Lentinus edodes peptide prepared based on Maillard reaction and application of lentinus edodes peptide in preparation of products with effects of reducing blood pressure, blood sugar, blood fat and uric acid and antioxidant activity

Abstract

The invention belongs to the technical field of protein peptide preparation, and particularly relates to mushroom peptide prepared based on Maillard reaction and application of the mushroom peptide to preparation of products with the effects of reducing blood pressure, blood sugar, blood fat and uric acid and antioxidant activity. The mushroom peptide disclosed by the invention has the effects of remarkably reducing blood pressure, blood sugar, blood fat and uric acid and resisting oxidation, and can be used as an auxiliary therapeutic agent or functional food for four high diseases such as hypertension, hyperlipidemia, diabetes mellitus and hyperuricemia and oxidative stress related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of protein peptide preparation, and specifically relates to a shiitake mushroom peptide prepared based on the Maillard reaction and its application in preparing products with blood pressure lowering, blood sugar lowering, blood lipid lowering, uric acid lowering and antioxidant activities. Background Art

[0002] With the change of modern lifestyle, metabolic diseases such as hypertension and hyperglycemia and health problems related to oxidative stress are becoming increasingly serious, and the search for natural, safe and effective functional food ingredients has become a research hotspot. As a traditional food ingredient, shiitake mushrooms are rich in a variety of bioactive ingredients, but their direct consumption has limited effects on lowering blood pressure, lowering blood sugar, lowering lipids, lowering uric acid and anti-oxidation.

[0003] In recent years, with the pursuit of food quality and health, the application of mushroom flavor substances has also received extensive attention and research. The flavor substances of mushrooms can be used in food additives, seasonings, functional foods and medicines. Especially in terms of functional foods and medicines, the flavor substances of mushrooms can be used to develop functional foods, such as functional foods with antioxidant and anti-aging functions, and can also be used to develop medicines, such as anti-tumor medicines.

[0004] There are a lot of peptide substances in shiitake mushrooms, and there are also some methods for extracting shiitake peptides from shiitake mushrooms in existing research. For example, some studies have shown that shiitake peptides extracted from shiitake mushrooms have the effect of reducing salt and increasing freshness; some studies have shown that shiitake peptides extracted from shiitake mushrooms have anti-tumor effects. However, shiitake peptides obtained with different peptide sequences and different preparation processes have different uses. At present, there is no shiitake peptide in the art that has the functions of lowering blood pressure, lowering blood sugar, lowering blood lipids, lowering uric acid and anti-oxidation. Summary of the invention

[0005] The purpose of the present invention is to provide a shiitake mushroom peptide prepared based on the Maillard reaction and its application in the preparation of products with blood pressure lowering, blood sugar lowering, blood lipid lowering, uric acid lowering and antioxidant activities. The shiitake mushroom peptide has significant blood pressure lowering, blood sugar lowering, blood lipid lowering, uric acid lowering and antioxidant activities, which provides possibilities for its use in the field of health care products and medicine preparation.

[0006] The present invention provides a shiitake peptide, which comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and / or an amino acid sequence as shown in SEQ ID NO.2.

[0007] The present invention also provides a method for preparing the shiitake mushroom peptide described in the above technical solution, comprising the following steps:

[0008] Mixing dried shiitake mushrooms, water and flavor protease for a first enzymolysis to obtain a first enzymolysis solution;

[0009] The first enzymatic hydrolysis solution is mixed with trypsin to perform a second enzymatic hydrolysis to obtain a second enzymatic hydrolysis solution;

[0010] The second enzymatic hydrolysate is subjected to solid-liquid separation, and the supernatant is taken to obtain a third enzymatic hydrolysate;

[0011] The third enzymatic hydrolysate, fructose and cysteine ​​are mixed to perform Maillard reaction to obtain a shiitake mushroom base material;

[0012] Desalting the shiitake mushroom base material to obtain a solution containing shiitake mushroom polypeptides;

[0013] The solution containing the shiitake mushroom polypeptide is subjected to liquid chromatography tandem mass spectrometry analysis to obtain the shiitake mushroom polypeptide.

[0014] Preferably, the desalination step comprises:

[0015] Dissolving the mushroom base material with a trifluoroacetic acid solution having a volume concentration of 0.1% to obtain a mushroom base material dissolved solution;

[0016] The mushroom base material solution is passed through C 18 Elution is performed using a microchromatographic column, and the eluate is collected to obtain a solution containing Lentinus edodes polypeptides;

[0017] The elution buffer for elution is an aqueous solution containing 60 vol% acetonitrile and 0.1 vol% trifluoroacetic acid.

[0018] Preferably, the step of liquid chromatography tandem mass spectrometry analysis comprises:

[0019] The solution containing shiitake mushroom polypeptide is vacuum dried to obtain shiitake mushroom polypeptide powder;

[0020] Dissolving the mushroom powder in an aqueous solution containing 0.1 vol% formic acid and 5 vol% acetonitrile, and performing solid-liquid separation on the obtained dissolved product to obtain a supernatant;

[0021] The supernatant is subjected to liquid chromatography tandem mass spectrometry detection to obtain the shiitake peptide;

[0022] The mobile phase for liquid chromatography tandem mass spectrometry detection includes mobile phase A and mobile phase B, wherein the mobile phase A is formic acid with a volume concentration of 0.1 vol%, and the mobile phase B is an aqueous solution containing 0.1 vol% formic acid and 80 vol% acetonitrile.

