Cascade extraction process of active ingredients of edible fungi

Abstract

The application discloses a kind of cascade extraction process of active ingredient of edible fungi, comprising the following steps: edible fungi ultrafine powder and ultrapure water are mixed to form suspension, after standing, centrifugal treatment is taken to supernatant and is concentrated;Concentrated liquid is adjusted to alkaline, after water bath, centrifugal treatment is obtained to supernatant A;Then supernatant A is adjusted to acid, standing makes that protein is precipitated, centrifugal treatment is obtained to supernatant B and precipitate A, precipitate A freeze-drying is obtained edible fungi protein;Supernatant B and ethanol are mixed, after standing, centrifugal treatment is obtained precipitate B, after washing, cooling and drying, edible fungi polysaccharide is obtained;Edible fungi protein is dissolved in water, with magnetic surface molecular imprinting polymer, after oscillation mixing at room temperature, elution is carried out, then rotary evaporation concentrated eluent is frozen and dried, realize the enrichment of ergothioneine.The application extracts various effective components in edible fungi, improves the utilization rate of edible fungi, reduces the loss rate of active ingredient of edible fungi.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of resource utilization, in particular to a cascade extraction process of active ingredients of edible fungi. BACKGROUND

[0002] Edible fungi protein accounts for about 19-37% of the dry weight of edible fungi, and is rich in essential amino acids for the human body. Studies have found that edible fungi protein has multiple functional activities such as anti-tumor, immune regulation, anti-virus, anti-inflammatory, and anti-oxidation. Common plant protein extraction methods include solvent extraction, salt dissolution, and enzyme method. Edible fungi polysaccharides have various biological activities, including anti-oxidation, anti-tumor, anti-inflammatory, immune regulation, blood sugar reduction, intestinal flora regulation, and anti-virus, and are often used for the prevention and treatment of various chronic diseases. The extraction methods of edible fungi polysaccharides include hot water extraction and water extraction-alcohol precipitation. Ergothioneine is a natural small molecule histidine thioether derivative, which is widely present in edible fungi fruiting bodies and has a content of more than 0.60 mg / g dry weight, and has good biological activities such as anti-oxidation, anti-inflammatory, and cell protection. The extraction methods of ergothioneine include reflux extraction and enzymatic extraction.

[0003] In the traditional development and application process of active ingredients of edible fungi, extraction and purification is one of the key steps. It not only affects the yield of active ingredients, but also has an impact on the environment, economy, and other aspects. In the extraction of active ingredients of edible fungi, most cases only extract a single active substance, which is difficult to achieve ideal results under the premise of long extraction time, high extraction temperature, and large energy consumption, which greatly limits the development and utilization of active substances. Only a small number of cases involve the composite extraction of active ingredients, such as the Chinese patent with the application number CN201410673654.0, which discloses a method for extracting or separating protein from the alcohol precipitation supernatant during the extraction of edible fungi polysaccharides, but there is no process for cascade extraction of multiple active ingredients from edible fungi. SUMMARY

[0004] The purpose of the present application is to provide a cascade extraction process of active ingredients of edible fungi, which simultaneously extracts edible fungi protein, edible fungi polysaccharides, and ergothioneine from edible fungi. The method has high utilization rate of raw materials, simple operation, and significantly reduces the loss rate of active ingredients of edible fungi.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] A cascade extraction process of active ingredients of edible fungi, comprising the following steps:

[0007] Step one: mixing edible fungi ultra-fine powder and ultrapure water according to a mass ratio of 1:(20-60) to form a suspension, and after standing, centrifuging to obtain supernatant, and concentrating the supernatant to 1 / 5 of the volume of the original suspension to obtain a concentrated solution;

[0008] Step 2: The concentrate is adjusted to alkaline, and then centrifuged after water bath to obtain supernatant A; then the supernatant A is adjusted to acidic, allowed to stand to allow protein to precipitate, and centrifuged to obtain supernatant B and precipitate A, and the precipitate A is freeze-dried to obtain edible fungus protein;

