An agaricus bisporus peptide prepared based on the Maillard reaction and its application in the preparation of products with antihypertensive, hypoglycemic, hypolipidemic, hypouricemic and antioxidant activities

Abstract

The present invention belongs to the technical field of protein peptide preparation, and particularly relates to a lentinan peptide prepared based on the Maillard reaction and its application in the preparation of products with antihypertensive, hypoglycemic, hypolipidemic, hypouricemic and antioxidant activities. The lentinan peptide of the present invention has significant effects of reducing blood pressure, blood sugar, blood lipids, uric acid and antioxidant, and can be used as an adjuvant therapeutic agent or functional food for four high diseases such as hypertension, hyperlipidemia, diabetes and hyperuricemia and diseases related to oxidative stress.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein peptide preparation, and particularly relates to a lentinan 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. Background Art

[0002] With the change of modern lifestyle, metabolic diseases such as hypertension and hyperglycemia and health problems related to oxidative stress have become increasingly serious. Searching for natural, safe and effective functional food ingredients has become a research hotspot. As a traditional food ingredient, Lentinus edodes is rich in various bioactive components, but the effects of directly consuming it on reducing blood pressure, blood sugar, blood lipid, uric acid and antioxidant are limited.

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

[0004] There are a large number of peptide substances in Lentinus edodes, and there are also some preparation methods for extracting lentinan peptides from Lentinus edodes in existing research. For example, some lentinan peptides extracted from Lentinus edodes have the effect of reducing salt and enhancing freshness; some lentinan peptides extracted from Lentinus edodes have anti-tumor effects. However, lentinan peptides with different peptide sequences and different preparation processes have different uses. At present, there is no lentinan peptide that takes into account the functions of reducing blood pressure, blood sugar, blood lipid, uric acid and antioxidant in this field. Summary of the Invention

[0005] The purpose of the present invention is to provide a lentinan 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 lentinan peptide 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 fields of health product and medicine preparation.

[0006] The present invention provides a lentinan peptide, which comprises a polypeptide with 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 preparation method of the lentinan peptide according to the above technical solution, comprising the following steps:

[0008] Mix dried Lentinus edodes, water and flavor protease for the first enzymatic hydrolysis to obtain a first enzymatic hydrolysate;

[0009] Mix the first enzymolysis solution with trypsin for a second enzymolysis to obtain a second enzymolysis solution;

[0010] Perform solid-liquid separation on the second enzymolysis solution, take the supernatant to obtain a third enzymolysis solution;

[0011] Mix the third enzymolysis solution, fructose and cysteine for a Maillard reaction to obtain a shiitake mushroom base material;

[0012] Perform desalting treatment on the shiitake mushroom base material to obtain a solution containing shiitake mushroom polypeptides;

[0013] Perform liquid chromatography-tandem mass spectrometry analysis on the solution containing shiitake mushroom polypeptides to obtain the shiitake mushroom peptides.

[0014] Preferably, the steps of the desalting treatment include:

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

[0016] Pass the dissolved solution of the shiitake mushroom base material through a C 18 Micro chromatography column for elution, collect the eluate to obtain a solution containing shiitake mushroom polypeptides;

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

[0018] Preferably, the steps of the liquid chromatography-tandem mass spectrometry analysis include:

[0019] Perform vacuum drying on the solution containing shiitake mushroom polypeptides to obtain shiitake mushroom peptide powder;

[0020] Dissolve the shiitake mushroom powder with an aqueous solution containing 0.1 vol % formic acid and 5 vol % acetonitrile, and perform solid-liquid separation on the obtained dissolved matter to obtain the supernatant;

[0021] Perform liquid chromatography-tandem mass spectrometry detection on the supernatant to obtain the shiitake mushroom peptides;

[0022] The mobile phase for the liquid chromatography-tandem mass spectrometry detection includes mobile phase A and mobile phase B. Mobile phase A is formic acid with a volume concentration of 0.1 vol %, and 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 mushrooms to the volume of water is 1 g: 30 mL; the dosage of the flavor protease is 1000 U / g of dried shiitake mushrooms;

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

[0025] Preferably, the dosage of trypsin is 2000 U / g of dried Lentinus edodes; 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 fructose to the volume of the third enzymatic hydrolysate is 2.5 g:100 mL; the mass ratio of cysteine to the volume of the third enzymatic hydrolysate 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 Lentinus edodes peptide described in the above technical solution or the Lentinus edodes peptide prepared by the preparation method described in the above technical solution in the preparation of health products and / or drugs.