[0023] Preferably, the mass ratio of the dried shiitake mushroom to the volume of water is 1 g:30 mL; the amount of the flavor protease is 1000 U / g dried shiitake mushroom;

[0024] The temperature of the first enzymatic hydrolysis is 50° C., the time is 45 min, and the pH value is 7.0.

[0025] Preferably, the dosage of the trypsin is 2000 U / g dried shiitake mushrooms; the temperature of the second enzymatic hydrolysis is 37° C., the time is 45 min, and the pH value is 8.0.

[0026] Preferably, the mass ratio of the fructose to the volume of the third enzymatic hydrolysis solution is 2.5 g:100 mL; the mass ratio of the cysteine ​​to the volume of the third enzymatic hydrolysis solution is 1.5 g:100 mL.

[0027] Preferably, the Maillard reaction is an oil bath reaction, the temperature of the oil bath reaction is 109° C., the time is 68 min, and the pH value is 7.0.

[0028] The present invention also provides the use of the shiitake peptide described in the above technical solution or the shiitake peptide prepared by the preparation method described in the above technical solution in the preparation of health products and / or medicines.

[0029] The present invention also provides the use of the shiitake peptide described in the above technical scheme or the shiitake peptide prepared by the preparation method described in the above technical scheme in the preparation of products having one or more functions of lowering blood pressure, lowering blood sugar, lowering blood lipids, lowering uric acid and antioxidant activity.

[0030] Beneficial effects:

[0031] The present invention provides a shiitake peptide, which comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and / or an amino acid sequence as shown in SEQ ID NO.2. The shiitake peptide of the present invention has significant effects of lowering blood pressure, lowering blood sugar, lowering blood lipids, lowering uric acid and anti-oxidation, and can be used as an auxiliary therapeutic agent or functional food for high blood pressure, high blood lipids, diabetes and high uric acid and diseases related to oxidative stress.

[0032] On this basis, the present invention also provides a method for preparing the shiitake mushroom peptide described in the above technical solution, comprising the following steps: mixing dried shiitake mushrooms, water and flavor protease for a first enzymolysis to obtain a first enzymolysis solution; mixing the first enzymolysis solution with trypsin for a second enzymolysis to obtain a second enzymolysis solution; performing solid-liquid separation on the second enzymolysis solution, taking the supernatant to obtain a third enzymolysis solution; mixing the third enzymolysis solution, fructose and cysteine ​​for Maillard reaction to obtain a shiitake mushroom base material; desalting the shiitake mushroom base material and performing liquid chromatography tandem mass spectrometry analysis to obtain the shiitake mushroom peptide. The present invention performs enzymolysis on shiitake mushrooms to obtain protein hydrolysates, and on this basis, modifies the protein hydrolysate molecules in combination with the Maillard reaction, so that the protein hydrolysate molecules are rearranged to produce new small molecular substances, effectively improving the biological activity of the protein hydrolysate (i.e., shiitake mushroom peptides), and obtaining the shiitake mushroom peptides with blood pressure lowering, blood sugar lowering, blood lipid lowering, uric acid lowering and anti-oxidation effects described in the present invention. In addition, the preparation process of the shiitake mushroom peptide is green and environmentally friendly, and no harmful by-products are produced, which meets the requirements of modern biopharmaceuticals and functional food production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0034] Figure 1 The structure diagram of the Lentinus edodes polypeptide having the amino acid sequence of APFDCKE in Example 1;

[0035] Figure 2 The structure diagram of the shiitake mushroom polypeptide having the amino acid sequence IPSKPMC in Example 1;

[0036] Figure 3 This is the docking diagram of the shiitake peptide APFDCKE and the ACE enzyme molecule in Example 1;

[0037] Figure 4 This is the docking diagram of the shiitake peptide IPSKPMC and the ACE enzyme molecule in Example 1;

[0038] Figure 5 This is the molecular docking diagram of Lentinus edodes peptide APFDCKE and α-glucosidase in Example 1;

[0039] Figure 6 This is the molecular docking diagram of Lentinus edodes peptide IPSKPMC and α-glucosidase in Example 1;

[0040] Figure 7 This is the molecular docking diagram of Lentinus edodes peptide APFDCKE and pancreatic lipase in Example 1;

[0041] Figure 8 This is the molecular docking diagram of Lentinus edodes peptide IPSKPMC and pancreatic lipase in Example 1;

[0042] Fig. 9 This is the molecular docking diagram of Lentinus edodes peptide APFDCKE and xanthine oxidase in Example 1;

[0043] Fig.10 This is the molecular docking diagram of Lentinus edodes peptide IPSKPMC and xanthine oxidase in Example 1;

[0044] Fig.11 This is the molecular docking diagram of Lentinus edodes peptide APFDCKE and Keap1 protein in Example 1;

[0045] Fig.12 This is the docking diagram of the shiitake peptide IPSKPMC and Keap1 protein molecules in Example 1. DETAILED DESCRIPTION

[0046] The present invention provides a shiitake peptide, which comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and / or an amino acid sequence as shown in SEQ ID NO.2.

[0047] In the present invention, as an embodiment, the shiitake peptide can be a polypeptide with an amino acid sequence such as SEQ ID NO. 1 or an amino acid sequence such as SEQ ID NO. 2. The amino acid sequence of SEQ ID NO. 1 of the present invention is APFDCKE, and the amino acid sequence of SEQ ID NO. 2 is IPSKPMC.

