Lentinus edodes residue proteoglycan as well as preparation method and application thereof

Abstract

The invention relates to the field of mushroom residue waste utilization, in particular to lentinus edodes mushroom residue proteoglycan and a preparation method and application thereof. The lentinus edodes residue proteoglycan provided by the invention is proteoglycan which is obtained from lentinus edodes residues, is high in purity and stable in quality, can be stored for a long time, does not deteriorate, has a remarkable anti-inflammatory effect, and can be applied to animal feeds or medicines.

Description

[0001] This application is a divisional application with the application date of September 19, 2022, application number 202211147384.0, and invention name "A kind of shiitake mushroom residue proteoglycan and its preparation method and application". Technical Field

[0002] The invention relates to the field of mushroom residue waste utilization, in particular to a shiitake mushroom residue proteoglycan and a preparation method and application thereof. Background Art

[0003] Shiitake mushrooms are the edible fungi with the highest cultivation volume and the largest output in my country. They have high nutritional value and have significant health-promoting effects. However, with the rapid development of the shiitake mushroom industry, a large amount of waste culture medium and mushroom residues are produced after the cultivation and harvest of shiitake mushrooms. After the shiitake mushrooms are harvested, most of the mushroom residues cannot be processed in time and effectively utilized, which not only causes waste of resources, but also easily goes moldy, pollutes water sources and land resources, and has a great impact on the environment. Shiitake mushroom residues usually contain complexes such as mycelium residues and crude fibers that have undergone enzymatic hydrolysis and structural changes, which are rich in nutritional active ingredients such as protein, amino acids, carbohydrates, vitamins and trace elements. Therefore, the scientific and effective use of shiitake mushroom residues is of great significance to environmental protection, waste recycling and sustainable development of agricultural economy.

[0004] Proteoglycan is a complex formed by a protein or peptide and a polysaccharide, that is, the protein or peptide is connected to a specific position of the polysaccharide side chain through a covalent bond, and the content of polysaccharide is significantly higher than that of protein or peptide. Proteoglycan has a variety of functions such as anti-tumor, antibacterial and anticoagulant, and has broad development and application prospects in the fields of food, medicine and biomaterials.

[0005] At present, the resource utilization of shiitake mushroom residue has received widespread attention, including its use in the preparation of organic fertilizer for crops or ecological environment restoration materials. However, the research on most uses is not in-depth enough, and the high-value utilization rate of shiitake mushroom residue is still relatively low. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a shiitake mushroom residue proteoglycan and its preparation method and application. The shiitake mushroom residue proteoglycan provided by the present invention is a high-purity and stable-quality proteoglycan obtained from shiitake mushroom residue, has significant anti-inflammatory effects, and can be used in animal feed or medicine.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing shiitake mushroom residue proteoglycan, comprising the following steps:

[0009] 1) Mixing mushroom residue and extractant, and subjecting to high temperature and high pressure treatment; the conditions of the high temperature and high pressure treatment include: temperature of 120-130° C., pressure of 0.05-0.25 MPa, and time of 15-30 min; the extractant includes buffer solution or water; the pH of the buffer solution is 4.5-6.0;

[0010] 2) hydrolyzing the bacterial residue treated with high temperature and high pressure with a composite enzyme to obtain a first extract and a filter residue;

[0011] The complex enzyme comprises cellulase, xylanase and β-glucanase; based on the mass of the mushroom residue, the addition amount of the cellulase is 1100-1500 U / g, the addition amount of the xylanase is 400-800 U / g, and the addition amount of the β-glucanase is 1100-1500 U / g;

[0012] 3) extracting the filter residue again with high temperature and high pressure assisted complex enzyme to obtain a second extract; combining the first extract and the second extract to obtain an extract; concentrating and centrifuging the extract in sequence to obtain a centrifugal supernatant; the centrifugal speed is 8000-10000 rpm, and the centrifugal time is 20-30 min;

[0013] The centrifugal supernatant and anhydrous ethanol are mixed and precipitated to obtain an alcohol precipitate; after the centrifugal supernatant and anhydrous ethanol are mixed, the volume concentration of ethanol in the mixed solution is 70% to 80%;

[0014] 4) performing a first dialysis on the alcohol precipitate to obtain primary proteoglycan; the molecular weight cutoff of the first dialysis is 3500 Daltons;

[0015] 5) separating the primary proteoglycan by chromatography to obtain an eluent; the gel column for the chromatography separation comprises an propylene dextran gel column; the eluent for the chromatography comprises an NH4HCO3 solution; the concentration of the NH4HCO3 solution is 0.1 to 0.3 mol / L;

[0016] 6) performing a second dialysis on the eluate to obtain the Lentinus edodes residue proteoglycan; the molecular weight cutoff of the second dialysis is 3500 Daltons.

[0017] Preferably, before the extract and the ethanol solution are mixed, the extract is concentrated and centrifuged in sequence; the centrifugal speed is 8000-10000 rpm, and the centrifugal time is 20-30 min.

[0018] Preferably, the solid-liquid ratio of the concentrated extract is 1 g: (6-10) mL.