[0009] Step 3: mixing the supernatant B with 50%-100% ethanol in a water-to-alcohol volume ratio of 1:(2-10), allowing the mixture to stand at a temperature of 0-10°C and then centrifuging to obtain a precipitate B, which is then washed, cooled, and dried to obtain an edible fungus polysaccharide;

[0010] Step 4: dissolving the edible fungus protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecularly imprinted polymer at a mass ratio of 1:1 under shaking at room temperature, eluting the mixture with an eluent prepared by mixing methanol and acetic acid at a volume ratio of 1:9, and then eluting the eluent by rotary evaporation and concentration, and then freeze-drying the eluent to achieve the enrichment of ergothioneine.

[0011] Furthermore, the centrifugal treatment in the above step is performed at a rotation speed of 4000-6000 rpm and a centrifugal time of 5-20 min.

[0012] Furthermore, the edible fungus in step 1 is one of Pleurotus eryngii, Pleurotus ostreatus or Enoki mushroom.

[0013] Furthermore, the concentrated solution in step 2 is adjusted to an alkaline pH value in the range of 9-13, the water bath temperature is 50-80° C., and the water bath time is 1-8 hours.

[0014] Furthermore, the supernatant A in step 2 is adjusted to an acidic pH value in the range of 2-4, and the standing time is 30-90 minutes.

[0015] Furthermore, the standing time in step 3 is 1-5 hours.

[0016] Furthermore, in step 3, 80% ethanol is used for washing three times.

[0017] Furthermore, the preparation process of the magnetic surface molecularly imprinted polymer in step 4 includes the following steps:

[0018] (1) Preparation of amino-functionalized Fe3O4@SiO2 nanoparticles:

[0019] Fe3O4 nanoparticles, ethanol, and water were mixed at a mass ratio of 1:4000:1000 to obtain a mixture A. Subsequently, a 28 wt% ammonia solution was mixed with the mixture A at a volume ratio of 1:(30-40) to obtain a mixture B. Finally, tetraethyl orthosilicate was thoroughly mixed with the mixture B at a volume ratio of 1:(30-40), and then recovered by adsorption using a magnet. After washing and vacuum drying, Fe3O4@SiO2 nanoparticles were obtained. Fe3O4@SiO2 nanoparticles, ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, washed, and vacuum dried to obtain amino-functionalized Fe3O4@SiO2 nanoparticles.

[0020] (2) Preparation of magnetic surface molecularly imprinted nanomaterials: using ergothioneine as a template molecule, methacrylic acid MAA and 4-vinylpyridine 4-VP as functional monomers, ethylene glycol acrylate EDMA as a crosslinker, amino-functionalized Fe3O4@SiO2 nanoparticles as a carrier, water, ethanol and tetrahydrofuran as porogens, and azobisisobutyronitrile as an initiator, water bath polymerization was performed at 65°C for 48h to obtain magnetic surface molecularly imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP was 1:(3-7):(3-7), the mass ratio of ergothioneine to ethylene glycol acrylate EDMA was 1:(26-30), the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles was 1:(4-9), the mass ratio of ergothioneine to azobisisobutyronitrile was 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran was 1:40:40:20;

[0021] (3) Removal of template molecules: The magnetic surface molecularly imprinted nanomaterials were eluted with an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer with an imprinting cavity complementary to the target molecule ergothioneine in terms of functional groups, size and shape.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Traditional extraction methods are usually extracted for a single active ingredient, while the cascade extraction process of the present invention can simultaneously extract edible fungus protein, polysaccharide and ergothioneine from edible fungi at one time, making the best use of edible fungus raw materials, improving the comprehensive utilization rate of raw materials, and reducing resource waste. This process has significant economic and environmental advantages and contributes to the comprehensive development of edible fungus multi-active ingredients. DETAILED DESCRIPTION

[0024] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is described in detail below with reference to specific embodiments.