[0048] The present invention also provides a method for preparing the shiitake mushroom peptide described in the above technical solution, comprising the following steps:

[0049] Mixing dried shiitake mushrooms, water and flavor protease for a first enzymolysis to obtain a first enzymolysis solution;

[0050] The first enzymatic hydrolysis solution is mixed with trypsin to perform a second enzymatic hydrolysis to obtain a second enzymatic hydrolysis solution;

[0051] The second enzymatic hydrolysate is subjected to solid-liquid separation, and the supernatant is taken to obtain a third enzymatic hydrolysate;

[0052] The third enzymatic hydrolysate, fructose and cysteine ​​are mixed to perform Maillard reaction to obtain a shiitake mushroom base material;

[0053] Desalting the shiitake mushroom base material to obtain a solution containing shiitake mushroom polypeptides;

[0054] The solution containing the shiitake mushroom polypeptide is subjected to liquid chromatography tandem mass spectrometry analysis to obtain the shiitake mushroom polypeptide.

[0055] The present invention mixes dried shiitake mushrooms, water and flavor protease for a first enzymatic hydrolysis to obtain a first enzymatic hydrolysis solution. In the present invention, as an embodiment, the dried shiitake mushrooms can be shiitake mushroom powder, and the particle size of the shiitake mushroom powder is less than 100 mesh. As an embodiment, the volume ratio of the mass of the dried shiitake mushrooms and water can be 1g:30mL. As an embodiment, the dosage of the flavor protease of the present invention can be 1000U / g dried shiitake mushrooms. As an embodiment, the temperature of the first enzymatic hydrolysis of the present invention can be 50°C; the time of the first enzymatic hydrolysis can be 45min; the pH value of the first enzymatic hydrolysis can be 7.0.

[0056] After obtaining the first enzymatic hydrolysis solution, the present invention mixes the first enzymatic hydrolysis solution with trypsin for second enzymatic hydrolysis to obtain a second enzymatic hydrolysis solution. As an embodiment, the amount of trypsin used in the present invention can be 2000U / g dried shiitake mushrooms. As an embodiment, the temperature of the second enzymatic hydrolysis of the present invention can be 37°C; the time of the second enzymatic hydrolysis can be 45min; and the pH value of the second enzymatic hydrolysis can be 8.0.

[0057] After obtaining the second enzymatic hydrolysate, as an embodiment, the present invention performs enzyme inactivation treatment on the second enzymatic hydrolysate to obtain an enzyme inactivation treated enzymatic hydrolysate. As an embodiment, the enzyme inactivation treatment may be performed by heating the second enzymatic hydrolysate in a 100° C. water bath for 10 minutes.

[0058] After obtaining the enzyme-inactivating hydrolyzate, the present invention performs solid-liquid separation on the enzyme-inactivating hydrolyzate, takes the supernatant, and obtains a third enzyme hydrolyzate. As an embodiment, the solid-liquid separation method of the present invention can be centrifugation. As an embodiment, the centrifugation speed can be 8000 rpm, and the time can be 20 min.

[0059] After obtaining the third enzymolysis solution, the present invention mixes the third enzymolysis solution, fructose and cysteine ​​to perform a Maillard reaction to obtain a Maillard reaction solution. As an embodiment, the mass ratio of fructose to the volume of the enzymolysis solution of the present invention can be 2.5g:100mL. As an embodiment, the mass ratio of cysteine ​​to the volume of the enzymolysis solution of the present invention can be 1.5g:100mL. As an embodiment, the Maillard reaction of the present invention can be an oil bath reaction; as another embodiment, the temperature of the oil bath reaction can be 109°C; the time of the oil bath reaction can be 68min; the pH value of the oil bath reaction is 7.0.

[0060] After obtaining the Maillard reaction solution, as an embodiment, the present invention can freeze-dry the Maillard reaction solution to obtain the shiitake mushroom base material. As an embodiment, the freeze-drying temperature of the present invention can be -70°C; and the freeze-drying time can be 48 hours.

[0061] After obtaining the shiitake mushroom base material, the present invention performs a desalting treatment on the shiitake mushroom base material. As an embodiment, the desalting treatment step of the present invention comprises: dissolving the shiitake mushroom base material with a trifluoroacetic acid solution having a volume concentration of 0.1% to obtain a shiitake mushroom base material solution; 18The micro-chromatographic column is used for elution, and the eluate is collected to obtain a solution containing shiitake mushroom polypeptides; the elution buffer for elution is an aqueous solution containing 60 vol% acetonitrile and 0.1 vol% trifluoroacetic acid. As an embodiment, the mass ratio of the shiitake mushroom base material to the volume ratio of the shiitake mushroom base material can be 1 mg: 3 μL. As an embodiment, the C 18 The microchromatography column can be ZipTip C 18 Microchromatography columns.