[0019] Preferably, the time for the alcohol precipitation of the mixture of the extract and anhydrous ethanol is 8 to 12 hours.

[0020] Preferably, the first dialysis and the second dialysis are performed for 48 to 72 hours respectively.

[0021] Preferably, the rotation speed of the enzymolysis is 100-200 r / min, the temperature of the enzymolysis is 40-55° C., and the time of the enzymolysis is 4-7 h.

[0022] The present invention provides a shiitake mushroom residue proteoglycan prepared by the preparation method described in the above technical solution, comprising a polysaccharide and a protein connected to the polysaccharide; the mass ratio of the protein to the polysaccharide is 8.5:88.9; the polysaccharide comprises 6 to 12 repeating units;

[0023] The main chain of the repeating unit contains an arabinose residue linked by an α-1,3-glycosidic bond, an arabinose residue linked by a β-1,2-glycosidic bond, a xylose residue linked by a β-1,4-glycosidic bond, a glucose residue linked by an α-1,6-glycosidic bond, a glucose residue linked by an α-1,4-glycosidic bond, a mannose residue linked by an α-1,4-glycosidic bond, a galactose residue linked by a β-1,3-glycosidic bond, and a xylose residue linked by an α-1,2-glycosidic bond;

[0024] The C-3 position of the mannose residue linked by the α-1,4-glycosidic bond is linked to a galactose side group, the C-6 position of the galactose residue linked by the β-1,3-glycosidic bond is linked to a xylose side group, and the C-6 position of the xylose residue linked by the α-1,2-glycosidic bond is linked to a mannose side group;

[0025] The amino acids in the protein include aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine;

[0026] The threonine in the protein is linked to the C-3 position of the α-1,6-linked glucose residue;

[0027] The serine in the protein is linked to the C-6 position of the α-1,4-linked glucose residue;

[0028] The average molecular weight of the shiitake mushroom residue proteoglycan is 11,000 to 13,000 Daltons.

[0029] Preferably, the molar ratio of the glucose residue, the arabinose residue, the galactose residue, the xylose residue and the mannose residue is 1.2:1.2:1.0:2.3:1.1.

[0030] The present invention also provides the use of the above-mentioned Lentinus edodes residue proteoglycan or the Lentinus edodes residue proteoglycan prepared by the above-mentioned preparation method in the preparation of anti-inflammatory drugs.

[0031] Preferably, the anti-inflammatory effect includes reducing the content of inflammatory factors and / or reducing the amount of NO released by inflammatory cells, and the inflammatory factors include one or more of TNF-α, IL-1β and IL-6.

[0032] Beneficial effects:

[0033] The invention provides a shiitake mushroom residue proteoglycan, comprising a polysaccharide and a protein connected to the polysaccharide; the mass ratio of the protein to the polysaccharide is 8.5:88.9; the polysaccharide comprises 6 to 12 repeating units; the main chain of the repeating unit contains an arabinose residue connected by an α-1,3-glycosidic bond, an arabinose residue connected by a β-1,2-glycosidic bond, a xylose residue connected by a β-1,4-glycosidic bond, a glucose residue connected by an α-1,6-glycosidic bond, a glucose residue connected by an α-1,4-glycosidic bond, a mannose residue connected by an α-1,4-glycosidic bond, a galactose residue connected by a β-1,3-glycosidic bond and a xylose residue connected by an α-1,2-glycosidic bond; the C-3 position of the mannose residue connected by an α-1,4-glycosidic bond is connected to a galactose residue; Side groups, the C-6 position of the galactose residue linked by the β-1,3-glycosidic bond is linked to a xylose side group, and the C-6 position of the xylose residue linked by the α-1,2-glycosidic bond is linked to a mannose side group; the amino acids in the protein include aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine; the threonine in the protein is linked to the C-3 position of the α-1,6-linked glucose residue; the serine in the protein is linked to the C-6 position of the α-1,4-linked glucose residue; the average molecular weight of the shiitake mushroom residue proteoglycan is 11,000 to 13,000 Daltons. The shiitake mushroom residue proteoglycan provided by the present invention is a proteoglycan obtained from shiitake mushroom residue with high purity and stable quality, which can be stored for a long time and will not deteriorate, has a significant anti-inflammatory effect, and can be used in animal feed or medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 is the chemical structural formula of Lentinus edodes residue proteoglycan, wherein n=6-12;

[0036] Figure 2 This is the infrared absorption spectrum of Lentinus edodes residue proteoglycan;

[0037] Figure 3 This is the H NMR spectrum of Lentinus edodes residue proteoglycan;

[0038] Figure 4 This is the C-NMR spectrum of Lentinus edodes residue proteoglycan;

[0039] Figure 5 Proteoglycan from Lentinus edodes residue 1 H- 13 C HSQC spectrum;

[0040] Figure 6 is the Griess standard curve;

[0041] Figure 7 The inhibitory effect of Lentinus edodes residue proteoglycan on LPS-induced NO release in macrophages RAW264.7;

[0042] Figure 8 The effect of Lentinus edodes residue proteoglycan on the release of TNF-α from macrophages RAW264.7 induced by LPS;

