWM (see “https: / / biochemia.uwm.edu.pl / biopep-uwm / ”), the amino acid combinations that play a key role in the inhibitory activity of ACE and DPP-IV in 15 peptides, among which 39 amino acid combinations play a key role in the inhibitory activity of ACE, and 42 amino acid combinations play a role in the inhibitory activity of DPP-IV. Most of these amino acid combinations are composed of hydrophobic and basic amino acids, such as PP, LP, PH and RP, where P represents proline (Pro), L represents leucine (Leu), H represents histidine (His), and R represents arginine (Arg). In addition, the amino acid combinations of proline-leucine (PL), arginine-proline (AP), leucine-proline-phenylalanine (LPF), proline-threonine (PT), threonine-proline (TP), histidine-proline (HP), phenylalanine-proline (FP), proline-proline-leucine (PPL), glycine-alanine (GA), glycine-leucine (GL), tryptophan-glycine (WG), glutamate-tyrosine (EY), isoleucine-proline (IP), glycine-isoleucine (GI), threonine-glycine (TG), glycine-proline (GP), glutamine-arginine (DR), alanine-tyrosine (AY) and tyrosine-proline (YP) may jointly inhibit ACE activity and DPP-IV activity. Therefore, these 15 candidate bioactive peptides were selected for further molecular docking analysis. The specific details of the 15 candidate bioactive peptides are shown in Table 1.

[0073] Example 2 Molecular docking of potential bioactive peptides with ACE and DPP-IV receptors

[0074] Using the MOE 2019 molecular docking tool, the three-dimensional structures of the screened potential bioactive peptides were successfully constructed and energy minimized. The crystal structure data of ACE (crystal structure number PDB ID: 1086) and DPP-IV (crystal structure number PDBID: 1X70) are derived from the RCSB protein database (https: / / www.rcsb.org). The DoGSiteScorer online tool (https: / / proteins.plus / ) was used to predict the protein receptor binding cavities and sites of 15 peptides with ACE and DPP-IV. Specifically:

[0075] In order to explore the binding mechanism of the 15 potential bioactive peptides obtained in Example 1 with ACE and DPP-IV receptors, molecular docking analysis was performed using MOE 2019 software. The final three-dimensional models of ACE receptor and DPP-IV receptor constructed by computer are shown in Figure 2. Figure 1As shown in A and B. The docking results of 15 peptides and ACE receptor and DPP-IV receptor are shown in Table 1. According to Table 1, the 15 peptides can enter the binding pockets of ACE and DPP-IV. The basic docking energy of 15 peptides with ACE receptor is between -13.1739 and -15.4686 kcal / mol, and the average docking energy is -14.4671 kcal / mol. The basic docking energy of 15 peptides with DPP-IV receptor is between -9.6474 and -11.5795 kcal / mol, and the average docking energy is -10.5944 kcal / mol. The stability of receptor-ligand complex can be judged by docking energy. The lower the docking energy, the more stable the binding. From the binding energy value, DAPLPHPNR (SEQ ID NO.1) and GSEDPLPGAK (SEQ ID NO.2) have low binding energy to ACE receptor and DPP-IV receptor, indicating that these two peptides may have strong ACE and DPP-IV inhibitory activity. Three-dimensional diagram of the interaction of 15 peptides with ACE receptor and DPP-IV receptor Figure 1 As shown in C and D. The main active sites of the 15 peptides binding to the ACE receptor are Glu403, Arg522, Asn150, Arg124, Tyr523, His387 and Glu411, among which Glu403 is the most connected, followed by Glu411, Arg124 and His387. The main active sites of the 15 peptides binding to the DPP-IV receptor are Arg125, Glu206, Lys554, Asp545, Glu205, Arg560 and His740, among which Glu206 is most frequently found in the docking of the DPP-IV receptor. Hydrogen bonds and electrostatic interactions are the main interaction modes between peptides and the ACE receptor and the DPP-IV receptor.