[0062] After obtaining the solution containing shiitake mushroom polypeptides, the present invention performs liquid chromatography tandem mass spectrometry analysis on the solution containing shiitake mushroom polypeptides to obtain the shiitake mushroom peptides. As an embodiment, the steps of liquid chromatography tandem mass spectrometry analysis of the present invention include: vacuum drying the solution containing shiitake mushroom polypeptides to obtain shiitake mushroom peptide powder; dissolving the shiitake mushroom powder with an aqueous solution containing 0.1 vol% formic acid and 5 vol% acetonitrile, and performing solid-liquid separation on the obtained dissolved substance to obtain a supernatant; performing liquid chromatography tandem mass spectrometry detection on the supernatant to obtain the shiitake mushroom peptides; the mobile phase of the liquid chromatography tandem mass spectrometry detection includes mobile phase A and mobile phase B, the mobile phase A is formic acid with a volume concentration of 0.1 vol%, and the mobile phase B is an aqueous solution containing 0.1 vol% formic acid and 80 vol% acetonitrile.

[0063] As an embodiment, the solid-liquid separation method of the present invention can be centrifugation; as another embodiment, the centrifugal speed can be 13500 rpm; the centrifugal temperature can be 4°C; and the centrifugal time can be 20 min.

[0064] As an embodiment, the liquid phase setting parameters for liquid chromatography tandem mass spectrometry detection of the present invention are shown in the following table.

[0065]

[0066] As an embodiment, the mass spectrometry setting parameters of the liquid chromatography tandem mass spectrometry detection of the present invention are shown in the following table.

[0067]

[0068] The present invention also provides the use of the shiitake peptide described in the above technical solution or the shiitake peptide prepared by the preparation method described in the above technical solution in the preparation of health products and / or medicines. As an embodiment, the application includes the use of the shiitake peptide described in the above technical solution or the shiitake peptide prepared by the preparation method described in the above technical solution in health products and medicines.

[0069] The present invention also provides the use of the shiitake peptide described in the above technical solution or the shiitake peptide prepared by the preparation method described in the above technical solution in the preparation of products having one or more functions of lowering blood pressure, lowering blood sugar, lowering blood lipids, lowering uric acid and antioxidant activity. As an embodiment, the shiitake peptide described in the present invention or the shiitake peptide prepared by the preparation method described in the above technical solution is used in the preparation of products having the functions of lowering blood pressure, lowering blood sugar, lowering blood lipids, lowering uric acid and antioxidant activity. As an embodiment, the product described in the present invention can be a health product and / or a medicine; as another embodiment, when the product is a medicine, the shiitake peptide or the shiitake peptide prepared by the preparation method described in the above technical solution can be used to prepare medicines having one or more functions of lowering blood pressure, lowering blood sugar, lowering blood lipids, lowering uric acid and antioxidant activity; when the product is a health product, the shiitake peptide or the shiitake peptide prepared by the preparation method described in the above technical solution can be used to prepare a health product having one or more functions of lowering blood pressure, lowering blood sugar, lowering blood lipids and antioxidant activity. As an embodiment, the shiitake peptide described in the present invention can be used as the only active ingredient or one of multiple active ingredients in the product.

[0070] 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.

[0071] Example 1

[0072] A method for preparing shiitake mushroom peptide, comprising the following steps:

[0073] (1) Raw material preparation: Dried shiitake mushrooms are selected as raw materials, and pulverized and sieved to obtain shiitake mushroom powder with a size of less than 100 mesh.

[0074] (2) Maillard reaction: Shiitake mushroom powder and water were mixed at a material-liquid ratio of 1 g:30 mL, and 1000 U / g flavor protease (enzyme activity / dry weight of shiitake mushroom powder, the enzyme activity of flavor protease was 250.NFU / mg, purchased from Beijing Solebow Technology Co., Ltd.) was first added to the mixture, and enzymatic hydrolysis was performed at 50°C and pH 7.0 for 45 min, and then 2000 U / g trypsin (enzyme activity / dry weight of shiitake mushroom powder, the enzyme activity of trypsin was 30000 U / g, purchased from Beijing Solebow Technology Co., Ltd.) was added, and enzymatic hydrolysis was performed at 37°C and pH 8.0 for 45 min. The obtained enzymatic hydrolyzate was inactivated in a 100°C water bath for 10 min, centrifuged at 8000 rpm for 20 min, and the supernatant was collected.

[0075] 2.5% fructose (mass volume ratio g / mL) and 1.5% cysteine ​​(mass volume ratio g / mL) were added to the supernatant, the pH was adjusted to 7.0, and the Maillard reaction was carried out at an oil bath reaction temperature of 109°C for 68 minutes. After the Maillard reaction, the reaction solution was freeze-dried at -70°C for 48 hours to obtain a base material prepared by the Maillard reaction of Lentinus edodes.

[0076] (3) Identification of Shiitake Mushroom Peptides: Mass spectrometry was used to identify peptide molecules in Shiitake Mushroom Maillard reaction base materials. ZipTip C 18 The base material was pretreated by desalting using a microchromatographic column (Merck-Millipore, Shanghai Anpu Laboratory Technology Co., Ltd.). The desalting method was as follows: 10 mg of the base material prepared by the Maillard reaction of Lentinus edodes was accurately weighed, and 30 μL of 0.1% (v / v) trifluoroacetic acid (TFA) was added to dissolve it to obtain a Lentinus edodes Maillard reaction base material solution;

[0077] The chromatography column was rinsed 10 times with 50 μL of a solution containing 60% (v / v) acetonitrile (ACN) and 0.1% TFA in pure water; the chromatography column was rinsed 10 times with 10 μL of 0.1% TFA; the base material solution prepared by the Maillard reaction of shiitake mushrooms was sucked and discharged through the chromatography column 20 times; the chromatography column was rinsed 5 times with 10 μL of 0.1% TFA; the chromatography column was eluted with 10 μL of a solution containing 60% ACN and 0.1% TFA in pure water, the eluate was collected, and the eluate was transferred to a polypropylene centrifuge tube and vacuum dried to obtain shiitake mushroom peptides.