[0043] Fig. 9 The effect of Lentinus edodes residue proteoglycan on the release of IL-1β from macrophages RAW264.7 induced by LPS;

[0044] Fig.10 The effect of Lentinus edodes residue proteoglycan on the release of IL-6 from macrophages RAW264.7 induced by LPS;

[0045] in Figures 7-10 LEP S1 is a proteoglycan from Lentinus edodes residue. DETAILED DESCRIPTION

[0046] The present invention provides a shiitake mushroom residue proteoglycan, comprising a polysaccharide and a protein connected to the polysaccharide; the mass ratio of the protein to the polysaccharide is 8.5:88.9. In the present invention, the average molecular weight of the shiitake mushroom residue proteoglycan is 11,000 to 13,000 Daltons, preferably 11,500 to 12,000 Daltons, and more preferably 11,800 Daltons.

[0047] In the present invention, the polysaccharide includes 6 to 12 repeating units; the main chain of the repeating unit contains arabinose residues linked by α-1,3-glycosidic bonds, arabinose residues linked by β-1,2-glycosidic bonds, xylose residues linked by β-1,4-glycosidic bonds, glucose residues linked by α-1,6-glycosidic bonds, glucose residues linked by α-1,4-glycosidic bonds, mannose residues linked by α-1,4-glycosidic bonds, galactose residues linked by β-1,3-glycosidic bonds and xylose residues linked by α-1,2-glycosidic bonds.

[0048] In the present invention, a galactose side group is connected to the C-3 position of the mannose residue linked by the α-1,4-glycosidic bond, a xylose side group is connected to the C-6 position of the galactose residue linked by the β-1,3-glycosidic bond, and a mannose side group is connected to the C-6 position of the xylose residue linked by the α-1,2-glycosidic bond.

[0049] In the present invention, the molar ratio of the glucose residue, the arabinose residue, the galactose residue, the xylose residue and the mannose residue is preferably 1.2:1.2:1.0:2.3:1.1.

[0050] In the present invention, the amino acids in the protein include aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine.

[0051] In the present invention, the shiitake mushroom residue proteoglycan preferably includes the following amino acids: aspartic acid 6.45 mg / g, threonine 6.55 mg / g, serine 6.59 mg / g, glutamic acid 7.71 mg / g, proline 4.26 mg / g, glycine 4.75 mg / g, alanine 3.61 mg / g, cysteine ​​0.84 mg / g, valine 2.91 mg / g, methionine 0.83 mg / g, isoleucine 1.58 mg / g, leucine 1.79 mg / g, tyrosine 0.80 mg / g, phenylalanine 1.32 mg / g, lysine 2.12 mg / g, histidine 1.25 mg / g and arginine 1.57 mg / g.

[0052] In the present invention, the threonine in the protein is linked to the C-3 position of the α-1,6-linked glucose residue; the serine in the protein is linked to the C-6 position of the α-1,4-linked glucose residue.

[0053] The chemical structural formula of the mushroom residue proteoglycan of the present invention is shown in Figure 1 , wherein the degree of polymerization n is preferably 6-12.

[0054] The shiitake mushroom residue proteoglycan provided by the present invention has stable quality, can be stored for a long time without deterioration, has a purity of more than 95%, has the effect of inhibiting LPS (bacterial lipopolysaccharide)-induced NO release from mouse macrophages RAW264.7 in vitro, and can also reduce the levels of inflammatory factors TNF-α, IL-1β and IL-6 produced by LPS-induced mouse macrophages RAW264.7, and can be used as an anti-inflammatory agent or a raw material in the development of anti-inflammatory related tumor drugs.

[0055] The present invention also provides a method for preparing the above-mentioned Lentinus edodes residue proteoglycan, comprising the following steps:

[0056] 1) Mixing mushroom residue and extractant, and subjecting to high temperature and high pressure treatment; the conditions of the high temperature and high pressure treatment include: temperature of 120-130° C., pressure of 0.05-0.25 MPa, and time of 15-30 min; the extractant includes buffer solution or water; the pH of the buffer solution is 4.5-6.0;

[0057] 2) hydrolyzing the fungus residue treated with high temperature and high pressure with a composite enzyme to obtain an extract;

[0058] The complex enzyme comprises cellulase, xylanase and β-glucanase; based on the mass of the mushroom residue, the addition amount of the cellulase is 1100-1500 U / g, the addition amount of the xylanase is 400-800 U / g, and the addition amount of the β-glucanase is 1100-1500 U / g;

[0059] 3) mixing the extract and anhydrous ethanol for alcohol precipitation to obtain an alcohol precipitate;

[0060] 4) performing a first dialysis on the alcohol precipitate to obtain primary proteoglycan; the molecular weight cutoff of the first dialysis is 3500 Daltons;

[0061] 5) separating the primary proteoglycan by chromatography to obtain an eluent; the gel column for the chromatography separation comprises an propylene dextran gel column; the eluent for the chromatography comprises an NH4HCO3 solution; the concentration of the NH4HCO3 solution is 0.1 to 0.3 mol / L;

[0062] 6) performing a second dialysis on the eluate to obtain the Lentinus edodes residue proteoglycan; the molecular weight cutoff of the second dialysis is 3500 Daltons.