[0076] Among them, DAPLPHPNR (-15.4686 kcal / mol) with the lowest binding energy interacts with the ACE receptor and can form hydrogen bonds with multiple binding sites (Tyr62, Arg124, Ala125, Glu384, Tyr523). Figure 2 As shown. His387 and Glu411 form ionic bonds with the ligand DAPLPHPNR. The oxygen atoms in the peptide bond with the Zn in ACE 2+ In addition, the 5th ring in the DAPLPHPNR ligand forms a cation-π bond with His410 in the ACE receptor, and Asn70 in ACE forms a Pi-H bond with the ligand. The two-dimensional diagram of GSEDPLPGAK binding to the ACE receptor is shown in Figure 3Asn66, Glu143, Met223, His353, Glu403, Ser516, Arg522 and Tyr523 in ACE form hydrogen bonds with the ligand GSEDPLPGAK, and Arg124, His353, His387 and Glu411 form ionic bonds with the ligand.

[0077] When binding to the DPP-IV receptor, the ligand GSEDPLPGAK has the lowest binding energy, as shown in Table 1 and Figure 5 As shown, the decapeptide interacts with Asp545, Lys554, Arg560, Asn562 and Ser577 of the DPP-IV backbone to form hydrogen bonds. The binding site Lys554 and Arg560 form ionic bonds with E3 and D4 in the ligand GSEDPLPGAK, and Tyr666 forms H-Pi interactions with the ligand. DAPLPHPNR mainly interacts with the DPP-IV receptor through hydrogen bonds, including Arg125, Glu206, Glu553, Lys554, Arg560 and His740, as shown Figure 4 As shown. Arg125, Glu205, Glu206, Lys554 and His740 in the DPP-IV receptor form ionic bonds with the ligand DAPLPHPNR. In addition, H-Pi interactions are formed between the ligand D1 and R9 and Tyr547 and Trp629 of the DPP-IV receptor, and Pi-H interactions exist between the ligand H6 and Lys554 of the receptor. The lower the molecular docking binding energy, the stronger the binding ability between the ligand and the receptor, and therefore it is easier to bind. It can be seen that the peptides with the strongest binding ability to ACE and DPP-IV receptors are DAPLPHPNR and GSEDPLPGAK, which have good stability, as shown in Table 1.

[0078] Table 1 Molecular docking binding energy of 15 potential bioactive peptides with ACE and DPP-IV receptors

[0079]

[0080] Example 3 Molecular dynamics simulation of potential bioactive peptides with ACE and DPP-IV receptors

[0081] In order to further explore the stability of bioactive peptides binding to ACE and DPP-IV receptors, the peptides with the strongest binding ability to ACE and DPP-IV receptors (DAPLPHPNR and GSEDPLPGAK) were selected for molecular dynamics simulation analysis.

[0082] The protein parameters were generated by the AMBER14SB force field. Sodium ions were added to the water tank of the TIP3P solvation model, and counterions were added to maintain the neutrality of the system. On the premise of ensuring the normal structure of the system, the fastest descent method and conjugate gradient method were used to minimize the energy of the solvent and solute structure. Then, the solution was heated to 10 kcal / min. The whole system was heated to 300K under the constraint of . All chemical bonds involving hydrogen atoms are suitable for SHAKE algorithm. After the system energy and density reached equilibrium, in order to further evaluate the interaction between these two potential bioactive peptides and ACE and DPP-IV receptors, the complex of DAPLPHPNR bound to ACE receptor, namely DAPLPHPNR-ACE, the complex of DAPLPHPNR bound to DPP-IV receptor, namely DAPLPHPNR-DPP-IV, the complex of GSEDPLPGAK bound to ACE receptor, namely GSEDPLPGAK-ACE, and the complex of GSEDPLPGAK bound to DPP-IV receptor, namely GSEDPLPGAK-DPP-IV, were obtained. The above four ACE-peptide complexes and DPP-IV-peptide complexes GSEDPLPGAK-ACE, GSEDPLPGAK-DPP-IV, DAPLPHPNR-DPP-IV, and DAPLPHPNR-ACE were imported into molecular dynamics simulation software for 50ns molecular dynamics simulation.