[0078] 20 μL of dissolving solution (containing 0.1% (v / v) formic acid and 5% (v / v) ACN pure water) was used to dissolve the shiitake peptides, vortexed, centrifuged at 13500rpm and 4°C for 20 minutes, and the supernatant was collected and transferred to the sample tube, with an injection volume of 8 μL for LC-MS / MS sequence analysis. The liquid chromatography mobile phase A was 0.1% formic acid, and the mobile phase B was a solution containing 0.1% formic acid and 80% (v / v) ACN pure water; the LC-MS / MS setting parameters are shown in Table 1.

[0079] Table 1 LC-MS / MS setting parameters

[0080]

[0081] (4) Screening of Lentinus edodes peptides: After LC-MS / MS identification, the database provided by PEAKS software was used to search the identification sequence database. Lentinus edodes peptides APFDCKE (SEQ ID NO.1) and IPSKPMC (SEQ ID NO.2) with high mass spectrum abundance were obtained. The structural diagrams of Lentinus edodes peptides APFDCKE and IPSKPMC are shown in the following table. Figures 1-2 shown.

[0082] (5) Molecular docking and functional activity analysis of shiitake mushroom peptides

[0083] The renin-angiotensin system is an important system for regulating blood pressure and electrolyte balance. When blood pressure decreases, the kidney releases renin (an aspartic protease) into the circulation, catalyzing the conversion of angiotensinogen into angiotensin I (Ang I); Ang I is a substrate of angiotensin converting enzyme (ACE), which converts it into biologically active angiotensin II (Ang II) by cutting off two amino acid fragments (His-Leu) at the free C-terminus of Ang I; Ang II has a strong pressor effect, which binds to the membrane-bound angiotensin II receptor and stimulates the adrenal cortex to secrete aldosterone, leading to sodium retention and vasoconstriction, and increasing blood pressure. ACE inhibitory peptides bind to the active site of ACE, blocking the catalytic effect of ACE on Ang I and reducing the generation of Ang II. At the same time, ACE inhibitory peptides may also reduce the hydrolysis of bradykinin, a peptide with vasodilatory effects, and its accumulation also helps to lower blood pressure. That is, ACE inhibitory peptides exert their hypotensive effects by inhibiting the activity of ACE, reducing the generation of Ang II and the hydrolysis of bradykinin.

[0084] α-Glucosidase is a key enzyme for carbohydrate digestion in the body. It is mainly located on the brush border of the small intestinal mucosal cells and plays an important role in the catabolism of carbohydrates in the body. It breaks down carbohydrates such as starch, sucrose, and maltose into monosaccharides (such as glucose), which can be absorbed by the small intestinal epithelial cells into the blood circulation, thereby causing postprandial blood sugar to rise. The structure of α-glucosidase inhibitory peptides is similar to oligosaccharides, glucose, or has an amino sugar structure, which can reversibly compete for the binding sites of carbohydrates and α-glucosidase. This competitive inhibition makes it impossible for α-glucosidase to effectively break down carbohydrates into monosaccharides, thereby delaying the absorption of glucose, and the increase in blood sugar becomes slow and stable, avoiding sharp fluctuations in blood sugar. The hypoglycemic effect of α-glucosidase inhibitors does not depend on insulin, and is suitable for Oriental diabetic people whose dietary structure is biased towards carbohydrates, and can be used for early intervention in patients with prediabetes. That is, α-glucosidase inhibitory peptides delay the decomposition of carbohydrates and the absorption of glucose by competitively inhibiting the activity of α-glucosidase, thereby exerting a hypoglycemic effect.

[0085] Pancreatic lipase is a proteolytic enzyme secreted by the pancreas. It mainly acts on the hydrolysis of dietary fat, especially triglycerides, breaking them down into glycerol and fatty acids. Pancreatic lipase inhibitory peptides are a class of polypeptides that can inhibit the activity of pancreatic lipase. After binding to pancreatic lipase receptor protein, pancreatic lipase inhibitory peptides may directly block the active center of pancreatic lipase, inhibit its hydrolysis of triglycerides and other fat substrates, reduce the decomposition and absorption of fat in the small intestine, and lower blood lipid levels.

[0086] Xanthine oxidase is a key enzyme in purine metabolism in the body, which can catalyze the conversion of hypoxanthine and xanthine into uric acid. When the activity of xanthine oxidase is too high, it will lead to excessive production of uric acid, which may cause diseases such as hyperuricemia and gout. Xanthine oxidase inhibitory peptides can specifically bind to xanthine oxidase, occupy its active site, prevent the binding of substrates (hypoxanthine and xanthine) to the enzyme, and inhibit the production of uric acid. At the same time, xanthine oxidase inhibitory peptides bind to xanthine oxidase, which can change the conformation of the enzyme, reduce its catalytic activity, and reduce the production of uric acid.