[0029] The present invention also provides the use of the Lentinus edodes peptide described in the above technical solution or the Lentinus edodes 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 lipid, lowering uric acid and antioxidant activity.

[0030] Beneficial effects:

[0031] The present invention provides a Lentinus edodes 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 Lentinus edodes peptide of the present invention has the remarkable effects of lowering blood pressure, lowering blood sugar, lowering blood lipid, lowering uric acid and antioxidant, and can be used as an adjuvant therapeutic agent for four high diseases such as hypertension, hyperlipidemia, diabetes and hyperuricemia and diseases related to oxidative stress.

[0032] On this basis, the present invention also provides a method for preparing the lentinan peptide described in the above technical solution, which includes the following steps: mixing dried lentinus edodes, water and flavor protease for the first enzymatic hydrolysis to obtain a first enzymatic hydrolysate; mixing the first enzymatic hydrolysate with trypsin for the second enzymatic hydrolysis to obtain a second enzymatic hydrolysate; performing solid-liquid separation on the second enzymatic hydrolysate, taking the supernatant to obtain a third enzymatic hydrolysate; mixing the third enzymatic hydrolysate, fructose and cysteine for Maillard reaction to obtain a lentinus edodes base material; performing desalting treatment and liquid chromatography-tandem mass spectrometry analysis on the lentinus edodes base material to obtain the lentinan peptide. The present invention performs enzymatic hydrolysis on lentinus edodes to obtain a protein hydrolysate, and on this basis, combines the Maillard reaction to modify the protein hydrolysate molecules, so that the protein hydrolysate molecules are rearranged to generate new small molecule substances, effectively improving the biological activity of the protein hydrolysate (i.e., lentinan peptide), and obtaining the lentinan peptide with the effects of lowering blood pressure, blood sugar, blood lipid, uric acid and antioxidant described in the present invention. Moreover, the preparation process of the lentinan peptide is green and environmentally friendly, without the generation of harmful by-products, meeting the requirements of modern biopharmaceuticals and functional food production. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0034] Figure 1 It is the structural diagram of the lentinan polypeptide with the amino acid sequence of APFDCKE in Example 1;

[0035] Figure 2 It is the structural diagram of the lentinan polypeptide with the amino acid sequence of IPSKPMC in Example 1;

[0036] Figure 3 It is the molecular docking diagram of the lentinan peptide APFDCKE and ACE enzyme in Example 1;

[0037] Figure 4 It is the molecular docking diagram of the lentinan peptide IPSKPMC and ACE enzyme in Example 1;

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

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

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

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

[0042] Figure 9 Molecular docking diagram of lentinopeptide APFDCKE and xanthine oxidase in Example 1;

[0043] Figure 10 Molecular docking diagram of lentinopeptide IPSKPMC and xanthine oxidase in Example 1;

[0044] Figure 11 Molecular docking diagram of lentinopeptide APFDCKE and Keap1 protein in Example 1;

[0045] Figure 12 Molecular docking diagram of lentinopeptide IPSKPMC and Keap1 protein in Example 1. Detailed implementation mode

[0046] The present invention provides a lentinopeptide, and the lentinopeptide comprises a polypeptide with 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 implementation mode, the lentinopeptide can be a polypeptide with an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence as shown in SEQ ID NO.2. The amino acid sequence of SEQ ID NO.1 in the present invention is APFDCKE, and the amino acid shown in SEQ ID NO.2 is IPSKPMC.