[0025] Example 1: A cascade extraction process for active ingredients of edible fungi is as follows:

[0026] Step 1: mixing the King Oyster Mushroom ultrafine powder and ultrapure water in a mass ratio of 1:20 to form a suspension, allowing the suspension to stand and then centrifuging at 5000 rpm for 10 minutes to obtain a supernatant, which was then concentrated to 1 / 5 of the volume of the original suspension to obtain a concentrated solution;

[0027] Step 2: The pH value of the concentrate was adjusted to 9, and after being incubated in a water bath at 50°C for 1 hour, the concentrate was centrifuged at 4000 rpm for 20 minutes to obtain supernatant A; the pH value of the supernatant A was then adjusted to 2, and the concentrate was allowed to stand for 30 minutes to allow protein precipitation, and the concentrate was centrifuged at 5000 rpm for 10 minutes to obtain supernatant B and precipitate A. The precipitate A was freeze-dried to obtain Pleurotus eryngii protein;

[0028] Step 3: The supernatant B was mixed with 50% ethanol in a water-to-alcohol volume ratio of 1:2, and the mixture was allowed to stand at 10°C for 1 hour, followed by centrifugation at 6000 rpm for 5 minutes to obtain a precipitate B. The precipitate B was then washed three times with 80% ethanol, cooled and dried to obtain Pleurotus eryngii polysaccharide.

[0029] Step 4: dissolving the Pleurotus eryngii protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecularly imprinted polymer at a mass ratio of 1:1 under shaking at room temperature, eluting the mixture with an eluent prepared by mixing methanol and acetic acid at a volume ratio of 1:9, concentrating the eluent by rotary evaporation and freeze-drying the mixture to achieve the enrichment of ergothioneine.

[0030] It should be noted that the magnetic surface molecularly imprinted polymer is prepared by the following steps:

[0031] (1) Preparation of amino-functionalized Fe3O4@SiO2 nanoparticles:

[0032] Fe3O4 nanoparticles, ethanol, and water were mixed at a mass ratio of 1:4000:1000 to obtain a mixture A. Subsequently, a 28wt% ammonia solution was mixed with the mixture A at a volume ratio of 1:30 to obtain a mixture B. Finally, tetraethyl orthosilicate was thoroughly mixed with the mixture B at a volume ratio of 1:35, and then recovered by adsorption using a magnet. After washing and vacuum drying, Fe3O4@SiO2 nanoparticles were obtained. Fe3O4@SiO2 nanoparticles, ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, washed, and vacuum dried to obtain amino-functionalized Fe3O4@SiO2 nanoparticles.

[0033] (2) Preparation of magnetic surface molecularly imprinted nanomaterials: using ergothioneine as a template molecule, methacrylic acid MAA and 4-vinylpyridine 4-VP as functional monomers, ethylene glycol acrylate EDMA as a crosslinker, amino-functionalized Fe3O4@SiO2 nanoparticles as a carrier, water, ethanol and tetrahydrofuran as porogens, and azobisisobutyronitrile as an initiator, water bath polymerization was performed at 65°C for 48h to obtain magnetic surface molecularly imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP was 1:3:4, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA was 1:26, the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles was 1:4, the mass ratio of ergothioneine to azobisisobutyronitrile was 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran was 1:40:40:20;

[0034] (3) Removal of template molecules: The magnetic surface molecularly imprinted nanomaterials were eluted with an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer with an imprinting cavity complementary to the target molecule ergothioneine in terms of functional groups, size and shape.

[0035] Example 2: A cascade extraction process for active ingredients of edible fungi is as follows:

[0036] Step 1: Mix the oyster mushroom ultrafine powder and ultrapure water in a mass ratio of 1:40 to form a suspension, let it stand, and then centrifuge it at 4600 rpm for 15 minutes to obtain the supernatant, and concentrate the supernatant to 1 / 5 of the volume of the original suspension to obtain a concentrated solution;

[0037] Step 2: The pH value of the concentrate was adjusted to 10, and the concentrate was incubated in a water bath at 60°C for 2 hours and centrifuged at 5000 rpm for 10 minutes to obtain supernatant A; the pH value of supernatant A was then adjusted to 2.8, and the concentrate was allowed to stand for 90 minutes to allow protein precipitation, and centrifuged at 5000 rpm for 10 minutes to obtain supernatant B and precipitate A. The precipitate A was freeze-dried to obtain oyster mushroom protein;

[0038] Step 3: Mix the supernatant B with 100% ethanol by volume in a water-to-alcohol ratio of 1:4, let it stand at 5°C for 4 hours, and then centrifuge at 5500 rpm for 18 minutes to obtain precipitate B. Wash the precipitate B three times with 80% ethanol, cool and dry it, and obtain oyster mushroom polysaccharide.