[0087] The Keap1-Nrf2 / ARE signaling pathway is one of the most important antioxidant signaling pathways in the body. Keap1, as a cytoplasmic protein, can bind to the transcription factor Nrf2 to form a Keap1-Nrf2 complex, thereby inhibiting the activity of Nrf2. Peptide molecules with antioxidant activity can bind to the Keap1 receptor protein, thereby interfering with the interaction between Keap1 and Nrf2, making the Keap1-Nrf2 complex unstable and releasing Nrf2. After being activated, Nrf2 can enter the cell nucleus and induce the expression of the antioxidant stress element (ARE) gene, thereby enhancing the antioxidant capacity of the cell. That is, antioxidant peptides activate the Keap1-Nrf2 / ARE signaling pathway by binding to the Keap1 receptor protein and exerting an antioxidant effect.

[0088] In the screening of active substances, molecular docking technology uses the specific binding between "receptor-ligand" to quickly screen out active small molecules that bind to specific targets, overcoming the shortcomings of traditional active ingredient screening methods such as long cycle, large workload, and low efficiency. By predicting the interactions between different small molecules and biological macromolecules, it reveals new drug action mechanisms and pathways, providing strong support for modern drug research, and is of great significance for clarifying the material basis of the efficacy of natural active substances.

[0089] The crystal structures of angiotensin converting enzyme (PDB: 1O86), α-glucosidase (PDB: 5NN8), pancreatic lipase (PDB: 1LPB), xanthine oxidase (PDB: 3NVZ), and Keap1 protein (PDB: 4L7B) were downloaded from the RCSB database (https: / / www.rcsb.org / ). MOE 2019 molecular docking software was used to optimize the crystal structure of the receptor protein, remove water molecules, and complete hydrogen atoms. MOE software constructed the 3D structure of shiitake peptide and performed molecular energy minimization. The Site Finder module of MOE was used to determine the active sites of amino acid residues of the receptor protein. The docking score, number of bonds, and binding bond energy of shiitake peptide binding to receptor protein were used as screening indicators to select the tightly bound complex of shiitake peptide and receptor protein. The binding sites and action modes between the peptide molecule and the receptor protein in the complex were analyzed by MOE software.

[0090] (6) Analysis results of inhibition of angiotensin converting enzyme (PDB: 1086)

[0091] The amino acid sites that can bind to the ACE receptor protein cavity pocket are composed of 133 amino acid residues, including the three active pockets S1 (ALA354, GLU384 and TYR523), S2 (GLN281, HIS353, HIS513, LYS511 and TYR520) and S1' (GLU162) reported in the literature and the divalent zinc ion ZN 2+ The HEXXH structure composed of zinc ion, HIS383, HIS387 and GLU411 is also the active center of ACE.

[0092] There are 6 hydrogen bonds, 1 metal bond and 5 ionic bonds between the shiitake peptide APFDCKE and the ACE enzyme molecule, and the bond energy is -63.4kcal / mol (the lower the bond energy, the higher the intermolecular bonding strength). 2+ The binding force with a binding energy of -24 kcal / mol was formed, destroying the binding bonds between zinc ions and GLU411, HIS387, and HIS383; the cysteine ​​residues bound by the Maillard reaction of the peptide molecule contributed a strong binding force to the ACE enzyme, with a binding energy of -27.8 kcal / mol. In summary, the peptide molecule binding to the ACE enzyme significantly inhibited the activity of the ACE enzyme ( Figure 3 , Table 2).

[0093] Table 2 Docking diagram of shiitake peptide APFDCKE and ACE enzyme molecule

[0094]

[0095]

[0096] There are 9 interaction bonds between the shiitake peptide IPSKPMC and the ACE enzyme molecule, including 5 hydrogen bonds, 1 metal bond and 3 ionic bonds, with a bond energy of -41.4 kcal / mol. 2+ The binding force with a binding energy of -21.6 kcal / mol was formed, destroying the binding bonds between zinc ions and GLU411, HIS387, and HIS383; the cysteine ​​residue bound by the Maillard reaction of the peptide molecule simultaneously bound the key active amino acid residue (ALA354) of the ACE enzyme and the zinc ion, with a binding energy of -22.1 kcal / mol. In summary, the peptide molecule binding to the ACE enzyme significantly inhibited the activity of the ACE enzyme ( Figure 4 , Table 3).

[0097] Table 3 Docking diagram of shiitake peptide IPSKPMC and ACE enzyme molecule

[0098]

[0099] (7) Analysis results of inhibition of α-glucosidase (PDB: 5NN8)

[0100] The MOE software retains the A-H molecular chain of α-glucosidase, and the docking results of the two shiitake peptides and α-glucosidase molecules are as follows.

[0101] Lentinus edodes peptide APFDCKE forms 6 hydrogen bonds and 7 ionic bonds with the A chain residues of α-glucosidase, with a bond energy of -59 kcal / mol. The cysteine ​​residues bound by the Maillard reaction of the peptide molecule contribute a strong binding force to the α-glucosidase, forming 5 bonds with a bond energy of -33.2 kcal / mol ( Figure 5 , Table 4).

[0102] Table 4 Docking results of Lentinus edodes peptide APFDCKE and α-glucosidase molecules

[0103]

[0104]

[0105] The shiitake peptide IPSKPMC formed 6 hydrogen bonds, 6 ionic bonds and 1 H-π interaction with the A chain residues of α-glucosidase, with a bond energy of -52.3 kcal / mol. The cysteine ​​residues bound by the Maillard reaction of the peptide molecule contributed a strong binding force to the α-glucosidase, forming 8 bonds with a bond energy of -26 kcal / mol ( Figure 6 , Table 5).