[0048] The present invention also provides a preparation method of the lentinopeptide according to the above technical solution, comprising the following steps:

[0049] Mix dried lentinus edodes, water and flavor protease for the first enzymatic hydrolysis to obtain a first enzymatic hydrolysate;

[0050] Mix the first enzymatic hydrolysate and trypsin for the second enzymatic hydrolysis to obtain a second enzymatic hydrolysate;

[0051] Perform solid-liquid separation on the second enzymatic hydrolysate, take the supernatant to obtain a third enzymatic hydrolysate;

[0052] Mix the third enzymatic hydrolysate, fructose and cysteine for the Maillard reaction to obtain a lentinus edodes base material;

[0053] Perform desalting treatment on the lentinus edodes base material to obtain a solution containing lentinus edodes polypeptide;

[0054] Perform liquid chromatography-tandem mass spectrometry analysis on the solution containing lentinus edodes polypeptide to obtain the lentinopeptide.

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

[0056] After obtaining the first enzymatic hydrolysate, the first enzymatic hydrolysate and trypsin of the present invention are mixed for the second enzymatic hydrolysis to obtain a second enzymatic hydrolysate. As an embodiment, the dosage of the trypsin in the present invention can be 2000 U / g of dried lentinula edodes. As an embodiment, the temperature of the second enzymatic hydrolysis in the present invention can be 37 °C; the time of the second enzymatic hydrolysis can be 45 min; 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 an enzyme inactivation treatment on the second enzymatic hydrolysate to obtain an enzyme-inactivated enzymatic hydrolysate. As an embodiment, the method of the enzyme inactivation treatment can be: heating the second enzymatic hydrolysate in a water bath at 100 °C for 10 min.

[0058] After obtaining the enzyme-inactivated enzymatic hydrolysate, the present invention performs solid-liquid separation on the enzyme-inactivated enzymatic hydrolysate, takes the supernatant to obtain a third enzymatic hydrolysate. As an embodiment, the method of the solid-liquid separation in the present invention can be centrifugation. As an embodiment, the rotation speed of the centrifugation can be 8000 rpm, and the time can be 20 min.

[0059] After obtaining the third enzymatic hydrolysate, the third enzymatic hydrolysate, fructose and cysteine of the present invention are mixed for Maillard reaction to obtain a Maillard reaction solution. As an embodiment, the mass ratio of the fructose to the volume of the enzymatic hydrolysate in the present invention can be 2.5 g: 100 mL. As an embodiment, the mass ratio of the cysteine to the volume of the enzymatic hydrolysate in the present invention can be 1.5 g: 100 mL. As an embodiment, the Maillard reaction in 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 68 min; 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 lyophilize the Maillard reaction solution to obtain a shiitake mushroom base material. As an embodiment, the temperature of the lyophilization can be -70 °C; the time of the lyophilization can be 48 h.

[0061] After obtaining the shiitake mushroom base material, the present invention performs desalting treatment on the shiitake mushroom base material. As an embodiment, the steps of the desalting treatment of the present invention include: 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 dissolution solution; passing the shiitake mushroom base material dissolution solution through a C 18 micro chromatography column for elution, collecting the eluate to obtain a solution containing shiitake mushroom polypeptides; the elution buffer for the elution is an aqueous solution containing 60 vol% acetonitrile and 0.1 vol% trifluoroacetic acid. As an embodiment, the mass-to-volume ratio of the shiitake mushroom base material of the present invention can be 1 mg: 3 μL. As an embodiment, the C 18 micro chromatography column can be a ZipTip C 18 micro chromatography column.

[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 the liquid chromatography-tandem mass spectrometry analysis of the present invention include: performing vacuum drying on 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 dissolution to obtain a supernatant; performing liquid chromatography-tandem mass spectrometry detection on the supernatant to obtain the shiitake mushroom peptides; the mobile phase for the liquid chromatography-tandem mass spectrometry detection includes mobile phase A and mobile phase B, the mobile phase A is formic acid having 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 method of the solid-liquid separation of the present invention can be centrifugation; as another embodiment, the rotation speed of the centrifugation can be 13,500 rpm; the temperature of the centrifugation can be 4 °C; the time of the centrifugation can be 20 min.