[0063] The invention mixes mushroom residues and an extractant and performs high temperature and high pressure treatment.

[0064] In the present invention, the mushroom residue preferably includes mushroom residue powder, and the particle size of the mushroom residue powder is preferably 50-100 meshes. The mushroom residue powder is more easily degraded by the composite enzyme, so that the active substances in the mushroom residue powder are fully released.

[0065] The present invention preferably further comprises mixing shiitake mushroom residue and an extractant before the high temperature and high pressure treatment. In the present invention, the extractant preferably comprises a buffer solution or water, and more preferably a sodium dihydrogen phosphate-citric acid buffer solution. In the present invention, the pH of the buffer solution is preferably 4.5 to 6.0, and more preferably 5.0. In the present invention, the concentration of the buffer solution is preferably 0.1 to 0.4 moL / L, and more preferably 0.1 to 0.3 moL / L. The present invention has no special requirements for the type of buffer solution, and it only needs to meet the above-mentioned pH and concentration requirements. The buffer solution in the embodiment of the present invention is preferably a sodium dihydrogen phosphate-citric acid buffer solution.

[0066] In the present invention, the concentration of disodium hydrogen phosphate in the extract is preferably 0.1-0.4 moL / L, more preferably 0.2 moL / L; the concentration of citric acid is preferably 0.1-0.3 moL / L, more preferably 0.1 moL / L. Under the conditions of the extractant of the present invention, the enzymatic activity of the complex enzyme is the strongest, which is beneficial to improving the enzymatic hydrolysis efficiency of the subsequent enzymatic hydrolysis step.

[0067] In the present invention, the volume ratio of the mass of the shiitake mushroom residue to the extractant is preferably 1g: (10-40) mL, further preferably 1g: (15-30) mL, and more preferably 1g: 20 mL. The specific solid-liquid ratio of the present invention can maximize the polysaccharide content and yield in the extract.

[0068] After mixing the mushroom residue and the extractant, the present invention performs high temperature and high pressure treatment. In the present invention, the temperature of the high temperature and high pressure treatment is preferably 120-130°C, more preferably 120-125°C, and more preferably 121°C; the pressure of the high temperature and high pressure treatment is preferably 0.05-0.0.25MPa, more preferably 0.1-0.2MPa, and more preferably 0.15MPa; the time of the high temperature and high pressure treatment is preferably 15-30min, more preferably 20-30min, and more preferably 30min. After the mushroom residue is treated under high temperature and high pressure conditions of the present invention, the structure of the mushroom residue becomes looser, which is more conducive to the enzymatic hydrolysis step and improves the release of active substances.

[0069] After high temperature and high pressure treatment, the present invention uses a composite enzyme to enzymolyze the mushroom residue treated with high temperature and high pressure to obtain an extract. In the present invention, the composite enzyme preferably includes cellulase, xylanase and β-glucanase; based on the mass of the mushroom residue, the addition amount of the cellulase is preferably 1100-1500U / g, more preferably 1150-1300U / g, and more preferably 1200U / g; the addition amount of the xylanase is preferably 400-800U / g, more preferably 450-650U / g, and more preferably 500U / g; the addition amount of the β-glucanase is preferably 1100-1500U / g, more preferably 1150-1300U / g, and more preferably 1200U / g. The specific composite enzyme combination of the present invention has the best enzymolysis efficiency for the mushroom residue, which improves the utilization rate of the mushroom residue. The present invention has no special requirements on the sources of cellulase, xylanase and beta-glucanase, and common commercial products can be used.

[0070] In the present invention, the rotation speed of the enzymatic hydrolysis is preferably 100-200 r / min, more preferably 150-180 r / min, more preferably 160 r / min; the temperature of the enzymatic hydrolysis is preferably 40-50°C, more preferably 43-48°C, more preferably 45°C; the time of the enzymatic hydrolysis is preferably 5-7h, more preferably 5.5-6.5h, more preferably 6h. The specific enzymatic hydrolysis conditions of the present invention maximize the enzymatic hydrolysis efficiency.

[0071] The present invention preferably further comprises enzyme inactivation and centrifugation after enzymolysis. The present invention preferably performs an enzyme inactivation step on the enzymolysis solution after enzymolysis to inactivate the complex enzyme in the enzymolysis solution. The enzyme inactivation of the present invention is preferably performed in a boiling water bath, and the boiling water bath time is preferably 5 to 30 minutes, more preferably 8 to 20 minutes, and more preferably 10 minutes.

[0072] After the enzyme is inactivated, the enzymatic hydrolyzate is centrifuged to obtain a filtrate and a filter residue. The centrifugal speed is preferably 6000-10000 r / min, more preferably 7000-9000 r / min, and more preferably 8000 r / min; the centrifugal time is preferably 10-30 min, more preferably 13-20 min, and more preferably 15 min.

