Mushroom peptide prepared based on thermal reaction and application of mushroom peptide in preparation of products capable of reducing blood pressure, blood fat, blood sugar and uric acid and having antioxidant activity

Abstract

The invention belongs to the technical field of active peptides, biological medicines and foods, and particularly relates to a shiitake mushroom peptide prepared based on thermal reaction and application of the shiitake mushroom peptide in preparation of products for reducing blood pressure, blood fat, blood sugar and uric acid and having antioxidant activity. The lentinus edodes peptide disclosed by the invention has the effects of remarkably reducing blood pressure, blood fat, blood sugar and uric acid and resisting oxidation, and provides a new natural medicine or functional food raw material for preventing and treating chronic diseases such as hypertension, hyperlipidemia, diabetes mellitus and hyperuricemia and oxidative stress related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of active peptides, biomedicine and food technology, and specifically relates to a shiitake mushroom peptide prepared based on thermal reaction and its application in preparing products with blood pressure lowering, blood lipid lowering, blood sugar lowering, uric acid lowering and antioxidant activity. Background Art

[0002] Shiitake mushrooms, also known as flower mushrooms and thick mushrooms, are a natural food ingredient that is both a medicine and a food. Not only are they delicious, they also contain many ingredients that are beneficial to health, such as shiitake peptides. These natural bioactive ingredients have multiple functions such as regulating immunity, anti-virus, and anti-tumor, so they have broad application prospects in the fields of health products and medicine.

[0003] In recent years, extracting and modifying the active ingredients in shiitake mushrooms by physical, chemical or biological methods to prepare products with specific health effects has become a research hotspot, such as preparing shiitake mushroom peptides by acid-base method or enzymatic method. At the same time, some existing studies have found that the functions of shiitake mushroom peptides with different amino acid sequences or prepared by different methods are not the same.

[0004] At present, there are some shiitake mushroom peptides with anti-tumor effects or anti-aging functions in existing research, but there are no reports of shiitake mushroom peptides that have the activities of lowering blood pressure, lowering blood sugar, lowering blood lipids, lowering uric acid and antioxidant activity. Summary of the invention

[0005] The purpose of the present invention is to provide a shiitake mushroom peptide prepared based on thermal reaction and its application in the preparation of products with blood pressure lowering, blood lipid lowering, blood sugar lowering, uric acid lowering and antioxidant activity. The shiitake mushroom peptide has significant effects on lowering blood pressure, blood lipid lowering, blood sugar lowering, uric acid lowering and antioxidant activity.

[0006] The present invention provides a shiitake peptide, which includes shiitake peptide I and / or shiitake peptide II. The amino acid sequences of the shiitake peptide I and shiitake peptide II are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[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] subjecting the third enzymatic hydrolyzate to a thermal reaction to obtain a shiitake mushroom thermal reaction base material;

[0012] Desalting the shiitake mushroom thermal reaction base material to obtain a solution containing shiitake mushroom peptides;

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

[0014] Preferably, the thermal reaction is carried out at a temperature of 110 to 130° C., a pressure of 0.1 to 0.2 MPa, and a time of 90 to 120 min.

[0015] Preferably, the desalination step comprises:

[0016] Dissolving the shiitake mushroom thermal reaction base material with a trifluoroacetic acid solution having a volume concentration of 0.1% to obtain a shiitake mushroom base material dissolved liquid;

[0017] 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 shiitake peptides;

[0018] The elution buffer for elution is an aqueous solution containing 60 vol% acetonitrile and 0.1 vol% trifluoroacetic acid.

[0019] Preferably, the step of liquid chromatography tandem mass spectrometry analysis comprises: vacuum drying the solution containing shiitake peptide to obtain shiitake peptide powder;

[0020] 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;

[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 liquid phase parameters of the liquid chromatography tandem mass spectrometry detection are shown in the following table:

[0024]

[0025] Preferably, the mass ratio of the dried shiitake mushroom product to the volume of water is 1 g: 28-32 mL; the amount of the flavor protease is 800-1000 U / g dried shiitake mushroom product;

[0026] The temperature of the first enzymatic hydrolysis is 45-55° C., the time is 40-50 min, and the pH value is 6.5-7.5.

[0027] Preferably, the dosage of the trypsin is 1800-2200 U / g dried shiitake mushrooms; the temperature of the second enzymatic hydrolysis is 32-42° C., the time is 40-45 min, and the pH value is 7.5-8.5.