[0064] As an embodiment, the liquid phase setting parameters of the 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 lentinan peptide described in the above technical solution or the lentinan peptide prepared by the preparation method described in the above technical solution in the preparation of health products and / or pharmaceuticals. As an embodiment, the use includes the use of the lentinan peptide described in the above technical solution or the lentinan peptide prepared by the preparation method described in the above technical solution in health products and pharmaceuticals.

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

[0070] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to 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 lentinan peptide comprises the following steps:

[0073] (1) Raw material preparation: Select dried lentinus edodes as the raw material, and obtain lentinus edodes powder with a particle size less than 100 mesh through the pretreatment steps of crushing and sieving.

[0074] (2) Maillard reaction: Mix the mushroom powder and water at a material-liquid ratio of 1 g: 30 mL. First, add 1000 U / g of flavor protease (enzyme activity / dry weight of mushroom powder, the enzyme activity of flavor protease is 250 N.F.U / mg, purchased from Beijing Solarbio Science & Technology Co., Ltd.) to the mixture. After enzymatic hydrolysis at 50 °C and pH 7.0 for 45 min, then add 2000 U / g of trypsin (enzyme activity / dry weight of mushroom powder, the enzyme activity of trypsin is 30000 U / g, purchased from Beijing Solarbio Science & Technology Co., Ltd.), and perform enzymatic hydrolysis at 37 °C and pH 8.0 for 45 min. Inactivate the enzyme in the obtained enzymatic hydrolysate in a water bath at 100 °C for 10 min, centrifuge at 8000 rpm for 20 min, and take the supernatant.

[0075] Add 2.5% (mass / volume ratio g / mL) of fructose and 1.5% (mass / volume ratio g / mL) of cysteine to the supernatant, adjust the pH to 7.0, and carry out the Maillard reaction at an oil bath reaction temperature of 109 °C for 68 min. After the Maillard reaction is completed, freeze-dry the reaction solution at -70 °C for 48 h to obtain the base material prepared by the Maillard reaction of mushrooms.

[0076] (3) Identification of mushroom peptides: Use mass spectrometry to identify peptide molecules in the mushroom Maillard reaction base material. Use a ZipTip C 18 microchromatography column (Merck-Millipore, Shanghai ANPEL Laboratory Technologies Inc.) to perform desalting pretreatment on the base material. The desalting method is as follows: Accurately weigh 10 mg of the base material prepared by the Maillard reaction of mushrooms, add 30 μL of 0.1% (v / v) trifluoroacetic acid (TFA) to dissolve it to obtain a solution of the base material prepared by the Maillard reaction of mushrooms;

[0077] Rinse the chromatography column 10 times with 50 μL of a solution prepared with 60% (v / v) acetonitrile (ACN) and 0.1% TFA pure water; rinse the chromatography column 10 times with 10 μL of 0.1% TFA; pass the solution of the base material prepared by the Maillard reaction of mushrooms through the chromatography column by suction and discharge 20 times; rinse the chromatography column 5 times with 10 μL of 0.1% TFA; elute the chromatography column with 10 μL of a solution prepared with 60% ACN and 0.1% TFA pure water, collect the eluate, transfer the eluate to a polypropylene centrifuge tube, and dry it in vacuo to obtain mushroom peptide segments.

[0078] Dissolve the lentinan peptide segments with 20 μL of dissolution solution (a solution prepared with pure water containing 0.1% (v / v) formic acid and 5% (v / v) ACN), vortex, centrifuge at 13,500 rpm and 4 °C for 20 min, collect the supernatant and transfer it to a sample injection tube, with an injection volume of 8 μL, and perform LC-MS / MS sequence analysis. The mobile phase A of the liquid chromatography is 0.1% formic acid, and the mobile phase B is a solution prepared with pure water containing 0.1% formic acid and 80% (v / v) ACN; the LC-MS / MS setting parameters are shown in Table 1.

