Needle mushroom sourced ACE and alpha-glucosidase inhibitory peptide and application thereof
Through enoki mushroom enzymatic method combined with molecular docking and artificial intelligence technology, peptides with ACE and α-glucosidase inhibitory activities were screened, solving the problems of cumbersome and low yields in the preparation of biologically active peptides in the prior art, and achieving efficient and rapid preparation and application of active peptides.
Patent Information
- Application Number
- CN202510029010.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-08
- Publication Date
- 2025-04-18
AI Technical Summary
The prior art methods for preparing bioactive peptides from food proteins are cumbersome, consume a lot of resources, and have low yields, making it difficult to efficiently discover and prepare large quantities of bioactive peptides.
Enoki mushroom enzymatic method combined with molecular docking and artificial intelligence technology, and peptides with ACE and α-glucosidase inhibitor treatment were screened through liquid nitrogen treatment, PMSF protease inhibitor treatment, papain and flavor protease combination, followed by ultrafiltration, protein extraction, reduction alkylation and desalination, and finally, peptides with ACE and α-glucosidase inhibitory activities were screened through liquid phase mass spectrometry analysis and De novo polypeptide sequence analysis.
A method of rapid screening and efficient preparation of biologically active peptides has been realized, which reduces the R&D cycle and cost, improves the accuracy and activity of active peptides, and has a wide range of application prospects in blood pressure and blood sugar-lowering products.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of bioactive peptides, and in particular to an ACE and alpha-glucosidase inhibitory peptide derived from Flammulina velutipes and uses thereof. Background Art
[0002] In the fast-paced modern life, the "three highs" problem - high blood pressure, high blood sugar, and high blood lipids - are increasingly becoming invisible killers threatening people's health. There is a close relationship between them and they affect each other. Hypertension is a standard chronic disease and the most critical risk factor for cardiovascular and cerebrovascular diseases. At the same time, hypertension is a cardiovascular disease syndrome, which is usually accompanied by a series of complications, such as myocardial infarction, atherosclerosis, heart failure, etc. At the same time, type 2 diabetes has become a global epidemic, with a significant risk of complications, including neuropathy, retinopathy, cardiovascular disease, kidney disease, etc. In addition, high blood sugar levels can trigger non-enzymatic glycation and oxidative stress of proteins, further accelerating the development of diabetic complications. Hypertensive patients are often accompanied by abnormal lipid metabolism, causing hyperlipidemia; hyperglycemic patients have disordered sugar metabolism, leading to disordered blood lipid metabolism. It can be seen that the three highs affect each other and are symptoms of each other. Solving hypertension and hyperglycemia is expected to reduce hyperlipidemia.
[0003] The pathogenesis of hypertension is related to the renin-angiotensin system (RAS). RAS contains two key enzymes: hypertensinase (renin) and ACE. Therefore, inhibiting the enzymatic activity of renin and ACE is one of the treatments for hypertension. ACE is a glycoprotein, and the substrate of renin is simple. Angiotensinogen is the only known substrate, so it is difficult to find an inhibitor of renin. Therefore, most of the research on antihypertensive peptides focuses on ACE. Angiotensin converting enzyme inhibitors (ACEI) prevent angiotensin I from converting to angiotensin II through ACE, thereby reducing the production of angiotensin II in the circulation and tissues. Angiotensin II is a strong vasoconstrictor that can increase blood pressure. Postprandial blood glucose level increases play a key role in the onset and progression of type II diabetes. As the disease progresses, the function of pancreatic β cells gradually declines, and significantly increased postprandial hyperglycemia and blood glucose fluctuations may further deteriorate β cell function. At the same time, postprandial blood glucose increase can increase platelet reactivity and activate platelets, which not only affects blood sugar control, but also increases the risk of multiple complications. High blood sugar levels trigger non-enzymatic glycation and oxidative stress of proteins, which further accelerate the development of diabetic complications. Inhibiting the activity of α-glucosidase, a key glucosyl hydrolase that catalyzes the last step of carbohydrate digestion in intestinal cells, can delay the release of glucose, thereby reducing postprandial blood sugar levels.