[0083] The dynamic stability of the ACE-peptide complex and the DPP-IV-peptide complex was analyzed by studying the root mean square deviation (RMSD) between the trunk and the initial docking structure throughout the trajectory. Figure 6 As shown in A, DAPLPHPNR-ACE, DAPLPHPNR-DPP-IV, GSEDPLPGAK-ACE, and GSEDPLPGAK-DPP-IV all reached equilibrium after 5 ns. Compared with the other three complexes, DAPLPHPNR-DPP-IV, GSEDPLPGAK-ACE, and GSEDPLPGAK-ACE, DAPLPHPNR-ACE showed higher stability in all kinetic simulations. The fluctuation of the RMSD value of DAPLPHPNR-DPP-IV in the kinetic simulation also means that the stability of DAPLPHPNR binding to the DPP-IV receptor is not as good as GSEDPLPGAK.

[0084] The root mean square fluctuation (RMSF) reflects the fluctuation of each atom relative to its average position and can be used to evaluate the overall flexibility of the protein complex. Figure 6 As shown in Figure 2B, the RMSF of the four complexes is low, and the RMSF of most sequences on the ACE and DPP-IV receptor proteins is lower than This indicates that the protein system is still relatively stable. It is worth noting that among the four complexes, the RMSF of DAPLPHPNR-DPP-IV is higher than the other three, indicating that the DPP-IV receptor fluctuates relatively greatly during the binding process with DAPLPHPNR. In contrast, DAPLPHPNR-ACE, GSEDPLPGAK-ACE, and GSEDPLPGAK-DPP-IV show lower RMSF in multiple places, which means that DAPLPHPNR can stabilize the ACE receptor. GSEDPLPGAK can stabilize the ACE receptor and the DPP-IV receptor more significantly.

[0085] The gyration of rotation (Rg) is a parameter used to measure the compactness of the overall structure during the simulation, which represents the distance from the center of mass to the end of all atoms in a certain period of time. Figure 6 As shown in C, the Rg value of DAPLPHPNR-DPP-IV fluctuates the most, ranging from GSEDPLPGAK-ACE Rg The fluctuation range is only This indicates that the structure of the ACE receptor protein is relatively stable during the molecular dynamics simulation. In general, during the simulation, the overall structural compactness of the two potential bioactive peptides and the ACE receptor protein may be better, and the resulting complex will be more stable.

[0086] Hydrogen bonding is a key driver between proteins and peptides, contributing to complex binding capacity and stability. Figure 6 D in the middle shows the number of hydrogen bonds in the complexes during the 50 ns molecular simulation interaction. During the molecular simulation interaction, the average number of hydrogen bonds for DAPLPHPNR-ACE, GSEDPLPGAK-ACE, DAPLPHPNR-DPP-IV and GSEDPLPGAK-DPP-IV were 5, 9, 9 and 7, respectively, which is basically consistent with the molecular docking results.

[0087] The above method can be used to obtain two shiitake mushroom flavor peptides with ACE and DPP-IV inhibitory effects. The two peptides (DAPLPHPNR and GSEDPLPGAK) show strong binding ability with ACE and DPP-IV receptors, and molecular dynamics simulation further verifies the stability of their complexes with the receptors.

[0088] Example 4

[0089] 1 Solid phase synthesis of potential bioactive peptides

[0090] Two peptides with potential biological activity were screened from the enzymatic hydrolyzed extract of Lentinus edodes: the first polypeptide: Asp-Ala-Pro-Leu-Pro-His-Pro-Asn-Arg (DAPLPHPNR) and the second polypeptide: Gly-Ser-Glu-Asp-Pro-Leu-Pro-Gly-Ala-Lys (GSEDPLPGAK). The first polypeptide and the second polypeptide were synthesized by Shanghai Jier Biochemical Co., Ltd. Based on the solid phase synthesis strategy of Fmoc, the length and sequence of the peptide chain were extended. The purity of the synthetic peptide was greater than 98%, and the amino acid sequence of the synthesized polypeptide was correct as identified by reverse phase high performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS).