[0028] The present invention also provides one or more uses 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, medicines and foods.

[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 lipids, lowering blood sugar, lowering uric acid and antioxidant activity.

[0030] Beneficial effects:

[0031] The present invention provides a shiitake peptide, which includes shiitake peptide I and / or shiitake peptide II, and the amino acid sequences of shiitake peptide I and shiitake peptide II are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The shiitake peptide of the present invention has significant blood pressure lowering, blood lipid lowering, blood sugar lowering, uric acid lowering and anti-oxidation activities, and provides a new natural medicine or functional food raw material for the prevention and treatment of chronic diseases such as hypertension, hyperlipidemia, diabetes, hyperuric acid and oxidative stress-related diseases.

[0032] The present invention also provides a method for preparing the above-mentioned shiitake mushroom peptide, 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; performing a thermal reaction on the third enzymolysis solution to obtain a shiitake mushroom thermal reaction base material; performing a desalting treatment on the shiitake mushroom thermal reaction base material to obtain a solution containing shiitake mushroom peptides; performing a liquid chromatography tandem mass spectrometry analysis on the solution containing shiitake mushroom peptides to obtain the shiitake mushroom peptides. Thermal reaction technology, as a simple and effective food processing method, can induce complex changes in the ingredients in the food materials and produce new bioactive substances. The preparation method of the present invention, on the basis of enzymolysis of dried shiitake mushrooms, uses thermal reaction to stimulate the enzymolysis product to produce new shiitake mushroom peptides, so that the prepared shiitake mushroom peptides have the effects of lowering blood pressure, lowering blood lipids, lowering blood sugar, lowering uric acid and anti-oxidation. At the same time, the preparation method of the present invention is simple, low-cost, and suitable for large-scale industrial 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 shiitake polypeptide having the amino acid sequence APVPIDQ in Example 1;

[0035] Figure 2 The structure diagram of the Lentinus edodes polypeptide having the amino acid sequence of IEPIKVP in Example 1;

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

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

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

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

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

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

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

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

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

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

[0046] The present invention provides a shiitake peptide, which includes shiitake peptide I and / or shiitake peptide II. The amino acid sequences of shiitake peptide I and shiitake peptide II are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0047] In the present invention, as an embodiment, the shiitake peptide of the present invention is shiitake peptide I or shiitake peptide II. The amino acid sequence shown in SEQ ID NO.1 of the present invention is APVPIDQ; the amino acid sequence shown in SEQ ID NO.2 is IEPIKVP.

[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] subjecting the third enzymatic hydrolyzate to a thermal reaction to obtain a shiitake mushroom thermal reaction base material;

[0053] Desalting the shiitake mushroom thermal reaction base material to obtain a solution containing shiitake mushroom peptides;

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

[0055] The present invention mixes dried shiitake mushrooms, water and flavor protease for first enzymolysis to obtain a first enzymolysis 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 meshes. As an embodiment, the mass of the dried shiitake mushrooms and the volume ratio of water can be 1g:28-32mL; as another embodiment, the mass of the dried shiitake mushrooms and the volume ratio of water can be 1g:30mL. As an embodiment, the amount of flavor protease in the present invention can be 800-1200U / g dried shiitake mushrooms; as another embodiment, the amount of flavor protease can be 1000U / g dried shiitake mushrooms. As an embodiment, the temperature of the first enzymolysis of the present invention can be 45-55°C; as another embodiment, the temperature of the first enzymolysis can be 50°C. As an embodiment, the time of the first enzymolysis of the present invention can be 40-50min; as another embodiment, the time of the first enzymolysis can be 45min. As an embodiment, the pH value of the first enzymatic hydrolysis of the present invention may be 6.5-7.5; as another embodiment, the pH value of the first enzymatic hydrolysis may be 7.0.