[0039] Step 4: dissolving the oyster mushroom protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecularly imprinted polymer at a mass ratio of 1:1 under shaking at room temperature, eluting the mixture with an eluent prepared by mixing methanol and acetic acid at a volume ratio of 1:9, concentrating the eluent by rotary evaporation and freeze-drying the mixture to achieve the enrichment of ergothioneine.

[0040] It should be noted that the magnetic surface molecularly imprinted polymer is prepared by the following steps:

[0041] (1) Preparation of amino-functionalized Fe3O4@SiO2 nanoparticles:

[0042] Fe3O4 nanoparticles, ethanol and water were mixed in a mass ratio of 1:4000:1000 to obtain a mixed solution A, then 28wt% ammonia solution was mixed with the mixed solution A in a volume ratio of 1:40 to obtain a mixed solution B; finally, tetraethyl orthosilicate was mixed with the mixed solution B in a volume ratio of 1:30, and then recovered by magnet adsorption, washed, and vacuum dried to obtain Fe3O4@SiO2 nanoparticles; the Fe3O4@SiO2 nanoparticles, ethanol and 3-aminopropyl triethoxysilane were mixed in a mass ratio of 1:2000:20, and then washed and vacuum dried to obtain amino-functionalized Fe3O4@SiO2 nanoparticles;

[0043] (2) Preparation of magnetic surface molecularly imprinted nanomaterial: with ergot alkaloids as a template molecule, methyl methacrylate MAA and 4-vinylpyridine 4-VP as functional monomers, ethylene glycol acrylate EDMA as a crosslinking agent, amino-functionalized Fe3O4@SiO2 nanoparticles as a carrier, water, ethanol and tetrahydrofuran as porogens, and azobisisobutyronitrile as an initiator, the magnetic surface molecularly imprinted nanomaterial was obtained by polymerization in a water bath at 65℃ for 48h, wherein the mass ratio of ergot alkaloids to MAA and 4-VP was 1:5:7, the mass ratio of ergot alkaloids to ethylene glycol acrylate EDMA was 1:30, the mass ratio of ergot alkaloids to Fe3O4@SiO2 nanoparticles was 1:9, the mass ratio of ergot alkaloids to azobisisobutyronitrile was 1:2, and the mass ratio of ergot alkaloids to water, ethanol and tetrahydrofuran was 1:40:40:20;

[0044] (3) Removal of template molecule: the magnetic surface molecularly imprinted nanomaterial was eluted with an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to remove ergot alkaloids, thereby obtaining a magnetic surface molecularly imprinted polymer with an imprinted cavity complementary to the target molecule ergot alkaloids in terms of functional groups, size and shape.

[0045] Example 3: A cascade extraction process for active ingredients of edible fungi is as follows:

[0046] Step one: mix the super-micro powder of golden needle mushroom and ultrapure water in a mass ratio of 1:30 to form a suspension, and then centrifuge the suspension at a speed of 4000 rpm for 20 min to obtain the supernatant; concentrate the supernatant to 1 / 5 of the volume of the original suspension to obtain a concentrated solution;

[0047] Step 2: The pH value of the concentrate was adjusted to 11, and the concentrate was incubated in a water bath at 80°C for 6 hours and centrifuged at 6000 rpm for 5 minutes to obtain supernatant A; the pH value of supernatant A was then adjusted to 3, and the concentrate was allowed to stand for 60 minutes to allow protein precipitation, and centrifuged at 5000 rpm for 10 minutes to obtain supernatant B and precipitate A, and the precipitate A was freeze-dried to obtain Enoki mushroom protein;

[0048] Step 3: The supernatant B was mixed with 70% ethanol in a water-to-alcohol volume ratio of 1:6, and the mixture was allowed to stand at 8°C for 2 hours, and then centrifuged at 5000 rpm for 15 minutes to obtain a precipitate B. The precipitate B was then washed three times with 80% ethanol, cooled and dried, and then obtained Flammulina velutipes polysaccharide.