[0004] Common ACE inhibitors such as captopril, benazepril, enalapril, lisinopril, etc., and α-glucosidase inhibitors such as acarbose and voglibose, etc., usually cause a series of side effects, such as cough, headache, weight gain, skin allergies, etc. Therefore, the development of safer drugs is necessary for such a severe situation. Food-derived bioactive peptides have many advantages, such as natural, mild, safe and easy to absorb. At the same time, these peptides often have antioxidant, anti-aging, anti-tumor and other functions. Therefore, food-derived bioactive peptides are becoming more and more popular and have broad prospects for development and application.
[0005] Flammulina velutipes has the effects of nourishing the liver, benefiting the stomach and intestines, and anti-cancer. It has a therapeutic effect on liver disease, gastrointestinal infection, ulcers, tumors and other diseases. "Compendium of Materia Medica" records: Flammulina velutipes is cool in nature, sweet in taste, and enters the spleen and large intestine meridians. Flammulina velutipes is rich in protein, which provides a rich amino acid resource for the preparation of bioactive peptides. At the same time, as a medicinal and edible fungus, its safety has been widely recognized. The prepared bioactive peptides also have high edible safety. Flammulina velutipes itself has the effects of anti-hypertension and cholesterol reduction, which helps to promote cardiovascular health. In addition, they also have anti-aging and antioxidant properties, which can help restore memory and overcome learning defects. In summary, the advantages of Flammulina velutipes in preparing bioactive peptides are its rich nutritional sources, diverse biological activities, high safety, and wide potential for health promotion and medical applications. These advantages make Flammulina velutipes an ideal raw material for the development of bioactive peptides. However, the common methods of preparing bioactive peptides from food proteins in the prior art are cumbersome, consume a lot of manpower and materials, and have low yields. Therefore, it is necessary to find a method for efficiently discovering active peptides to achieve large-scale preparation. Summary of the invention
[0006] In view of the problems existing in the prior art, the present invention provides a method for preparing ACE and α-glucosidase inhibitory peptides derived from Flammulina velutipes and their application. The present invention screened bioactive peptides with hypotensive and hypoglycemic activities by enzymatic hydrolysis of Flammulina velutipes. Molecular docking and artificial intelligence technology have been used to virtually screen peptides based on the structure and function of peptides. Compared with equivalent molecular technologies, the processing time is greatly reduced, thus providing a new strategy for the rapid development and production of natural bioactive peptides.
[0007] The technical solution to be adopted in the present invention is:
[0008] An ACE and α-glucosidase inhibitory peptide derived from Flammulina velutipes, the amino acid sequence of which is Ala-Phe-Asn-Trp-Gln (AFNWQ) as shown in SEQ ID NO.1, Gly-Pro-Val-Val-Tyr-Ala-Phe-Ala-Val-Phe (GPVVYAFAVF) as shown in SEQ ID NO.2, Val-Ala-Leu-Pro-Leu-Pro (VALPLP) as shown in SEQ ID NO.3, Pro-His-Leu-Ala-Pro-Pro-Thr-Ala (PHLAPPTA) as shown in SEQ ID NO.4, and Tyr-Tyr-Glu-Glu-Trp-Trp-Trp (YYEEWWW) as shown in SEQ ID NO.5.
[0009] Furthermore, the molecular weights of the ACE and α-glucosidase inhibitory peptides are both <1 KDa.
[0010] Furthermore, the ACE and α-glucosidase inhibitory peptides are easily soluble in water.
[0011] Furthermore, the ACE and α-glucosidase inhibitory peptides are derived from Flammulina velutipes protein.
[0012] The present invention also discloses the use of the ACE and α-glucosidase inhibitory peptides derived from Flammulina velutipes in preparing products for lowering blood pressure and blood sugar, and products for preventing or treating hypertension and hyperglycemia.