[0073] After obtaining the filtrate, the present invention preferably subjects the filter residue to high temperature and high pressure treatment and enzymolysis treatment 1-2 times according to the above scheme, and combines the filtrates of multiple treatments to obtain the extract. The present invention can improve the raw material utilization rate of the mushroom residue by subjecting the treated filter residue to high temperature and high pressure treatment and enzymolysis again.

[0074] After obtaining the extract, the present invention preferably concentrates and centrifuges the extract in sequence to obtain a supernatant as the extract after centrifugation. In the present invention, the concentration preferably includes reduced pressure concentration; the solid-liquid ratio of the concentrated extract is preferably 1g: (6-10) mL, more preferably 1g: (8-10) mL, and more preferably 1g: 8 mL. The present invention can facilitate the subsequent preparation of pure sediments by concentrating the extract to an appropriate solid-liquid ratio, thereby improving the purity of shiitake mushroom residue proteoglycans.

[0075] In the present invention, the centrifugal speed is preferably 8000-10000rpm, more preferably 8500-9500rpm, and more preferably 9000rpm; the centrifugal time is preferably 20-30min, more preferably 25-30min, and more preferably 30min. The present invention can separate the insoluble matter in the extract by centrifuging at an appropriate speed and time, thereby improving the purity of the shiitake mushroom residue proteoglycan.

[0076] After obtaining the centrifuged extract, the centrifuged extract is mixed with numerous ethanol to perform alcohol precipitation to obtain an alcohol precipitate.

[0077] In the present invention, the mixed alcohol precipitation method preferably includes: slowly adding anhydrous ethanol to the centrifuged extract, stirring while adding, until the volume concentration of ethanol in the mixed solution reaches 70% to 80%, standing at room temperature for 8 to 12 hours, centrifuging, and obtaining a precipitate as the alcohol precipitate; the volume concentration of ethanol in the mixed solution is further preferably 75% to 80%, more preferably 80%; the temperature of the room temperature is preferably 20 to 25°C; the standing time is further preferably 10 to 12 hours, more preferably 12 hours; the speed and time of the centrifugation are the same as above, and are not repeated here. The present invention can obtain proteoglycans of a specific molecular weight by mixing an appropriate amount of anhydrous ethanol with the centrifuged extract, and the proteoglycans in the extract can be precipitated as much as possible by standing for a suitable time, and the prepared proteoglycans have better anti-inflammatory effects.

[0078] After obtaining the alcohol precipitate, the present invention preferably adds distilled water to the alcohol precipitate to fully dissolve it, and after centrifugation, the supernatant is taken for the first dialysis to obtain a dialysate; the speed and time of the centrifugation are the same as above, and are not repeated here; the molecular weight cutoff of the first dialysis is 3500 Daltons. In the present invention, the time of the first dialysis is preferably 48 to 72 hours, more preferably 60 to 72 hours, and more preferably 72 hours. The present invention performs dialysis by a suitable molecular weight cutoff, which can separate small molecule sugars and proteoglycans, thereby further improving the purity of proteoglycans.

[0079] After obtaining the dialysate, the present invention preferably freeze-dries the dialysate to obtain the primary proteoglycan. The present invention has no special requirements for the freeze-drying method, and any method known to those skilled in the art can be used.

[0080] After obtaining the primary proteoglycan, the present invention preferably dissolves the primary proteoglycan in distilled water, centrifuges, takes the supernatant for chromatographic separation, and collects the eluate at the peak of the signal peak according to the ultraviolet and parallax signals; the speed and time of the centrifugation are the same as above and will not be repeated here. The gel column for chromatographic separation of the present invention includes an propylene dextran gel column, preferably a Sephacryl S-300HR gel column; the eluent during the chromatography includes an NH4HCO3 solution; the concentration of the NH4HCO3 solution is 0.1 to 0.3 mol / L, preferably 0.2 mol / L. The present invention can further improve the purity of proteoglycans through appropriate chromatographic separation.

[0081] After obtaining the eluate, the present invention performs a second dialysis on the eluate to obtain the shiitake mushroom residue proteoglycan; the molecular weight cutoff of the second dialysis is 3500 Daltons. In the present invention, the time of the second dialysis is preferably 48 to 72 hours, more preferably 60 to 72 hours, and more preferably 72 hours.

[0082] The shiitake mushroom residue proteoglycan prepared by the method provided by the present invention has stable quality, can be stored for a long time without deterioration, has a purity of more than 95%, has the effect of inhibiting the release of NO by mouse macrophages RAW264.7 induced by LPS (bacterial lipopolysaccharide) in vitro, and can also reduce the levels of inflammatory factors TNF-α, IL-1β and IL-6 produced by mouse macrophages RAW264.7 induced by LPS, and can be used as an anti-inflammatory agent or a raw material in the development of anti-inflammatory related tumor drugs.

[0083] The present invention also provides the use of the above-mentioned Lentinus edodes residue proteoglycan or the Lentinus edodes residue proteoglycan prepared by the above-mentioned preparation method in the preparation of anti-inflammatory drugs.