[0056] After obtaining the first enzymatic hydrolysis solution, the present invention mixes the first enzymatic hydrolysis solution with trypsin for a second enzymatic hydrolysis to obtain a second enzymatic hydrolysis solution. As an embodiment, the amount of trypsin used in the present invention can be 1800-2200U / g dried shiitake mushrooms; as another embodiment, the amount of trypsin used can be 2000U / g dried shiitake mushrooms. As an embodiment, the temperature of the second enzymatic hydrolysis of the present invention can be 32-42°C; as another embodiment, the temperature of the second enzymatic hydrolysis can be 37°C. As an embodiment, the time of the second enzymatic hydrolysis of the present invention can be 40-45min; as another embodiment, the time of the second enzymatic hydrolysis can be 45min. As an embodiment, the pH value of the second enzymatic hydrolysis of the present invention can be 7.5-8.5; as another embodiment, 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 enzymatic hydrolysate, the present invention conducts a thermal reaction on the third enzymatic hydrolysate to obtain a shiitake mushroom thermal reaction base material solution. As an embodiment, the thermal reaction of the present invention can be carried out in a high-pressure reactor. The source and model of the high-pressure reactor of the present invention are not particularly limited, and a high-pressure reactor of conventional source in the art can be used. As an embodiment, the temperature of the thermal reaction of the present invention can be 110-130°C; as another embodiment, the temperature of the thermal reaction can be 120°C. As an embodiment, the pressure of the thermal reaction of the present invention can be 0.1-0.2MPa; as another embodiment, the pressure of the thermal reaction can be 0.1MPa. As an embodiment, the time of the thermal reaction can be 90-120min; as another embodiment, the time of the thermal reaction can be 120min. The thermal reaction of the present invention can induce complex changes in the components of the shiitake mushroom enzymatic hydrolysate, produce new bioactive substances, and then based on the thermal reaction of the present invention, the shiitake mushroom enzymatic hydrolysate can produce shiitake mushroom peptides with blood pressure lowering, blood lipid lowering, blood sugar lowering, uric acid lowering and antioxidant activities.

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

[0061] After obtaining the shiitake mushroom heat reaction 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 heat reaction base material with a trifluoroacetic acid solution having a volume concentration of 0.1% to obtain a shiitake mushroom base material dissolved liquid; 18 The micro-chromatographic column is used for elution, and the eluate is collected to obtain a solution containing shiitake peptides; 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 base material of the present invention to the volume ratio of the shiitake 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 peptides, the present invention performs liquid chromatography tandem mass spectrometry analysis on the solution containing shiitake peptides to obtain the shiitake peptides. As an embodiment, the steps of liquid chromatography tandem mass spectrometry analysis of the present invention include: vacuum drying the solution containing shiitake peptides to obtain shiitake peptide powder; dissolving the shiitake 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 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 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 shiitake peptide described in the above technical solution or the shiitake peptide prepared by the preparation method described in the above technical solution in one or more of the preparation of health products, medicines and foods. As an embodiment, the application described in the present invention is the use of the shiitake peptide described in the above technical solution in health products, medicines and foods. As an embodiment, when the shiitake peptide is applied to food, the shiitake peptide can be used as a food additive.

[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 of the following activities: lowering blood pressure, lowering blood lipids, lowering blood sugar, 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 lipids, lowering blood sugar, 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 lipids, lowering blood sugar, 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 lipids, lowering blood sugar 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 peptides based on thermal reaction, the steps are as follows:

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

[0075] The supernatant was placed in a high pressure reactor and reacted for 120 min at 120° C. and 0.1 MPa. After the thermal reaction was completed, the reaction liquid was freeze-dried at -70° C. for 48 h to obtain a shiitake mushroom thermal reaction base material.

[0076] (3) Identification of Shiitake Mushroom Peptides: Mass spectrometry was used to identify peptide molecules in Shiitake mushroom thermal reaction base. ZipTipC 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 heat 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 solution of the base material prepared by the heat reaction of Lentinus edodes;

[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 thermal 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 setup parameters

[0080]

[0081]

[0082] (4) Screening of shiitake peptides: After LC-MS / MS identification, the database provided by PEAKS software was used to search the identification sequence database. The shiitake peptides APVPIDQ (SEQ ID NO.1) and IEPIKVP (SEQ ID NO.2) with high mass spectrum abundance were obtained. The structural diagrams of the shiitake peptides APFDCKE and IPSKPMC are shown in the following table. Figures 1-2 shown.

[0083] (5) Molecular docking and functional activity analysis of shiitake mushroom peptides

[0084] 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.