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

[0080]

[0081] (4) Screening of lentinan peptides: Identified by LC-MS / MS, the in-built database of the PEAKS software was used for identification sequence database retrieval. The lentinan peptides APFDCKE (SEQ ID NO.1) and IPSKPMC (SEQ ID NO.2) with high mass spectrometry abundances were obtained. The structural diagrams of the lentinan peptides APFDCKE and IPSKPMC are respectively as Figures 1 - 2 shown.

[0082] (5) Analysis of the functional activity of lentinan peptide molecular docking

[0083] The renin-angiotensin system is an important system for regulating blood pressure and electrolyte balance. When blood pressure drops, the kidney releases renin (an aspartic protease) into the circulation, catalyzing the conversion of angiotensinogen to angiotensin I (Ang I); Ang I is the substrate of angiotensin-converting enzyme (ACE). ACE converts it into biologically active angiotensin II (Ang II) by cleaving two amino acid fragments (His-Leu) at the free C-terminus of Ang I; Ang II has a strong blood pressure-raising effect. It binds to the membrane-bound angiotensin II receptor, stimulates the adrenal cortex to secrete aldosterone, resulting in sodium retention and vasoconstriction, and increases blood pressure. The ACE inhibitory peptide binds to the active site of ACE, blocks the catalytic action of ACE on Ang I, and reduces the production of Ang II. At the same time, the ACE inhibitory peptide may also reduce the hydrolysis of bradykinin. Bradykinin is a peptide with vasodilatory effects, and its accumulation also helps to lower blood pressure. That is, the ACE inhibitory peptide exerts a blood pressure-lowering effect by inhibiting the activity of ACE, reducing the production 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. It 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. As a cytoplasmic protein, Keap1 can bind to the transcription factor Nrf2 to form the Keap1-Nrf2 complex, thereby inhibiting the activity of Nrf2. Peptide molecules with antioxidant activity can bind to the Keap1 receptor protein, thus interfering with the interaction between Keap1 and Nrf2, making the Keap1-Nrf2 complex unstable, and then releasing Nrf2. After being activated, Nrf2 can enter the nucleus and induce the expression of antioxidant response element (ARE) genes, thereby enhancing the antioxidant capacity of cells. That is, antioxidant peptides play an antioxidant role by binding to the Keap1 receptor protein and activating the Keap1-Nrf2 / ARE signaling pathway.

[0088] In the screening of active substances, the molecular docking technology uses the specific binding between "receptor-ligand" to quickly screen out active small molecule substances that bind to specific targets, overcoming the disadvantages 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, provides strong support for modern drug research, and is of great significance for clarifying the pharmacodynamic material basis of natural active substances.

[0089] Download 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) from the RCSB database (https: / / www.rcsb.org / ). Use the MOE 2019 molecular docking software to optimize the crystal structure of the receptor protein, remove water molecules, and complete hydrogen atoms. Use the MOE software to construct the 3D structure of Lentinus edodes peptide and perform molecular energy minimization. Use the Site Finder module of MOE to determine the amino acid residue active sites of the receptor protein. Taking the docking score, the number of formed bonds, and the binding bond energy of the binding of Lentinus edodes peptide to the receptor protein as screening indicators, select the complex of Lentinus edodes peptide and the receptor protein that binds tightly, and use the MOE software to analyze the binding sites and interaction modes between the peptide molecule and the receptor protein in the complex.

[0090] (6)Analysis results of inhibiting angiotensin-converting enzyme (PDB: 1O86)

[0091] The amino acid sites that can bind within the cavity pocket of the ACE receptor protein consist 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] Six hydrogen bonds, one metal bond, and five ionic bond interaction forces are formed between the lentinopeptide APFDCKE and the ACE enzyme molecule, with a binding energy of -63.4 kcal / mol (the lower the binding energy, the higher the intermolecular binding strength). The peptide molecule forms a binding force with the active zinc ion ZN of the ACE enzyme 2+ with a binding energy of -24 kcal / mol, disrupting the binding bonds between the zinc ion and GLU411, HIS387, and HIS383; the cysteine residue bound by the Maillard reaction of the peptide molecule contributes a strong binding force to the binding of the ACE enzyme, with a binding energy of -27.8 kcal / mol. In summary, the peptide molecule binding to the ACE enzyme significantly inhibits the activity of the ACE enzyme ( Figure 3 , Table 2).