[0084] In order to further illustrate the present invention, a shiitake mushroom residue proteoglycan and its preparation method and application provided by the present invention are described in detail below in combination with examples and drawings, but they should not be construed as limiting the scope of protection of the present invention.

[0085] Example 1

[0086] Preparation of Proteoglycan from Lentinus edodes Residue

[0087] 1. Pretreatment of mushroom residue: The discarded mushroom residue after picking mushrooms is dried, crushed, and passed through a 60-mesh sieve to obtain mushroom residue powder, wherein the mushroom residue comes from Shanghai Chengying Agricultural Development Co., Ltd.;

[0088] 2. High temperature and high pressure assisted complex enzyme extraction of shiitake mushroom residue: weigh 10.0g of shiitake mushroom residue powder into a conical flask, add 200mL of extractant, wherein the extractant is disodium hydrogen phosphate-citric acid buffer, the concentration of disodium hydrogen phosphate is 0.2moL / L, the concentration of citric acid is 0.1moL / L, pH=5.0; shake well and place in a high pressure sterilizer, set the temperature to 121℃, 0.15MPa, 30min, after the reaction is completed, take out and cool to room temperature. Add complex enzyme for enzymolysis, wherein the complex enzyme is cellulase, xylanase and β-glucanase, all of which are purchased from Shanghai Yuanye Biotechnology Co., Ltd.; based on the mass of shiitake mushroom residue, the addition amount of cellulase is 1500U / g, the addition amount of xylanase is 1000U / g, and the addition amount of β-glucanase is 1500U / g. The enzyme was hydrolyzed for 6 hours at 160 r / min and 45°C in a shaker. After the enzymatic hydrolysis reaction, the enzyme was inactivated by boiling water bath for 10 minutes, and then centrifuged at 8000 r / min for 15 minutes to obtain the filtrate and residue.

[0089] 3. The filter residue obtained in step 2 was subjected to high temperature and high pressure assisted complex enzyme extraction again according to the method described in step 2, the two filtrates were combined and concentrated under reduced pressure to a solid-liquid ratio of 1 g: 8 mL; the concentrate was centrifuged at 9000 rpm for 30 min to obtain the supernatant.

[0090] 4. Slowly add anhydrous ethanol to the supernatant obtained in step 3, stirring while adding, until the ethanol content reaches 80% (volume fraction), stand at room temperature for 12 hours, centrifuge at 9000rpm to remove the alcohol precipitate supernatant, and collect the precipitate. Add water to fully dissolve the precipitate, centrifuge at 8000rpm for 30min to take the supernatant. Use a 3500 Dalton dialysis bag at room temperature, dialyze with distilled water for 72 hours, and freeze-dry the dialysate to obtain 857.4mg of gray solid sample (yield 28.58%).

[0091] 5. Take 1 g of the solid sample obtained in step 4, add 20 mL of water to dissolve it, centrifuge it, and use the supernatant The sample was separated by primeFPLC chromatography system, and separated by gel column chromatography on a Sephacryl S-300HR gel column (Φ4.6cm×100cm), and eluted with 0.2mol / LNH4HCO3 solution of 2 times the column volume. According to the signal of the parallax detector, the sample was collected and concentrated at the signal peak, and dialyzed with distilled water for 48 hours at room temperature using a 3500 Dalton dialysis bag. After freeze-drying, Lentinus edodes residue proteoglycan (850mg) was obtained. The polysaccharide content was 88.9% by the phenol-sulfuric acid method, and the protein content was 8.5% by the BCA method.

[0092] The samples were separated by high performance gel chromatography (Waters 2695 pump separation system) in series with TSK-GEL series G3000PWXL and G4000PWXL columns (7.8 mm × 300 mm, TOSOH, Japan), and analyzed by an octagonal laser light scattering detector (MALLS, Wyatt) and a Waters 2414 differential detector (RI). The results are shown in Figure 2 to Figure 5 .

[0093] Depend on Figure 2 It can be seen that at 3418.39cm -1 There is a strong and broad absorption peak at 2933.14 cm, which is the stretching vibration of OH and NH. -1 The weak absorption peak at 1714.43 cm was assigned to the stretching vibration of CH. -1 The weak absorption peak at 1638.80cm is the absorption peak of carbonyl. -1 and 1402.41cm -1 The characteristic absorption peaks at 1070.29cm are the bending vibration absorption peaks of amide and amino groups. -1 and 1044.05cm -1 The characteristic absorption peak at 897.51cm indicates the presence of pyranoside in proteoglycan. -1 The absorption peak at is attributed to the CH stretching vibration, which indicates the existence of β configuration in the proteoglycan component.