[0013] Furthermore, the product is a medicine or a functional food.
[0014] The present invention also provides a method for preparing the ACE and α-glucosidase inhibitory peptide derived from Flammulina velutipes, which comprises the following steps:
[0015] 1) Treating Flammulina velutipes with liquid nitrogen;
[0016] 2) treating the Flammulina velutipes obtained in step 1) with RIPA lysis buffer containing PMSF protease inhibitor to obtain a Flammulina velutipes polypeptide matrix;
[0017] 3) using papain and flavor protease to enzymatically hydrolyze the Flammulina velutipes polypeptide matrix;
[0018] 4) ultrafiltration of the enzymatic hydrolyzate obtained in step 3) and collection of components to obtain Flammulina velutipes ACE and α-glucosidase inhibitory peptide components;
[0019] 5) extracting protein, reducing alkylating and desalting the Flammulina velutipes ACE and α-glucosidase inhibitory peptide components obtained in step 4);
[0020] 6) The components obtained in step 5) are subjected to liquid phase mass spectrometry analysis, and the original mass spectrometry files are subjected to peptide sequence analysis using a De novo method;
[0021] 7) The components obtained in step 6) are subjected to molecular docking and database analysis to obtain Flammulina velutipes ACE and α-glucosidase inhibitory peptide components.
[0022] Furthermore, in the step 1), the processing time is 5 minutes.
[0023] Furthermore, in the step 2), the final concentration of PMSF protease inhibitor is 1 mM, and the lysis system is substrate: lysis solution (w / v) = 1:10.
[0024] Furthermore, in the step 3), the enzymolysis time is 3 hours, the temperature is 50° C., and the enzyme: substrate = 5000 U / g.
[0025] Furthermore, in step 3), after the enzymatic hydrolysis is completed, the enzyme activity is inactivated at 100° C. for 10 minutes.
[0026] Furthermore, in the step 4), the ultrafiltration is specifically: ultrafiltration is performed on the enzymatic hydrolysate of Flammulina velutipes using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain a component with MW<3 kDa.
[0027] Furthermore, in the step 5), the components obtained in the above step 4) are subjected to protein extraction, reduction alkylation, desalting using a C18 stage-tip, and vacuum drying at 45°C.
[0028] Furthermore, in step 6), the liquid phase mass spectrometry analysis method is: mobile phase A is water containing 0.1% trifluoroacetic acid; mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; flow rate: 300 nL / min; analysis time: 66 min.
[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0030] 1) Compared with the existing technology of preparing active peptides by enzymatic hydrolysis, the present invention combines computer assistance and uses the database in peptide omics for screening, which reduces the screening intensity, reduces the screening cost, shortens the research and development cycle, and can obtain high-precision and high-activity potential bioactive peptides, which can provide a new method for rapid screening of food-borne bioactive peptides;
[0031] 2) AFNWQ, GPVVYAFAVF, VALPLP, PHLAPPTA, and YYEEWWW described in the present invention can bind to the amino acid residues in the active pocket of ACE (PDBID: 1O8A) and Saccharomyces cerevisiae α-D-glucose-binding isomaltase (PDB ID: 3A4A), and the docking results show that they have strong ACE inhibition and α-glucosidase inhibition effects;
[0032] 3) The peptide of the present invention can be prepared in large quantities through solid phase chemical synthesis, and has good application prospects as a health product and medicine for auxiliary treatment of hypertension and hyperglycemia. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 This is a diagram showing the inhibitory effect of the bioactive peptide component prepared in the present invention on ACE and α-glucosidase;
[0034] Figure 2 This is the molecular docking diagram of AFNWQ and 1O8A of the present invention;
[0035] Figure 3 This is the molecular docking diagram of AFNWQ and 3A4A of the present invention;
[0036] Figure 4 This is the molecular docking diagram of GPVVYAFAVF and 1O8A of the present invention;
[0037] Figure 5 This is the molecular docking diagram of GPVVYAFAVF and 3A4A of the present invention;
[0038] Figure 6 This is the molecular docking diagram of VALPLP and 1O8A of the present invention;
[0039] Figure 7 This is the molecular docking diagram of VALPLP and 3A4A of the present invention;
[0040] Figure 8 This is the molecular docking diagram of PHLAPPTA and 1O8A of the present invention;
[0041] Fig. 9 This is the molecular docking diagram of PHLAPPTA and 3A4A of the present invention;
[0042] Fig.10 This is the molecular docking diagram of YYEEWWW and 1O8A of the present invention;
[0043] Fig.11 This is the molecular docking diagram of YYEEWWW and 3A4A of the present invention. DETAILED DESCRIPTION
[0044] The present invention is further described in detail below in conjunction with embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments.