[0049] Step 4: dissolving the enoki mushroom protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecularly imprinted polymer at a mass ratio of 1:1 under shaking at room temperature, eluting the mixture with an eluent prepared by mixing methanol and acetic acid at a volume ratio of 1:9, concentrating the eluent by rotary evaporation and freeze-drying the mixture to achieve the enrichment of ergothioneine.

[0050] It should be noted that the magnetic surface molecularly imprinted polymer with an imprinted cavity complementary to the target molecule thioneine in functional groups, size and shape is prepared by the following steps:

[0051] (1) Preparation of amino-functionalized Fe3O4@SiO2 nanoparticles:

[0052] Fe3O4 nanoparticles, ethanol, and water were mixed at a mass ratio of 1:4000:1000 to obtain a mixture A. Subsequently, a 28wt% ammonia solution was mixed with the mixture A at a volume ratio of 1:32 to obtain a mixture B. Finally, tetraethyl orthosilicate was thoroughly mixed with the mixture B at a volume ratio of 1:40, and then recovered by adsorption using a magnet. After washing and vacuum drying, Fe3O4@SiO2 nanoparticles were obtained. Fe3O4@SiO2 nanoparticles, ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, washed, and vacuum dried to obtain amino-functionalized Fe3O4@SiO2 nanoparticles.

[0053] (2) Preparation of magnetic surface molecularly imprinted nanomaterials: using ergothioneine as a template molecule, methacrylic acid MAA and 4-vinylpyridine 4-VP as functional monomers, ethylene glycol acrylate EDMA as a crosslinker, amino-functionalized Fe3O4@SiO2 nanoparticles as a carrier, water, ethanol and tetrahydrofuran as porogens, and azobisisobutyronitrile as an initiator, water bath polymerization was performed at 65°C for 48h to obtain magnetic surface molecularly imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP was 1:7:3, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA was 1:28, the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles was 1:7, the mass ratio of ergothioneine to azobisisobutyronitrile was 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran was 1:40:40:20;

[0054] (3) Removal of template molecules: The magnetic surface molecularly imprinted nanomaterials were eluted with an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer with an imprinting cavity complementary to the target molecule ergothioneine in terms of functional groups, size and shape.

[0055] Example 4: A cascade extraction process for active ingredients of edible fungi is as follows:

[0056] Step 1: Mix the enoki mushroom ultrafine powder and ultrapure water in a mass ratio of 1:50 to form a suspension, let it stand, and then centrifuge it at a speed of 5000 rpm for 10 minutes to obtain the supernatant, and concentrate the supernatant to 1 / 5 of the volume of the original suspension to obtain a concentrated solution;

[0057] Step 2: The pH value of the concentrate was adjusted to 13, and the concentrate was incubated in a water bath at 70°C for 4 hours and centrifuged at 5000 rpm for 10 minutes to obtain supernatant A; the pH of supernatant A was then adjusted to 4, and the concentrate was allowed to stand for 45 minutes to allow protein precipitation, and centrifuged at 5000 rpm for 10 minutes to obtain supernatant B and precipitate A, and the precipitate A was freeze-dried to obtain Enoki mushroom protein;

[0058] Step 3: The supernatant B was mixed with 80% ethanol in a water-to-alcohol volume ratio of 1:8, and the mixture was allowed to stand at 0°C for 3 hours, followed by centrifugation at 5000 rpm for 10 minutes to obtain a precipitate B. The precipitate B was then washed three times with 80% ethanol, cooled and dried to obtain Flammulina velutipes polysaccharide.