[0094] Depend on Figures 2 to 5 It can be seen that the molecular weight of the proteoglycan is 11,800 Daltons. The monosaccharide composition was analyzed by high performance anion chromatography, and the results showed that the polysaccharide part of the proteoglycan contained glucose, arabinose, galactose, xylose and mannose residues with a molar ratio of 1.2:1.2:1.0:2.3:1.1. NMR, IR and methylation analysis proved that the main chain of the polysaccharide part of the proteoglycan contained α-1,3-linked arabinose residues ( Figure 1 A in), β-1,2-linked arabinose residues ( Figure 1 B), β-1,4-linked xylose residues ( Figure 1 C), α-1,6-linked glucose residues ( Figure 1 D in), α-1,4-linked glucose residues ( Figure 1 E in), α-1,3,4-linked mannose residues ( Figure 1 F in), β-1,3,6-linked galactose residues ( Figure 1 G) and α-1,2,4-linked xylose residues ( Figure 1 H in the figure), galactose residues, galactose residues and xylose residues are also connected to galactose ( Figure 1I), xylose ( Figure 1 J) and mannose side groups ( Figure 1 The amino acid analysis results showed that the protein part of the proteoglycan was composed of amino acids such as aspartic acid, threonine, serine, glutamic acid, proline and glycine. The β-elimination reaction confirmed that the protein part was connected to the C-3 position and C-6 position of the α-1,6-linked glucose residue and the α-1,4-linked glucose residue through threonine and serine, respectively. Based on the above results, the structural formula of the proteoglycan was obtained as follows Figure 1 shown.

[0095] Example 2

[0096] Analysis of the inhibitory activity of Lentinus edodes residue proteoglycan on NO release from mouse macrophages RAW264.7 induced by LPS in vitro

[0097] 1. Sample preparation

[0098] Accurately weigh 4 mg of the solid sample prepared in step 5 of Example 1, add 1 mL of PBS solution to fully dissolve it, centrifuge at 18000 g for 30 min to sterilize, transfer the supernatant to a sterile tube using a sterile filter, dilute to 100 μg / mL, 200 μg / mL and 400 μg / mL solutions for later use.

[0099] 2. Analysis of the inhibitory activity of NO release from LPS-induced mouse macrophage RAW264.7

[0100] Preparation of Griess reagent: 0.1wt.% N-naphthylethylenediamine hydrochloride aqueous solution and 1wt.% p-aminobenzenesulfonic acid solution were mixed in equal volumes. Use NaNO2 solution to draw the Griess standard curve, see Figure 6 .

[0101] Inhibition of LPS-induced NO release from RAW264.7 macrophages: RAW264.7 was diluted to 5×10 5 / mL cell suspension, 160μL per group, cultured at 37℃ until cells were completely attached, added 20μL LPS (LPS concentration 2μg / mL) and 20μL test sample (the test sample concentrations were 100μg / mL, 200μg / mL, 400μg / mL), added 20μL LPS (LPS concentration 2μg / mL) and 20μL PBS (pH=7) to each well of the LPS group, added 40μL PBS to each well of the PBS group, cultured at 37℃ for 48h, aspirated 100μL of the supernatant, added 50μL Griess reagent, reacted for 10min, measured the OD value at 543nm, and substituted into the standard curve to calculate the NO release. Results are shown in Figure 7 and Table 1.

[0102] Table 1 NO release in different groups

[0103]

[0104] Note: “-” indicates PBS group, and the same applies to the following tables.

[0105] from Figure 7 As shown in Table 1, the proteoglycan from Lentinus edodes residue can inhibit the release of NO from LPS-induced mouse macrophages RAW264.7, and play a certain anti-inflammatory role.

[0106] Example 3

[0107] Analysis of Lentinus edodes residue proteoglycans in reducing the levels of inflammatory factors TNF-α, IL-1β and IL-6 produced by mouse macrophages RAW264.7 induced by LPS

[0108] 1. Sample preparation

[0109] Accurately weigh about 4 mg of the sample, add a certain amount of PBS solution to fully dissolve it, centrifuge at 18000g for 30 min to sterilize, filter the supernatant with a sterile filter, transfer to a sterile tube, and dilute it into 100μg / mL, 200μg / mL and 400μg / mL solutions for later use.

[0110] 2. Determination and comparison of TNF-α, IL-1β and IL-6 contents in the supernatant of LPS-induced macrophage RAW264.7 cells

[0111] RAW264.7 was diluted to 5×10 5 Each group was treated with 160 μL of cell suspension of 1 μg / mL, and the cells were cultured at 37°C until they were completely attached. Then, 20 μL of LPS (the concentration of LPS was 2 μg / mL) and 20 μL of the sample to be tested (the concentrations of the sample to be tested were 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively) were added to the sample group. 20 μL (the concentration of LPS was 2 μg / mL) and 20 μL of PBS (pH = 7) were added to each well of the LPS group. 40 μL of PBS was added to each well of the PBS group. After culturing at 37°C for 48 hours, the supernatant was aspirated and the TNF-α, IL-1β and IL-6 determination ELISA kits produced by Beijing Zhengsibai Company were used to determine the contents of TNF-α, IL-1β and IL-6 in the supernatant. The results are shown in Figures 8 to 10 and Table 2.

[0112] Table 2 Results of TNF-α, IL-1β and IL-6 contents in the supernatant of different groups

[0113]

[0114] from Figures 8 to 10 As can be seen from the results in Table 2, Lentinus edodes residue proteoglycan has a good activity in inhibiting the release of inflammatory factors TNF-α, IL-1β and IL-6 by LPS-stimulated macrophages RAW264.7, and has significant in vitro anti-inflammatory activity.