[0106] Table 5 Docking results of Lentinus edodes peptide IPSKPMC and α-glucosidase molecule

[0107]

[0108] In summary, the two shiitake mushroom peptides exert their inhibitory effect on α-glucosidase by forming multiple binding bonds with low binding energy.

[0109] (8) Combined with pancreatic lipase (PDB: 1LPB) lipid-lowering analysis results

[0110] The MOE software retains the A and B molecular chains of pancreatic lipase, including COLIPASE (pancreatic lipase coenzyme A, A chain) and LIPASE (lipase, B chain). The docking results of the two shiitake mushroom peptides with pancreatic lipase molecules are as follows.

[0111] The shiitake peptide APFDCKE interacted with the amino acid residues in the A and B molecular chains of pancreatic lipase by forming 6 hydrogen bonds and 4 ionic bonds, with a bond energy of -44.5 kcal / mol; the binding strength between the peptide molecule and the amino acid residues in the A chain of pancreatic lipase was higher than that between the peptide molecule and the B chain, and the binding energy between the peptide molecule and the A chain amino acid residues was twice that of the B chain amino acid residues; the cysteine ​​residues bound by the Maillard reaction of the peptide molecule formed 3 bonds with the amino acid residues in the B chain of pancreatic lipase ( Figure 7 , Table 6).

[0112] Table 6 Docking results of Lentinus edodes peptide APFDCKE and pancreatic lipase

[0113]

[0114] The shiitake peptide IPSKPMC interacted with the amino acid residues in the A and B molecular chains of pancreatic lipase by forming 4 hydrogen bonds and 6 ionic bonds, with a bond energy of -50.4 kcal / mol; the peptide molecule had a similar binding strength with the amino acid residues in the A and B molecular chains of pancreatic lipase, and the bond energy was not much different; the cysteine ​​residues bound by the Maillard reaction of the peptide molecule formed 6 bonds with the amino acid residues in the B chain of pancreatic lipase, with a bond energy of -26.7 kcal / mol ( Figure 8 , Table 7).

[0115] Table 7 Docking results of Lentinus edodes peptide IPSKPMC and pancreatic lipase

[0116]

[0117] The two shiitake mushroom peptides bind to the residues in the A and B molecular chains of pancreatic lipase in a complementary manner, and may have the characteristics of synergistic inhibition of pancreatic lipase activity; and the multiple low-energy bonds formed by the cysteine ​​residues and the amino acid residues in the B chain of pancreatic lipase contribute an important role in inhibiting the activity of pancreatic lipase and thus exerting a lipid-lowering effect.

[0118] (8) Combined with xanthine oxidase (PDB: 3NVZ) uric acid lowering analysis results

[0119] The MOE software retains the A to F molecular chains of xanthine oxidase, including xanthine dehydrogenase / oxidase. The docking results of the two shiitake mushroom peptides and xanthine oxidase molecules are as follows.

[0120] The number of bonds between the shiitake peptide APFDCKE and the amino acid residues in the E and F chains of xanthine oxidase is the same (8 each), including 7 hydrogen bonds, 7 ionic bonds and 2 H-π / π-H interactions, with a bond energy of -57.1 kcal / mol; the binding strength of the amino acid residues in the F chain of xanthine oxidase is higher than that of the peptide molecule and the amino acid residues in the E chain of xanthine oxidase; and the cysteine ​​residues bound by the Maillard reaction of the peptide molecule form 4 bonds with the amino acid residues in the F chain of pancreatic lipase ( Fig. 9 , Table 8).

[0121] Table 8 Docking results of Lentinus edodes peptide APFDCKE and xanthine oxidase

[0122]

[0123] The shiitake peptide IPSKPMC formed 6 hydrogen bonds, 3 ionic bonds and 1 H-π interaction with the amino acid residues in the E and F chains of xanthine oxidase, with a binding energy of -32.2 kcal / mol; among them, the cysteine ​​residues bound by the Maillard reaction of the peptide molecule contributed more to the binding of xanthine oxidase, forming 6 binding bonds with a binding energy of -16.2 kcal / mol ( Fig.10 , Table 9).

[0124] Table 9 Docking results of Lentinus edodes peptide IPSKPMC and xanthine oxidase

[0125]

[0126] In summary, the two shiitake mushroom peptides bind to the amino acid residues in multiple molecular chains of xanthine oxidase to form low-bond energy and large-quantity interaction forces, thereby exerting the uric acid-lowering effect of the peptide molecules by inhibiting xanthine oxidase.

[0127] (9) Antioxidant analysis results of Keap1 protein (PDB: 4L7B)

[0128] The MOE software retains the A and B molecular chains of Keap1 protein. The docking results of the two shiitake mushroom peptides and Keap1 protein molecules are as follows.

[0129] The shiitake peptide APFDCKE formed 9 hydrogen bonds, 7 ionic bonds and 1 cation-π interaction with Keap1 protein, with a binding energy of -49.9 kcal / mol. The number of amino acid residues in the B molecular chain of the peptide molecule binding to Keap1 protein (14) and the binding strength (binding energy -39.4 kcal / mol) were much higher than the number and binding strength of the amino acid residues in the A molecular chain of the peptide molecule binding to Keap1 protein. The cysteine ​​residues bound by the Maillard reaction of the peptide molecule formed 2 binding bonds with the amino acid residues in the A chain of Keap1 protein, with a binding energy of -9 kcal / mol ( Fig.11 , Table 10).