[0059] Step 4: dissolving the enoki mushroom protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecularly imprinted polymer at a mass ratio of 1:1 under shaking at room temperature, eluting the mixture with an eluent prepared by mixing methanol and acetic acid at a volume ratio of 1:9, concentrating the eluent by rotary evaporation and freeze-drying the mixture to achieve the enrichment of ergothioneine.

[0060] It should be noted that the magnetic surface molecularly imprinted polymer with an imprinted cavity complementary to the target molecule thioneine in functional groups, size and shape is prepared by the following steps:

[0061] (1) Preparation of amino-functionalized Fe3O4@SiO2 nanoparticles:

[0062] Fe3O4 nanoparticles, ethanol, and water were mixed at a mass ratio of 1:4000:1000 to obtain a mixture A. Subsequently, a 28wt% ammonia solution was mixed with the mixture A at a volume ratio of 1:38 to obtain a mixture B. Finally, tetraethyl orthosilicate was thoroughly mixed with the mixture B at a volume ratio of 1:31, and then recovered by adsorption using a magnet. After washing and vacuum drying, Fe3O4@SiO2 nanoparticles were obtained. Fe3O4@SiO2 nanoparticles, ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, washed, and vacuum dried to obtain amino-functionalized Fe3O4@SiO2 nanoparticles.

[0063] (2) Preparation of magnetic surface molecularly imprinted nanomaterials: using ergothioneine as a template molecule, methacrylic acid MAA and 4-vinylpyridine 4-VP as functional monomers, ethylene glycol acrylate EDMA as a crosslinker, amino-functionalized Fe3O4@SiO2 nanoparticles as a carrier, water, ethanol and tetrahydrofuran as porogens, and azobisisobutyronitrile as an initiator, water bath polymerization was performed at 65°C for 48h to obtain magnetic surface molecularly imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP was 1:4:6, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA was 1:27, the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles was 1:5, the mass ratio of ergothioneine to azobisisobutyronitrile was 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran was 1:40:40:20;

[0064] (3) Removal of template molecules: The magnetic surface molecularly imprinted nanomaterials were eluted with an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer with an imprinting cavity complementary to the target molecule ergothioneine in terms of functional groups, size and shape.

[0065] Example 5: A cascade extraction process for active ingredients of edible fungi is as follows:

[0066] Step 1: Mix the enoki mushroom ultrafine powder and ultrapure water in a mass ratio of 1:60 to form a suspension, let it stand, and then centrifuge it at a speed of 5000 rpm for 10 minutes to obtain the supernatant, and concentrate the supernatant to 1 / 5 of the volume of the original suspension to obtain a concentrated solution;

[0067] Step 2: The pH value of the concentrate was adjusted to 12, and the concentrate was incubated in a water bath at 70°C for 4 hours and centrifuged at 5000 rpm for 10 minutes to obtain supernatant A; the pH value of supernatant A was then adjusted to 3.6, and the concentrate was allowed to stand for 60 minutes to allow protein precipitation, and centrifuged at 5000 rpm for 10 minutes to obtain supernatant B and precipitate A. The precipitate A was freeze-dried to obtain Enoki mushroom protein;

[0068] Step 3: Mix the supernatant B with 90% ethanol by volume in a water-alcohol ratio of 1:10, let it stand at 2°C for 5 hours, and then centrifuge it at 5000 rpm for 10 minutes to obtain precipitate B. Wash the precipitate B three times with 80% ethanol, cool and dry it, and obtain Flammulina velutipes polysaccharide.

[0069] Step 4: dissolving the enoki mushroom protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecularly imprinted polymer at a mass ratio of 1:1 under shaking at room temperature, eluting the mixture with an eluent prepared by mixing methanol and acetic acid at a volume ratio of 1:9, concentrating the eluent by rotary evaporation and freeze-drying the mixture to achieve the enrichment of ergothioneine.