[0085] 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 decomposes carbohydrates such as starch, sucrose, and maltose to form 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. Competitive inhibition makes it impossible for α-glucosidase to effectively decompose carbohydrates into monosaccharides, delays 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 play a hypoglycemic effect by competitively inhibiting the activity of α-glucosidase, delaying the decomposition of carbohydrates and the absorption of glucose.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] Molecular docking technology predicts the binding mode and interaction strength between small molecules and receptor proteins through computer simulation, predicts the activity of compounds, and thus screens out compounds with potential pharmacological activity, improving the efficiency and success rate of drug screening, reducing the ineffective detection of a large number of compound samples in the traditional drug screening process, and reducing the cost of drug research and development. Molecular docking technology can be used to predict the structural information of drug-receptor binding and analyze the mechanism of action of drug molecules, thereby improving the efficacy and treatment effect of drugs.

[0090] 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.

[0091] (6) Combined with angiotensin converting enzyme (PDB: 1O86) blood pressure analysis results

[0092] The shiitake peptide APVPIDQ forms 10 binding bonds with the ACE enzyme molecule, including hydrogen bonds and ionic bonds, with a binding energy of -46.7 kcal / mol (the lower the binding energy, the higher the intermolecular binding strength). 2+Strong binding occurs, with a bond energy of -35.4 kcal / mol, which distorts the tetrahedral geometry of the enzyme active center formed by zinc ions, GLU411, HIS387, and HIS383, significantly inhibiting ACE enzyme activity and thus exerting a hypotensive effect ( Figure 3 , Table 2).

[0093] Table 2 Docking results of Lentinus edodes peptide APVPIDQ and ACE enzyme molecule

[0094]

[0095] There are 5 hydrogen bond interactions, 1 metal bond, 5 ionic bond interactions and 1 H-pi interaction between the shiitake peptide IEPIKVP and the ACE enzyme molecule, and the binding energy is -52.4 kcal / mol. 2+ Strong binding occurs, with a bond energy of -22 kcal / mol, destroying the bonds between zinc ions and GLU411, HIS387, and HIS383, inhibiting ACE enzyme activity and exerting a hypotensive effect ( Figure 4 , Table 3).

[0096] Table 3 Docking results of Lentinus edodes peptide IEPIKVP and ACE enzyme molecule

[0097]

[0098]

[0099] (7) Combined with α-glucosidase (PDB: 5NN8) glucose-lowering analysis results

[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 APVPIDQ formed 6 hydrogen bonds and 3 ionic bonds with the A chain residues of α-glucosidase, with a bond energy of -28.8 kcal / mol ( Figure 5 , Table 4).

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

[0103]

[0104] Lentinus edodes peptide IEPIKVP formed 6 hydrogen bonds and 5 ionic bonds with the A chain residues of α-glucosidase, with a bond energy of -41.3 kcal / mol ( Figure 6 , Table 5).

[0105] Table 5 Docking results of Lentinus edodes peptide IEPIKVP and α-glucosidase molecules

[0106]

[0107] It can be seen that the two shiitake mushroom peptides have different binding residues to the A chain of α-glucosidase, and have a synergistic inhibitory effect on the activity of α-glucosidase to exert a hypoglycemic effect.

[0108] (8) Combined with pancreatic lipase (PDB: 1LPB) lipid-lowering analysis results

[0109] 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.

[0110] Lentinus edodes peptide APVPIDQ forms 6 bonds with amino acid residues in the molecular chains of pancreatic lipase A and B, including hydrogen bonds and ionic bonds, with a bond energy of -29.5 kcal / mol ( Figure 7 , Table 6).

[0111] Table 6 Docking results of Lentinus edodes peptide APVPIDQ and pancreatic lipase

[0112]

[0113] Lentinus edodes peptide IEPIKVP forms 9 bonds with the amino acid residues in the molecular chains of pancreatic lipase A and B, including 4 hydrogen bonds and 6 ionic bonds, with a bond energy of -40.2 kcal / mol ( Figure 8 , Table 7).

[0114] Table 7 Docking results of Lentinan peptide IEPIKVP and pancreatic lipase

[0115]

[0116] It can be seen that both shiitake mushroom peptide molecules mainly bind to the amino acid residues of the B chain of pancreatic lipase, forming multiple bonds with low bond energy, thereby exerting the inhibitory effect of the peptide molecules on pancreatic lipase and further exerting a lipid-lowering effect.

[0117] (9) Combined with xanthine oxidase (PDB: 3NVZ) uric acid lowering analysis results

[0118] 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.

[0119] Lentinus edodes peptide APVPIDQ formed 5 hydrogen bonds and 5 ionic bonds with the B and C chain amino acid residues of xanthine oxidase, with a bond energy of -43 kcal / mol; the peptide molecule had a higher binding strength with the B chain amino acid residues, with a bond energy of -37.3 kcal / mol ( Fig. 9 , Table 8).