[0130] Table 10 Docking results of Lentinus edodes peptide APFDCKE and Keap1 protein molecule

[0131]

[0132]

[0133] The shiitake peptide IPSKPMC formed 8 hydrogen bonds, 4 ionic bonds and 1 H-π interaction with Keap1 protein, with a binding energy of -41.3 kcal / mol. The number of amino acid residues in the B molecular chain of the peptide molecule binding to Keap1 protein (11) and the binding strength (binding energy -34.3 kcal / mol) were also much higher than the number and binding strength of the amino acid residues in the A molecular chain of the peptide molecule binding to Keap1 protein. The cysteine ​​residues bound by the Maillard reaction of the peptide molecule formed 7 binding bonds with the amino acid residues in the A and B chains of the Keap1 protein, with a binding energy of -23.1 kcal / mol ( Fig.12 , Table 11).

[0134] Table 11 Docking results of Lentinus edodes peptide IPSKPMC and Keap1 protein molecule

[0135]

[0136] In summary, peptide molecules can form low-energy and multiple binding bonds with Keap1 protein to exert antioxidant effects.

[0137] It can be concluded from the above examples that the shiitake mushroom peptide of the present invention has significant blood pressure lowering, blood sugar lowering, blood lipid lowering, uric acid lowering and antioxidant activities, which provides possibilities for its application in the field of health care products and medicine preparation.

[0138] 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 shiitake mushroom peptide, characterized in that: The shiitake peptide includes a polypeptide having an amino acid sequence as shown in SEQ ID NO.1 and / or an amino acid sequence as shown in SEQ ID NO.

2.

2. The method for preparing the shiitake mushroom peptide according to claim 1, characterized in that: The steps include: Mixing dried shiitake mushrooms, water and flavor protease for a first enzymolysis to obtain a first enzymolysis solution; The first enzymatic hydrolysis solution is mixed with trypsin to perform a second enzymatic hydrolysis to obtain a second enzymatic hydrolysis solution; The second enzymatic hydrolysate is subjected to solid-liquid separation, and the supernatant is taken to obtain a third enzymatic hydrolysate; The third enzymatic hydrolysate, fructose and cysteine ​​are mixed to perform Maillard reaction to obtain a shiitake mushroom base material; Desalting the shiitake mushroom base material to obtain a solution containing shiitake mushroom polypeptides; The solution containing the shiitake mushroom polypeptide is subjected to liquid chromatography tandem mass spectrometry analysis to obtain the shiitake mushroom polypeptide.

3. The preparation method according to claim 2, characterized in that: The steps of the desalination treatment include: Dissolving the mushroom base material with a trifluoroacetic acid solution having a volume concentration of 0.1% to obtain a mushroom base material dissolved solution; The mushroom base material solution is passed through C 18 Elution is performed using a microchromatographic column, and the eluate is collected to obtain a solution containing Lentinus edodes polypeptides; The elution buffer for elution is an aqueous solution containing 60 vol% acetonitrile and 0.1 vol% trifluoroacetic acid.

4. The preparation method according to claim 2, characterized in that: The steps of liquid chromatography tandem mass spectrometry analysis include: The solution containing shiitake mushroom polypeptide is vacuum dried to obtain shiitake mushroom polypeptide powder; Dissolving the shiitake peptide powder in an aqueous solution containing 0.1 vol% formic acid and 5 vol% acetonitrile, and performing solid-liquid separation on the obtained dissolved product to obtain a supernatant; The supernatant is subjected to liquid chromatography tandem mass spectrometry detection to obtain the shiitake peptide; The mobile phase for liquid chromatography tandem mass spectrometry detection includes mobile phase A and mobile phase B, wherein the mobile phase A is formic acid with a volume concentration of 0.1 vol%, and the mobile phase B is an aqueous solution containing 0.1 vol% formic acid and 80 vol% acetonitrile.

5. The preparation method according to claim 2, characterized in that: The mass ratio of the dried shiitake mushroom product to water is 1 g:30 mL; the amount of the flavor protease is 1000 U / g of dried shiitake mushroom product; The temperature of the first enzymatic hydrolysis is 50° C., the time is 45 min, and the pH value is 7.

0.

6. The preparation method according to claim 2, characterized in that: The dosage of the trypsin is 2000U / g dried shiitake mushrooms; the temperature of the second enzymolysis is 37°C, the time is 45min, and the pH value is 8.

0.

7. The preparation method according to claim 2, characterized in that: The mass ratio of the fructose to the volume of the third enzymolysis solution is 2.5 g:100 mL; the mass ratio of the cysteine ​​to the volume of the third enzymolysis solution is 1.5 g:100 mL.

8. The preparation method according to claim 2 or 7, characterized in that: The Maillard reaction is an oil bath reaction, the temperature of the oil bath reaction is 109° C., the time is 68 min, and the pH value is 7.

0.

9. Use of the shiitake peptide according to claim 1 or the shiitake peptide prepared by the preparation method according to any one of claims 2 to 8 in the preparation of health products and / or medicines.

10. Use of the shiitake peptide according to claim 1 or the shiitake peptide prepared by the preparation method according to any one of claims 2 to 8 in preparing products having one or more functions of lowering blood pressure, lowering blood sugar, lowering blood lipids, lowering uric acid and having antioxidant activity.

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