[0070] It should be noted that the magnetic surface molecularly imprinted polymer with an imprinted cavity complementary to the target molecule thioneine in functional groups, size and shape is prepared by the following steps:

[0071] (1) Preparation of amino-functionalized Fe3O4@SiO2 nanoparticles:

[0072] Fe3O4 nanoparticles, ethanol, and water were mixed at a mass ratio of 1:4000:1000 to obtain a mixture A. Subsequently, a 28wt% ammonia solution was mixed with the mixture A at a volume ratio of 1:36 to obtain a mixture B. Finally, tetraethyl orthosilicate was thoroughly mixed with the mixture B at a volume ratio of 1:30, and then recovered by adsorption using a magnet. After washing and vacuum drying, Fe3O4@SiO2 nanoparticles were obtained. Fe3O4@SiO2 nanoparticles, ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, washed, and vacuum dried to obtain amino-functionalized Fe3O4@SiO2 nanoparticles.

[0073] (2) Preparation of magnetic surface molecularly imprinted nanomaterials: Using ergothioneine as a template molecule, methacrylic acid MAA and 4-vinylpyridine 4-VP as functional monomers, ethylene glycol acrylate EDMA as a crosslinker, amino-functionalized Fe3O4@SiO2 nanoparticles as a carrier, water, ethanol and tetrahydrofuran as porogens, and azobisisobutyronitrile as an initiator, a magnetic surface molecularly imprinted nanomaterial was obtained by water bath polymerization at 65°C for 48h, wherein the mass ratio of ergothioneine to MAA and 4-VP was 1:5:4, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA was (1:29), the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles was (1:6), the mass ratio of ergothioneine to azobisisobutyronitrile was 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran was 1:40:40:20;

[0074] (3) Removal of template molecules: The magnetic surface molecularly imprinted nanomaterials were eluted with an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer with an imprinting cavity complementary to the target molecule ergothioneine in terms of functional groups, size and shape.

[0075] Experimental part:

[0076] The freeze-dried edible fungus protein, edible fungus polysaccharide and thioneine in Example 1-5 were weighed, and the yield was calculated according to the formula: Calculate the yield of the corresponding component, where W1 is the weight of the polysaccharide (protein, thioneine) after freeze drying, in g; W2 is the weight of the edible fungus powder added during the water bath, in g. The specific yield is shown in Table 1:

[0077] Table 1

[0078]

[0079] It can be seen that the present invention is enriched in edible fungus protein, edible fungus polysaccharide and thioneine simultaneously from edible fungus, wherein polysaccharide yield is 2.21%-5.32%, protein yield is 8.02%-13.59%, and thioneine maximum yield is 0.302%, compared with single extraction method yield, no significant difference. Wherein embodiment 2 (water bath temperature is 60 DEG C, water alcohol volume ratio 1:4) edible fungus protein yield is the highest. When temperature is relatively low, cell wall can not be completely destroyed, and intracellular protein and polysaccharide can not be separated out, and yield is relatively low, and yield reaches the highest at 60 DEG C, and then with the rising of temperature, easily causes the denaturation and inactivation of protein, reduces yield;After water bath temperature exceeds 80 DEG C, most of protein denaturation and inactivation, are difficult to extract. Simultaneously, water alcohol volume ratio is too low to make polysaccharide precipitation incomplete, and the subsequent operation such as rotary evaporation that too high can affect polysaccharide consumes more time and resources, causes waste, and if ethanol is not removed in addition, subsequent freeze drying has a greater impact. In summary, the present invention extracts multiple effective components from edible fungi, improves the utilization rate of edible fungi, and reduces the loss rate of active components of edible fungi.