[0120] Table 8 Docking results of Lentinus edodes peptide APVPIDQ and xanthine oxidase

[0121]

[0122] Lentinus edodes peptide IEPIKVP formed 6 hydrogen bonds, 7 ionic bonds and 1 H-π interaction force with the D, E and F chain residues of xanthine oxidase, with a binding bond energy of -49.3 kcal / mol; the peptide molecule had a higher binding strength with the F chain amino acid residues, with a binding bond energy of -26.2 kcal / mol, which was more than twice the binding bond energy between the peptide molecule and the D and E chain amino acid residues. The binding bond energy between the peptide molecule and the D and E chain amino acid residues was equivalent ( Fig.10 , Table 9).

[0123] Table 9 Docking results of Lentinan peptide IEPIKVP and xanthine oxidase

[0124]

[0125]

[0126] In summary, the two shiitake peptide molecules bind to different molecular chain amino acid residues of xanthine oxidase, synergistically inhibiting the activity of xanthine oxidase and exerting a lipid-lowering effect.

[0127] (10) 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 APVPIDQ formed 7 hydrogen bonds, 6 ionic bonds and 1 H-π interaction with Keap1 protein, with a binding energy of -33 kcal / mol, and the binding energy between the peptide molecule and the amino acid residues in the two protein links was not much different ( Fig.11 , Table 10).

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

[0131]

[0132] Lentinus edodes peptide IEPIKVP formed 9 hydrogen bonds and 8 ionic bonds with Keap1 protein, with a binding energy of -58.2 kcal / mol. The peptide molecule had a high strength in binding to the amino acid residues in the A molecular chain of Keap1 protein ( Fig.12 , Table 11).

[0133] Table 11 Docking results of Lentinus edodes peptide IEPIKVP and Keap1 protein molecule

[0134]

[0135]

[0136] In summary, both shiitake peptide molecules can bind to Keap1 receptor 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 effects on lowering blood pressure, lowering blood lipids, lowering blood sugar, lowering uric acid and anti-oxidation.

[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 peptides include shiitake peptide I and / or shiitake peptide II, and the amino acid sequences of the shiitake peptide I and shiitake peptide II are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

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; subjecting the third enzymatic hydrolyzate to a thermal reaction to obtain a shiitake mushroom thermal reaction base material; Desalting the shiitake mushroom thermal reaction base material to obtain a solution containing shiitake mushroom peptides; The solution containing the shiitake peptide is subjected to liquid chromatography tandem mass spectrometry analysis to obtain the shiitake peptide.

3. The preparation method according to claim 2, characterized in that: The temperature of the thermal reaction is 110-130° C., the pressure is 0.1-0.2 MPa, and the time is 90-120 min.

4. The preparation method according to claim 2, characterized in that: The steps of the desalination treatment include: Dissolving the shiitake mushroom thermal reaction base material with a trifluoroacetic acid solution having a volume concentration of 0.1% to obtain a shiitake mushroom base material dissolved liquid; 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 shiitake peptides; The elution buffer for elution is an aqueous solution containing 60 vol% acetonitrile and 0.1 vol% trifluoroacetic acid.

5. The preparation method according to claim 2, characterized in that: The steps of liquid chromatography tandem mass spectrometry analysis include: vacuum drying the solution containing shiitake peptide to obtain shiitake peptide 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.

6. The preparation method according to claim 5, characterized in that: The liquid phase parameters of the liquid chromatography tandem mass spectrometry detection are shown in the following table:

7. The preparation method according to claim 2, characterized in that: The mass ratio of the dried shiitake mushroom product to water is 1 g: 28-32 mL; the amount of the flavor protease is 800-1000 U / g of the dried shiitake mushroom product; The temperature of the first enzymatic hydrolysis is 45-55° C., the time is 40-50 min, and the pH value is 6.5-7.

5.

8. The preparation method according to claim 2, characterized in that: The dosage of the trypsin is 1800-2200 U / g dried shiitake mushrooms; the temperature of the second enzymolysis is 32-42° C., the time is 40-45 minutes, and the pH value is 7.5-8.

5.

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 one or more of the preparation of health products, medicines and foods.

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 lipids, lowering blood sugar, lowering uric acid and having antioxidant activity.

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