[0091] 2 Electronic tongue assay of synthetic peptides

[0092] The synthesized first polypeptide and the second polypeptide were prepared into 0.5 mg / mL solutions, respectively, and the 6 sensors in the SA-402B taste analysis system were used to measure the acidity (sensor type, CAO), sweetness (GL1), bitterness (COO), saltiness (CTO), umami (AAE) and astringency (AE1). The samples were poured into a specific measuring cup and placed in the sample tank for measurement. Each sample was tested 4 times, the sample test time was 120s, and the data of the last 3 times were taken for analysis.

[0093] The sensory evaluation results showed that (see Figure 7 In Figure A), the two peptides GSEDPLPGAK and DAPLPHPNR have similar taste characteristics, with stronger umami and salty tastes and weaker sour and bitter tastes.

[0094] 3 Sensory evaluation analysis of synthetic peptides

[0095] The sensory evaluation team consisted of 11 laboratory members, 5 men and 6 women, aged 20 to 30 years old, without taste or smell disorders. Basic taste training was conducted for sensory evaluators. The standard samples of sour, sweet, bitter, salty and fresh were 0.08% citric acid solution, 1% sucrose solution, 1% isoleucine solution, 0.35% sodium chloride solution and 0.35% monosodium glutamate solution, respectively. The sensory evaluation experiment was conducted in a sensory analysis room at (25±1)℃. The samples of the first polypeptide and the second polypeptide to be tested were placed in a 30mL tasting cup and randomly numbered with three digits. The evaluator randomly selected a sample with a sample volume of not less than 5mL, stirred it with the tongue for 10s, and then spit it out. To avoid fatigue and residual effects, sensory evaluators were required to rinse their mouths with ultrapure water and rest for 2min between tastings. The samples of the first polypeptide and the second polypeptide were prepared into peptide solutions with a mass concentration of 1 mg / mL using ultrapure water, and the team members scored the samples using a 10-point scoring method. The sensory scores of the five standard substances of sour, sweet, bitter, salty, and umami were set to 5 points, with 0 points for none and 10 points for the strongest. The sour, sweet, bitter, salty, and umami tastes of the first polypeptide and the second polypeptide samples were scored, and the evaluation results were displayed using a radar chart.

[0096] The sensory evaluation results were consistent with the electronic tongue evaluation results. GSEDPLPGAK showed more pleasant taste characteristics than DAPLPHPNR, with more prominent umami and salty tastes (see Figure 7 (middle B).

[0097] 4 In vitro activity assay

[0098] (1) Determination of the in vitro ACE inhibitory activity of synthetic peptides

[0099] 0.1 mol / L borate buffer contains 0.3 mol / L sodium chloride and has a pH of 8.3;

[0100] The first polypeptide was dissolved in 0.1 mol / L borate buffer to obtain first polypeptide solutions with concentrations of 0.016, 0.08, 0.24, 0.4, and 2.0 mg / mL, respectively.

[0101] The second polypeptide was dissolved in 0.1 mol / L borate buffer to obtain second polypeptide solutions with concentrations of 0.016, 0.08, 0.24, 0.4, and 2.0 mg / mL, respectively.

[0102] Dissolve hippuryl-histidyl-leucine (HHL) in 0.1 mol / L borate buffer to obtain a 4.7 mmol / L HHL solution;

[0103] ACE was dissolved in 0.1 mol / L borate buffer (containing 0.3 mol / L sodium chloride, pH 8.3) to obtain an ACE solution with a concentration of 0.025 U / mL.

[0104] Determination of the first polypeptide solution: Treatment 1: 25 μL of HHL solution was mixed with 12.5 μL of the first polypeptide solution of different concentrations, kept at 37°C for 5 min, then 25 μL of ACE solution was added, kept at 37°C for 30 min, and then 375 μL of NaOH solution with a concentration of 0.3 mol / L was added to stop the reaction. Then 25 μL of 2% mass concentration of o-phthalaldehyde (OPA) solution was added to the system, and it was allowed to stand in the dark at 25°C for 10 min, and finally 50 μL of 3.0 mol / L hydrochloric acid was added to the system to stop the reaction. The excitation wavelength of the fluorescence intensity detection was 340 nm, the emission wavelength was 455 nm, and the results were measured at the emission wavelength.