Claims

1. A cascade extraction process for active ingredients of edible fungi, characterized in that: The steps include: Step 1: Mix the edible fungus ultrafine powder and ultrapure water in a mass ratio of 1: (20-60) to form a suspension, let it stand and then centrifuge to obtain the supernatant, and concentrate the supernatant to 1 / 5 of the volume of the original suspension to obtain a concentrated solution; Step 2: The concentrate is adjusted to alkaline, and then centrifuged after water bath to obtain supernatant A; then the supernatant A is adjusted to acidic, allowed to stand to allow protein to precipitate, and centrifuged to obtain supernatant B and precipitate A, and the precipitate A is freeze-dried to obtain edible fungus protein; Step 3: mixing the supernatant B with 50%-100% ethanol in a water-to-alcohol volume ratio of 1:(2-10), allowing the mixture to stand at a temperature of 0-10°C and then centrifuging to obtain a precipitate B, which is then washed, cooled, and dried to obtain an edible fungus polysaccharide; Step 4: dissolving the edible fungus protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecularly imprinted polymer at a mass ratio of 1:1 under shaking at room temperature, eluting the mixture with an eluent prepared by mixing methanol and acetic acid at a volume ratio of 1:9, and then eluting the eluent by rotary evaporation and concentration, and then freeze-drying the eluent to achieve the enrichment of ergothioneine.

2. The cascade extraction process for active ingredients of edible fungi according to claim 1, characterized in that: The centrifugal treatment in the above steps is performed at a speed of 4000-6000 rpm and a centrifugal time of 5-20 min.

3. The cascade extraction process for active ingredients of edible fungi according to claim 1, characterized in that: The edible fungus in step 1 is one of king oyster mushroom, oyster mushroom or golden needle mushroom.

4. The cascade extraction process for active ingredients of edible fungi according to claim 1, characterized in that: In step 2, the concentrated solution is adjusted to an alkaline pH value in the range of 9-13, the water bath temperature is 50-80° C., and the water bath time is 1-8 hours.

5. The cascade extraction process for active ingredients of edible fungi according to claim 1, characterized in that: In step 2, the supernatant A is adjusted to an acidic pH value in the range of 2-4 and the standing time is 30-90 minutes.

6. The cascade extraction process for active ingredients of edible fungi according to claim 1, characterized in that: The standing time in step 3 is 1-5 hours 。 7. The cascade extraction process for active ingredients of edible fungi according to claim 1, characterized in that: The washing in step 3 was performed with 80% ethanol for three times.

8. The cascade extraction process for active ingredients of edible fungi according to claim 1, characterized in that: The preparation process of the magnetic surface molecularly imprinted polymer in step 4 includes the following steps: (1) Preparation of amino-functionalized Fe3O4@SiO2 nanoparticles: Fe3O4 nanoparticles, ethanol, and water were mixed at a mass ratio of 1:4000:1000 to obtain a mixed solution A. Subsequently, a 28 wt% ammonia solution was mixed with the mixed solution A at a volume ratio of 1:(30-40) to obtain a mixed solution B. Finally, tetraethyl orthosilicate was thoroughly mixed with the mixed solution B at a volume ratio of 1:(30-40), and then recovered by adsorption using a magnet. After washing and vacuum drying, Fe3O4@SiO2 nanoparticles were obtained. Fe3O4@SiO2 nanoparticles, ethanol, and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, and after washing and vacuum drying, amino-functionalized Fe3O4@SiO2 nanoparticles were obtained. (2) Preparation of magnetic surface molecularly imprinted nanomaterials: ergothioneine was used as a template molecule, methacrylic acid MAA and 4-vinylpyridine 4-VP were used as functional monomers, ethylene glycol acrylate EDMA was used as a crosslinker, amino-functionalized Fe3O4@SiO2 nanoparticles were used as carriers, water, ethanol and tetrahydrofuran were used as porogens, and azobisisobutyronitrile was used as an initiator. The magnetic surface molecularly imprinted nanomaterials were obtained by water bath polymerization at 65°C for 48h, wherein the mass ratio of ergothioneine to MAA and 4-VP was 1:(3-7):(3-7), the mass ratio of ergothioneine to ethylene glycol acrylate EDMA was 1:(26-30), the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles was 1:(4-9), and the mass ratio of ergothioneine to azobisisobutyronitrile was 1:

2. ， The mass ratio of ergothioneine to water, ethanol and tetrahydrofuran is 1:40:40:20; (3) Removal of template molecules: The magnetic surface molecularly imprinted nanomaterials were eluted with an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer with an imprinting cavity complementary to the target molecule ergothioneine in terms of functional groups, size and shape.

Images