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

[0094]

[0095] Nine interaction bonds are formed between the lentinopeptide IPSKPMC and the ACE enzyme molecule, including five hydrogen bonds, one metal bond, and three ionic bond interaction forces, with a binding energy of -41.4 kcal / mol. The peptide molecule forms a binding force with the active zinc ion ZN of the ACE enzyme 2+ with a binding energy of -21.6 kcal / mol, disrupting the binding bonds between the zinc ion and GLU411, HIS387, and HIS383; the cysteine residue bound by the Maillard reaction of the peptide molecule binds both the key active amino acid residue (ALA354) and the zinc ion of the ACE enzyme, with a binding energy of -22.1 kcal / mol. In summary, the peptide molecule binding to the ACE enzyme significantly inhibits the activity of the ACE enzyme ( Figure 4 , Table 3).

[0096] Table 3 Docking diagram of lentinopeptide IPSKPMC and ACE enzyme molecule

[0097]

[0098] Analysis results of inhibiting α-glucosidase (PDB: 5NN8)

[0099] The MOE software retains the A - H molecular chains of α-glucosidase, and the docking results of two lentinan peptides with α-glucosidase molecules are as follows.

[0100] Lentinan peptide APFDCKE formed 6 hydrogen bonds and 7 ionic bond interactions with the residues of the A chain of α-glucosidase, with a binding energy of -59 kcal / mol. Among them, the cysteine residue formed by the Maillard reaction of the peptide molecule contributed a strong binding force in binding to α-glucosidase, forming 5 binding bonds, and the binding energy was -33.2 kcal / mol ( Figure 5 , Table 4).

[0101] Table 4 Docking results of lentinan peptide APFDCKE with α-glucosidase molecules

[0102]

[0103] Lentinan peptide IPSKPMC formed 6 hydrogen bonds, 6 ionic bonds and 1 H-π interaction with the residues of the A chain of α-glucosidase, with a binding energy of -52.3 kcal / mol. Among them, the cysteine residue formed by the Maillard reaction of the peptide molecule contributed a strong binding force in binding to α-glucosidase, forming 8 binding bonds, and the binding energy was -26 kcal / mol ( Figure 6 , Table 5).

[0104] Table 5 Docking results of lentinan peptide IPSKPMC with α-glucosidase molecules

[0105]

[0106] In summary, the two lentinan peptides play an inhibitory role on α-glucosidase by forming multiple binding bonds with α-glucosidase and having a low binding energy.

[0107] (8)Analysis results of lipid-lowering by binding to pancreatic lipase (PDB: 1LPB)

[0108] 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 two lentinan peptides with pancreatic lipase molecules are as follows.

[0109] The amino acid residues in the molecular chains of Lentinan peptide APFDCKE and pancreatic lipase A and B formed 6 hydrogen bonds and 4 ionic bond interactions, with a binding energy of -44.5 kcal / mol; the binding strength of the peptide molecule to the amino acid residues in the A chain of pancreatic lipase was higher than that to the amino acid residues in the B chain, and the binding energy of the peptide molecule to the amino acid residues in the A chain was twice that of the B chain; the cysteine residues conjugated by the Maillard reaction of the peptide molecule formed 3 binding bonds with the amino acid residues in the B chain of pancreatic lipase ( Figure 7 , Table 6).