[0045] Example 1: Preparation of bioactive peptides using Flammulina velutipes enzymatic hydrolysis
[0046] The specific preparation steps of the bioactive peptide are as follows:
[0047] Step (1): grinding Flammulina velutipes (selected from commercially available Flammulina velutipes) with liquid nitrogen for 5 minutes to obtain Flammulina velutipes powder.
[0048] Step (2): Take the Flammulina velutipes powder obtained in step (1), add lysis solution containing a final concentration of 1 mM PMSF protease inhibitor according to the ratio of substrate: lysis solution (w / v) = 1:10, repeatedly shake on ice for sufficient lysis for 10 minutes, and then collect by centrifugation to obtain the Flammulina velutipes polypeptide matrix.
[0049] Step (3): The Flammulina velutipes polypeptide matrix obtained in step (2) is subjected to combined enzymolysis using papain and flavor protease at an enzyme:substrate ratio of 5000 U / g, and the enzymolysis time is 3 hours at 50° C. After the enzymolysis is completed, the enzyme activity is inactivated at 100° C. for 10 minutes, and the Flammulina velutipes enzymolysis solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain ACE and α-glucosidase inhibitory peptide components with a MW of Flammulina velutipes <3 kDa.
[0050] Example 2: Preparation of bioactive peptides using Flammulina velutipes hydrolysis method
[0051] The difference from Example 1 is that: in a method for preparing ACE and α-glucosidase inhibitory peptides derived from Flammulina velutipes, no enzyme is added in step 3), and the components are directly collected after hydrolysis.
[0052] Example 3: Sequence Identification of Bioactive Peptides
[0053] The sequencing of bioactive peptides was performed by Beijing Bio-Tech Biotechnology Co., Ltd.
[0054] 1. Pre-experimental treatment: The sample is the component obtained in step (3) of the above Example 1, which is drained at 45 degrees, used for reductive alkylation with DTT and IAM, and then desalted and dried.
[0055] 2. Sequence identification: Liquid phase mass spectrometry analysis method is: mobile phase A is water containing 0.1% trifluoroacetic acid; mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; flow rate: 300 nL / min; analysis time: 66 min.
[0056] The specific chromatographic conditions are:
[0057] Time (min) Phase A Phase B 0 96％ 4％ 2 92％ 8％ 35 72％ 28％ 55 60％ 40％ 56 5％ 95％ 66 5％ 95％
[0058] 3. Sequence analysis: The original mass spectrometry file was analyzed for peptide sequence using the PEAKSDe novo method.
[0059] Example 4: Database prediction and molecular docking screening of bioactive peptides
[0060] 1. Database prediction: Further analysis of shared peptides using BIOPEP
[0061] ( https: / / biochemia.uwm.edu.pl / biopep / peptide_data.php ) The obtained sequences were screened and compared to determine whether potential new bioactive peptides were produced; then Peptide Ranker
[0062] ( http: / / distilldeep.ucd.ie / PeptideRanker / ) program was used to evaluate the bioactivity potential of the screened active peptide fragments and select peptides with high scores; the online software PepCalc ( https: / / pepcalc.com / )'s pI tool and solubility tool to predict peptide isoelectric point and solubility; the software ToxinPred 2
[0063] ( https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php ) predict peptide toxicity.