[0115] In summary, the shiitake mushroom residue proteoglycan provided by the present invention is a high-purity and stable-quality proteoglycan obtained from shiitake mushroom residue, has a significant anti-inflammatory effect, and can be applied to animal feed or medicine.

[0116] Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing shiitake mushroom residue proteoglycan, characterized in that: The following steps are involved: 1) mixing shiitake mushroom residue and an extractant, and subjecting the mixture to high temperature and high pressure treatment; The conditions of the high temperature and high pressure treatment include: temperature 120-130°C, pressure 0.05-0.25 MPa, time 15-30 min; the extractant includes buffer solution or water; the pH of the buffer solution is 4.5-6.0; 2) hydrolyzing the bacterial residue treated with high temperature and high pressure with a composite enzyme to obtain a first extract and a filter residue; The complex enzyme comprises cellulase, xylanase and β-glucanase; based on the mass of the mushroom residue, the added amount of the cellulase is 1500U / g, the added amount of the xylanase is 1000U / g, and the added amount of the β-glucanase is 1500U / g; 3) extracting the filter residue again with high temperature and high pressure assisted complex enzyme to obtain a second extract; combining the first extract and the second extract to obtain an extract; concentrating and centrifuging the extract in sequence to obtain a centrifugal supernatant; the centrifugal speed is 8000-10000 rpm, and the centrifugal time is 20-30 min; 4) mixing the centrifugal supernatant and anhydrous ethanol for alcohol precipitation to obtain an alcohol precipitate; after the centrifugal supernatant and anhydrous ethanol are mixed, the volume concentration of ethanol in the mixed solution is 70% to 80%; 5) performing a first dialysis on the alcohol precipitate to obtain primary proteoglycan; the molecular weight cutoff of the first dialysis is 3500 Daltons; 6) separating the primary proteoglycan by chromatography to obtain an eluent; the gel column for the chromatography separation comprises an propylene dextran gel column; the eluent for the chromatography comprises an NH4HCO3 solution; the concentration of the NH4HCO3 solution is 0.1 to 0.3 mol / L; 7) The eluate is dialyzed for the second time to obtain the Lentinus edodes residue proteoglycan; the molecular weight cut-off of the second dialysis is 3500 Daltons.

2. The preparation method according to claim 1, characterized in that: The solid-liquid ratio of the concentrated extract is 1 g: (6-10) mL.

3. The preparation method according to claim 1, characterized in that: The time for the mixed alcohol precipitation of the extract and anhydrous ethanol is 8 to 12 hours.

4. The preparation method according to claim 1, characterized in that: The duration of the first dialysis and the second dialysis is 48 to 72 hours respectively.

5. The preparation method according to claim 1, characterized in that: The rotation speed of the enzymolysis is 100-200 r / min, the temperature of the enzymolysis is 40-55° C., and the time of the enzymolysis is 4-7 hours.

6. The shiitake mushroom residue proteoglycan prepared by the preparation method according to any one of claims 1 to 5, characterized in that: It comprises a polysaccharide and a protein connected to the polysaccharide; the mass ratio of the protein to the polysaccharide is 8.5:88.9; the polysaccharide comprises 6 to 12 repeating units; The main chain of the repeating unit contains an arabinose residue linked by an α-1,3-glycosidic bond, an arabinose residue linked by a β-1,2-glycosidic bond, a xylose residue linked by a β-1,4-glycosidic bond, a glucose residue linked by an α-1,6-glycosidic bond, a glucose residue linked by an α-1,4-glycosidic bond, a mannose residue linked by an α-1,4-glycosidic bond, a galactose residue linked by a β-1,3-glycosidic bond, and a xylose residue linked by an α-1,2-glycosidic bond; The C-3 position of the mannose residue linked by the α-1,4-glycosidic bond is linked to a galactose side group, the C-6 position of the galactose residue linked by the β-1,3-glycosidic bond is linked to a xylose side group, and the C-6 position of the xylose residue linked by the α-1,2-glycosidic bond is linked to a mannose side group; The amino acids in the protein include aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine; The threonine in the protein is linked to the C-3 position of the α-1,6-linked glucose residue; The serine in the protein is linked to the C-6 position of the α-1,4-linked glucose residue; The average molecular weight of the shiitake mushroom residue proteoglycan is 11,000 to 13,000 Daltons.

7. The shiitake mushroom residue proteoglycan according to claim 6, characterized in that: The molar ratio of the glucose residue, the arabinose residue, the galactose residue, the xylose residue and the mannose residue is 1.2:1.2:1.0:2.3:1.

1.

8. Use of the Lentinus edodes residue proteoglycan prepared by the preparation method according to any one of claims 1 to 5 or the Lentinus edodes residue proteoglycan according to claim 6 or 7 in the preparation of anti-inflammatory drugs.

9. The use according to claim 8, characterized in that: The anti-inflammatory effect includes reducing the content of inflammatory factors and / or reducing the NO release of inflammatory cells. The inflammatory factors include one or more of TNF-α, IL-1β and IL-6.

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