[0110] Table 6 Docking results of Lentinan peptide APFDCKE and pancreatic lipase molecules

[0111]

[0112] The amino acid residues in the molecular chains of Lentinan peptide IPSKPMC and pancreatic lipase A and B formed 4 hydrogen bonds and 6 ionic bond interactions, with a binding energy of -50.4 kcal / mol; the binding strength of the peptide molecule to the amino acid residues in the molecular chains of pancreatic lipase A and B was comparable, and the binding energy difference was small; the cysteine residues conjugated by the Maillard reaction of the peptide molecule formed 6 binding bonds with the amino acid residues in the B chain of pancreatic lipase, with a binding energy of -26.7 kcal / mol ( Figure 8 , Table 7).

[0113] Table 7 Docking results of Lentinan peptide IPSKPMC and pancreatic lipase molecules

[0114]

[0115] The residues of the two Lentinan peptides binding to the A and B molecular chains of pancreatic lipase were complementary, and there may be a characteristic of synergistically inhibiting the activity of pancreatic lipase; and the multiple low-energy binding bonds formed by the cysteine residues and the amino acid residues in the B chain of pancreatic lipase played an important role in inhibiting the activity of pancreatic lipase and thus exerting a lipid-lowering effect.

[0116] (8) Analysis results of uric acid reduction by binding to xanthine oxidase (PDB: 3NVZ)

[0117] The MOE software retained the A - F molecular chains of xanthine oxidase, including Xanthine dehydrogenase / oxidase. The docking results of the two Lentinan peptides and xanthine oxidase molecules are as follows.

[0118] The number of bonds formed between the amino acid residues in the E and F chains of lentinan peptide APFDCKE and xanthine oxidase is the same (both are 8), including 7 hydrogen bonds, 7 ionic bonds and 2 H-π / π-H interactions, and the binding energy is -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 residue bound by the Maillard reaction of the peptide molecule forms 4 binding bonds with the amino acid residues in the F chain of pancreatic lipase ( Figure 9 , Table 8).

[0119] Table 8 Molecular docking results of lentinan peptide APFDCKE and xanthine oxidase

[0120]

[0121] Lentinan peptide IPSKPMC forms 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, and the binding energy is -32.2 kcal / mol; among them, the cysteine residue bound by the Maillard reaction of the peptide molecule makes a greater contribution to binding xanthine oxidase, forming 6 binding bonds with a binding energy of -16.2 kcal / mol ( Figure 10 , Table 9).

[0122] Table 9 Molecular docking results of lentinan peptide IPSKPMC and xanthine oxidase

[0123]

[0124] In summary, the two lentinan peptides bind to the amino acid residues in multiple molecular chains of xanthine oxidase, forming low bond energy and multiple interactions, and the peptide molecules play a role in inhibiting xanthine oxidase to reduce uric acid.

[0125] (9) Combining the antioxidant analysis results of Keap1 protein (PDB: 4L7B)

[0126] The A and B molecular chains of Keap1 protein are retained by the MOE software. The molecular docking results of the two lentinan peptides and Keap1 protein are as follows.

[0127] The mushroom peptide APFDCKE formed 9 hydrogen bonds, 7 ionic bonds and 1 cation-π interaction with the Keap1 protein, with a binding energy of -49.9 kcal / mol. The number of amino acid residues (14) and the binding strength (binding energy -39.4 kcal / mol) of the peptide molecule binding to the B molecular chain of the Keap1 protein are much higher than the number and binding strength of the amino acid residues bound by the peptide molecule to the A molecular chain of the Keap1 protein; the cysteine residues bound by the peptide molecule through the Maillard reaction formed 2 binding bonds with the amino acid residues in the A chain of the Keap1 protein, with a binding energy of -9 kcal / mol ( Figure 11 , Table 10).

[0128] Table 10 Docking results of the mushroom peptide APFDCKE and the Keap1 protein

[0129]

[0130] The mushroom peptide IPSKPMC formed 8 hydrogen bonds, 4 ionic bonds and 1 H-π interaction with the Keap1 protein, with a binding energy of -41.3 kcal / mol. The number of amino acid residues (11) and the binding strength (binding energy -34.3 kcal / mol) of the peptide molecule binding to the B molecular chain of the Keap1 protein are also much higher than the number and binding strength of the amino acid residues bound by the peptide molecule to the A molecular chain of the Keap1 protein; the cysteine residues bound by the peptide molecule through the Maillard reaction 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 ( Figure 12 , Table 11).