[0105] Treatment 2: Same as treatment 1, the only difference is that the first polypeptide solution is replaced by the second polypeptide solution;

[0106] Treatment 3: Same as treatment 1, the only difference is that the first polypeptide solution is replaced with captopril (a commercial antihypertensive drug), and captopril is used as a positive control;

[0107] The calculation formula of ACE inhibition rate (%) is: ACE inhibition rate (%) = [1-(A3-A4) / (A1-A2)] × 100;

[0108] A1 is the control experimental group; A2 is the control blank group; A3 is the sample experimental group, which is the measurement result of treatment 1 or treatment 2. A4 is the sample blank group. The specific addition method of A1 to A4 is shown in Table 2.

[0109] Table 2 Sampling method for determining ACE inhibition rate by OPA method

[0110]

[0111] (2) Determination of the inhibitory activity of synthetic peptides on DPP-IV in vitro

[0112] Determination of the first polypeptide solution: Treatment 4: The first polypeptide solution sample was dissolved in 100mmol / L Tris-HCl (pH 8.0) buffer to obtain synthetic peptide solutions with concentrations of 0.016, 0.08, 0.24, 0.4, and 2.0mg / mL, respectively. 25μL of the synthetic peptide solutions of different concentrations and 25μL of 1.6mmol / L Gly-Pro-pNA were added to the 96-well plate. The plates were allowed to stand at 37°C for 10min, and then 50μL of 0.025U / mL DPP-IV was added and incubated at 37°C for 1h. Finally, 100μL of sodium acetate buffer (1mol / L, pH 4.0) was added to stop the reaction, and the absorbance at 405nm was measured using an enzyme marker.

[0113] Treatment 5: Same as treatment 4, the only difference is that the first polypeptide solution is replaced by the second polypeptide solution;

[0114] Treatment 6: Same as treatment 4, the only difference is that the first peptide solution is replaced with sitagliptin, which serves as a positive control;

[0115] Treatment 7: Same as treatment 4, the only difference is that the first peptide solution is replaced with Tris-HCl;

[0116] Treatment 8: Same as treatment 4, the only difference is that the first polypeptide solution and DPP-IV are replaced with Tris-HCl;

[0117] Treatment 9: Same as treatment 4, except that the first polypeptide solution was not added and Tris-HCl was used instead of DPP-IV.

[0118] The half inhibition rate was calculated according to the DPP-IV activity inhibition rate curve. The DPP-IV inhibition rate was calculated as follows:

[0119] DPP-IV inhibition rate (%) = [1-(cd) / (ab)] × 100;

[0120] Wherein a is the negative control group, which is the measurement result of treatment 10; b is the negative control group, which is the measurement result of treatment 11; c is the polypeptide sample group, which is the result of treatment 7 or treatment 8; d is the blank group, which is the measurement result of treatment 12. When calculating the DPP-IV inhibition rate of the first polypeptide, the polypeptide samples are all the first polypeptide; when calculating the DPP-IV inhibition rate of the second polypeptide, the polypeptide samples are all the second polypeptide.

[0121] The inhibitory activities of the two potential bioactive peptides, the first polypeptide and the second polypeptide, on ACE and DPP-IV were determined in vitro. The results are shown in Table 3.

[0122] According to Table 3, when the concentration was 2 mg / mL, the inhibition rates of the two peptides were both above 50%. Captopril ACE half-inhibitory concentration IC in the positive control group 50 Compared with captopril, the ACE half-inhibitory concentrations of the synthetic peptides DAPLPHPNR and GSEDPLPGAK were 0.08 and 0.61 mg / mL, respectively. These results indicate that both peptides exhibit ACE inhibition.