[0064] 2. Molecular docking screening: Using AutoDock Vina software, PDB ID: 1O8A and PDB ID: 3A4A were used as target proteins for molecular docking with the above-screened peptides, respectively, to screen peptides that can bind tightly to them and analyze the molecular mechanism of interaction.
[0065] 3. The screening results are shown in Table 1:
[0066] Table 1 Physicochemical properties, property prediction and molecular docking scores of peptides
[0067]
[0068] 4. After molecular docking, the above active peptides can form relatively tight connections with 3A4A and 1O8A, among which PHLAPPTA has the tightest connection. The molecular docking results are as follows Figure 2 to Figure 11 shown.
[0069] 5. The peptide sequences shown in Table 1 above have good ACE inhibitory activity and α-glucosidase inhibitory activity, and can be used in the preparation of new antihypertensive and hypoglycemic drugs.
[0070] Example 5: ACE inhibitory activity test and α-glucosidase inhibitory activity test of bioactive peptides
[0071] 1. The above peptides were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (https: / / www.sangon.com / ) for the next step of activity testing.
[0072] 2. ACE inhibitory activity test method:
[0073] Preparation of reagents: 1.0 mmol·L-1 FAPGG (N-[3-(2-furanacryloyl)]-L-phenylpropionyl-glycine-glycine) was dissolved in Tris-HCl (50 mmol·L-1) containing 0.3 mol·L-1 NaCl, the pH was adjusted to 7.5, and placed in a 37°C water bath for preparation of substrate solution; 0.25 U ACE was dissolved in 1 mL of ultrapure water and stored at -20°C for future use.
[0074] Determination of ACE inhibitory activity: Set the microplate reader to 37°C, add reactants to the 96-well plate according to the design in Table 1, and measure the absorbance of the blank wells and sample wells at 340 nm for 30 minutes.
[0075] The ACE inhibitory activity was calculated as follows:
[0076]
[0077] Table 2 ACE inhibitory activity assay
[0078] Sample hole / ul Blank hole / ul 0.25U / MLACE 10 10 1mmol / LFAPGG 150 150 ACE-I (1 mg / mL) 10 0 Buffer 0 10
[0079] 3. α-glucosidase inhibition activity test method:
[0080] Preparation of reagents: α-glucosidase solution is 0.34U / mL, prepare 50× stock solution, dilute to 1× with PBS buffer before use; PNPG solution 5mM; sodium carbonate solution 0.2M; PBS buffer (10×), dilute to 1× before use;
[0081] Determination of α-glucosidase inhibitory activity: Add reactants to a 96-well plate according to the design in Table 2, in the following order: add PBS buffer (pH 6.5), sample solution (1 mg / mL) and α-glucosidase solution, place in an incubator at 37°C for 15 minutes, then add PNPG solution, react at 37°C for 15 minutes, then add sodium carbonate solution to terminate the reaction. Measure the absorbance of the blank well and the sample well at 405 nm.
[0082] The α-glucosidase inhibitory activity was calculated as follows:
[0083]
[0084] Table 3 α-glucosidase inhibitory activity assay
[0085] Addition volume μL Enzyme blank group Enzyme activity group Sample hole Sample background set PBS buffer 140 120 110 130 sample 0 0 10 10 Enzyme solution 20 20 20 20 PNPG 0 20 20 0 Sodium carbonate solution 80 80 80 80
[0086] 4. Inhibitory activity results
[0087] The activity of the synthesized peptides was tested, and the results are shown in Table 4. It was found that some peptide sequences had both ACE inhibition and α-glucosidase inhibition effects, and some peptide sequences had only one activity, which shows that molecular docking can only be an auxiliary means to screen bioactive peptides.