[0131] Table 11 Docking results of the mushroom peptide IPSKPMC and the Keap1 protein

[0132]

[0133] In summary, it can be seen that the peptide molecule can form binding bonds with low binding energy and multiple binding bonds with the Keap1 protein to exert antioxidant effects.

[0134] From the above examples, it can be concluded that the mushroom peptide described in the present invention has significant blood pressure-lowering, blood sugar-lowering, blood lipid-lowering, uric acid-lowering and antioxidant activities, providing possibilities for its application in the fields of health product and drug preparation.

[0135] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An Lentinan peptide, characterized in that, The Lentinula edodes peptide is a polypeptide with 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 preparation method of the lentinan peptide according to claim 1, characterized in that, It includes the following steps: Mix dried Lentinula edodes, water and flavor protease for the first enzymatic hydrolysis to obtain a first enzymatic hydrolysate; the temperature of the first enzymatic hydrolysis is 50 °C, the time is 45 min, and the pH value is 7.0; Mix the first enzymatic hydrolysate with trypsin for the second enzymatic hydrolysis to obtain a second enzymatic hydrolysate; the temperature of the second enzymatic hydrolysis is 37 °C, the time is 45 min, and the pH value is 8.0; Perform solid-liquid separation on the second enzymatic hydrolysate, take the supernatant to obtain a third enzymatic hydrolysate; Mix the third enzymatic hydrolysate, fructose and cysteine for the Maillard reaction to obtain a Lentinula edodes base material; Perform desalting treatment on the Lentinula edodes base material to obtain a solution containing Lentinula edodes polypeptide; Perform liquid chromatography-tandem mass spectrometry analysis on the solution containing Lentinula edodes polypeptide to obtain the Lentinula edodes peptide.

3. The preparation method according to claim 2, characterized in that, The steps of the desalting treatment include: Dissolve the Lentinula edodes base material with a trifluoroacetic acid solution with a volume concentration of 0.1% to obtain a Lentinula edodes base material dissolution solution; Elute the Lentinula edodes base material dissolution solution through a C18 microchromatography column, collect the eluate to obtain a solution containing Lentinula edodes polypeptide; The elution buffer for the 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 the liquid chromatography-tandem mass spectrometry analysis include: Perform vacuum drying on the solution containing Lentinula edodes polypeptide to obtain Lentinula edodes peptide powder; Dissolve the Lentinula edodes peptide powder with an aqueous solution containing 0.1 vol% formic acid and 5 vol% acetonitrile, and perform solid-liquid separation on the obtained dissolution to obtain the supernatant; Perform liquid chromatography-tandem mass spectrometry detection on the supernatant to obtain the Lentinula edodes peptide; The mobile phase for the liquid chromatography-tandem mass spectrometry detection includes mobile phase A and mobile phase B. Mobile phase A is formic acid with a volume concentration of 0.1 vol%, and 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 Lentinula edodes to the volume of water is 1 g: 30 mL; the dosage of the flavor protease is 1000 U / g of dried Lentinula edodes.

6. The preparation method according to claim 2, wherein The dosage of the trypsin is 2000 U / g of dried Lentinula edodes.

7. The preparation method according to claim 2, characterized in that, The mass ratio of the fructose to the volume of the third enzymatic hydrolysate is 2.5 g: 100 mL; the mass ratio of the cysteine to the volume of the third enzymatic hydrolysate 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 Lentinula edodes peptide according to claim 1 in the preparation of a drug having any one or more functions of lowering blood pressure, lowering blood sugar, lowering blood lipid, lowering uric acid and antioxidant.

10. Use of the Lentinula edodes peptide according to claim 1 in the preparation of a health product having any one or more functions of lowering blood pressure, lowering blood sugar, lowering blood lipid and antioxidant.

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