[0123] As a positive control group, sitagliptin showed strong DPP-IV inhibitory activity (IC 50 =0.33 mg / mL). At a concentration of 2 mg / mL, the inhibition rate of DAPLPHPNR on DPP-IV was 62.13%, and the half inhibition concentration IC 50 The inhibition rate of GSEDPLPGAK on DPP-IV was 56.68%, and the half inhibition concentration was 1.32 mg / mL. At the same time, from the molecular docking results, the binding ability of these two peptides to DPP-IV receptors is generally lower than that to ACE receptors, which is consistent with the results verified in vitro that ACE inhibitory activity is higher than DPP-IV inhibitory activity.

[0124] IC 50 The IC value is calculated by converting the original mass concentration data into a logarithmic form and plotting a curve, and then fitting the curve. 50 The reference for the value calculation method is the existing technology ("Wang Yangduo, Su Yongchang, Wang Xiaoyan, et al. Analysis of ACE inhibitory activity in different parts of sea cucumber and optimization of active peptide preparation process [J]. Food Industry Science and Technology, 2024, 45(10): 187-197.).

[0125] Table 3 Determination of ACE and DPP-IV inhibitory activity of DAPLPHPNR and GSEDPLPGAK in vitro

[0126]

[0127]

[0128] In summary, the present invention provides two shiitake mushroom flavor peptides with ACE and DPP-IV inhibitory effects. Both peptides (DAPLPHPNR and GSEDPLPGAK) show strong binding ability with ACE and DPP-IV receptors, and molecular dynamics simulation further verifies the stability of their receptor complexes. The half inhibition rate IC of DAPLPHPNR and GSEDPLPGAK on ACE 50 The half inhibition rate IC of DPP-IV was 0.08 and 0.61 mg / mL respectively. 50The values ​​were 0.89 and 1.32 mg / mL respectively, with good inhibitory effect.

[0129] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A peptide, characterized in that The peptide comprises a first polypeptide and / or a second polypeptide; The amino acid sequence of the first polypeptide is shown in SEQ ID NO.1; The amino acid sequence of the second polypeptide is shown in SEQ ID NO.

2.

2. Use of the peptide according to claim 1 in the preparation of angiotensin converting enzyme and / or dipeptidyl peptidase IV inhibitors.

3. The use according to claim 2, characterized in that: The active ingredient of the inhibitor includes the peptide according to claim 1; or the only active ingredient of the inhibitor is the peptide according to claim 1.

4. Use of the peptide according to claim 1 in the preparation of at least one of the products described in 1) and 2); 1) Products for the prevention and / or treatment of cardiovascular diseases; 2) Products for the prevention and / or treatment of metabolic diseases.

5. The use according to claim 4, characterized in that: The product types include medicines and / or health products.

6. An angiotensin converting enzyme inhibitor and / or a dipeptidyl peptidase IV inhibitor, characterized in that: The active ingredient of the inhibitor includes the peptide according to claim 1; or the only active ingredient of the inhibitor is the peptide according to claim 1.

7. A product, characterized in that The active ingredient of the product includes the peptide according to claim 1; or the only active ingredient of the product is the peptide according to claim 1; The product is at least one of the following Ⅰ) and Ⅱ); Ⅰ) Products for the prevention and / or treatment of cardiovascular diseases; II) Products for the prevention and / or treatment of metabolic diseases.

8. Use of the peptide according to claim 1 in preparing fresh products.

9. A fresh product, characterized in that: The active ingredient of the fresh-looking product includes the peptide according to claim 1; or the only active ingredient of the fresh-looking product is the peptide according to claim 1.

10. The peptide screening method according to claim 1, characterized in that: include: Use the PeptideRanker tool to score the bioactive peptides to be screened, and obtain active peptides with scores > 0.5; The obtained active peptides were screened by BIOPEP-UWM, and the bioactive peptides with angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities were obtained by molecular docking and molecular dynamics simulation methods; The toxicity, sensitization, water solubility and stability of the bioactive polypeptides were screened by ToxinPred tool, AllerTOP v.2.0 tool, Innovagen tool and Expasy ProtParam tool in sequence to obtain non-toxic, non-allergenic, water-soluble and stable peptides.

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