[0088] Table 4 Results of ACE inhibition and α-glucosidase inhibition activities of peptides
[0089] Peptide sequence ACE inhibitory activity α-Glucosidase inhibitory activity QUR 36.76％ 42.94％ GPVVYAFAVF 0 99.47％ VALPLP 87.73％ 31.49％ PHLAPPTA 84.97％ 0 YYEEWWW 89.88％ 0
[0090] In addition, it should be noted that the shapes and names of the parts and components of the specific embodiments described in this specification may be different. Any equivalent or simple changes made based on the structure, features and principles described in the patent concept of the present invention are included in the protection scope of the patent of the present invention. The technicians in the technical field of the present invention can make various modifications or supplements to the specific embodiments described or replace them in a similar manner, as long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.
Claims
1. An ACE and α-glucosidase inhibitory peptide derived from Flammulina velutipes, characterized in that: The amino acid sequences of the ACE and α-glucosidase inhibitory peptides are shown in SEQ OD NO.1, SEQ OD NO.2, SEQ OD NO.3, SEQ OD NO.4 and SEQ OD NO.
5.
2. Use of the ACE and α-glucosidase inhibitory peptide derived from Flammulina velutipes according to claim 1 in the preparation of products for lowering blood pressure and blood sugar, and products for preventing or treating hypertension and hyperglycemia.
3. Use of the ACE and α-glucosidase inhibitory peptide derived from Flammulina velutipes according to claim 2, wherein the product is a medicine or a functional food.
4. The ACE and α-glucosidase inhibitory peptide derived from Flammulina velutipes according to claim 1, characterized in that: The peptide sequence is derived from enzymatically hydrolyzed Flammulina velutipes.
5. The method for preparing an ACE and α-glucosidase inhibitory peptide derived from Flammulina velutipes according to claim 1, characterized in that: The steps include: 1) Treating Flammulina velutipes with liquid nitrogen; 2) treating the Flammulina velutipes obtained in step 1) with RIPA lysis buffer containing PMSF protease inhibitor to obtain a Flammulina velutipes polypeptide matrix; 3) using papain and flavor protease to enzymatically hydrolyze the Flammulina velutipes polypeptide matrix; 4) ultrafiltration of the enzymatic hydrolysate obtained in step 3) and collection of components to obtain Flammulina velutipes ACE and α-glucosidase inhibitory peptides.
6. The method for preparing Flammulina velutipes ACE and α-glucosidase inhibitory peptides derived from Flammulina velutipes according to claim 5, characterized in that: In the step 1), the processing time is 5 minutes.
7. The method for preparing Flammulina velutipes ACE and α-glucosidase inhibitory peptides derived from Flammulina velutipes according to claim 5, characterized in that: In the step 2), the final concentration of PMSF protease inhibitor is 1 mM, and the lysis system is substrate: lysis solution (w / v) = 1:
10.
8. The method for preparing Flammulina velutipes ACE and α-glucosidase inhibitory peptides derived from Flammulina velutipes according to claim 5, characterized in that: In the step 3), the enzymatic hydrolysis time is 3 hours, the temperature is 50° C., and the enzyme: substrate ratio is 5000 U / g. After the enzymatic hydrolysis is completed, the enzyme activity is inactivated at 100° C. for 10 minutes.
9. The method for preparing Flammulina velutipes ACE and α-glucosidase inhibitory peptides derived from Flammulina velutipes according to claim 5, characterized in that: In the step 4), the ultrafiltration is specifically: ultrafiltration is performed on the enzymatic hydrolysate of Flammulina velutipes using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain a component with MW<3 kDa.
10. The method for preparing Flammulina velutipes ACE and α-glucosidase inhibitory peptides derived from Flammulina velutipes according to claim 5, characterized in that: The inhibition rates of the Flammulina velutipes ACE and α-glucosidase inhibitory peptide components are both greater than 90%.