Cascade extraction process for active ingredients of edible mushrooms

Abstract

The invention discloses a cascade extraction process of active ingredients of edible mushrooms, which comprises the following steps: mixing edible mushroom superfine powder and ultrapure water to form turbid liquid, standing, centrifuging, taking supernate, and concentrating; adjusting the concentrated solution to be alkaline, performing water bath, and then performing centrifugal treatment to obtain supernate A; adjusting the supernate A to be acidic, standing to separate out protein, performing centrifugal treatment to obtain supernate B and precipitate A, and performing freeze drying on the precipitate A to obtain edible mushroom protein; mixing the supernate B with ethanol, standing, centrifuging to obtain a precipitate B, washing the precipitate B, and cooling and drying to obtain the edible fungus polysaccharide; the method comprises the following steps: dissolving edible mushroom protein in water, oscillating and mixing with a magnetic surface molecularly imprinted polymer at room temperature, eluting, carrying out rotary evaporation and concentration on an eluent, and freeze-drying to realize enrichment of ergothioneine. Various effective components in the edible mushrooms are extracted, the utilization rate of the edible mushrooms is increased, and the loss rate of the active components of the edible mushrooms is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of resource utilization, and in particular to a cascade extraction process of active ingredients of edible fungi. Background Art

[0002] Edible fungus 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 fungus protein has multiple functional activities such as anti-tumor, immunomodulatory, antiviral, anti-inflammatory, and antioxidant. Common plant protein extraction methods include solvent extraction, salt dissolution, and enzyme methods. Edible fungus polysaccharides have a variety of biological activities, including antioxidant, anti-tumor, anti-inflammatory, immunomodulatory, hypoglycemic, regulating intestinal flora, and antiviral activities, and are often used to prevent and treat various chronic diseases. The extraction methods of edible fungus polysaccharides include hot water extraction, water extraction and alcohol precipitation, etc. Ergothioneine is a natural small molecule histidine thiourea derivative that is widely present in edible fungus fruiting bodies. The content can exceed 0.60 mg / g dry weight and has good antioxidant, anti-inflammatory, cell protection and other biological activities. The extraction methods of ergothioneine include reflux extraction, enzymatic extraction, etc.

[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, in most cases, only a single active substance is extracted. It is difficult to achieve the ideal effect under the premise of long extraction time, high extraction temperature and high 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 application number CN201410673654.0, which discloses a method of extracting or separating proteins from the alcohol precipitation supernatant during the extraction of edible fungi polysaccharides, but there is no process for cascading extraction of multiple active ingredients from edible fungi. Summary of the invention

[0004] The invention aims to provide a cascade extraction process of active ingredients of edible fungi, which can simultaneously extract edible fungi protein, edible fungi polysaccharide and ergothioneine from edible fungi, and the method has high raw material utilization rate, is simple to operate, and significantly reduces the loss rate of active ingredients of edible fungi.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

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

[0007] 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 a supernatant, and concentrate the supernatant to 1 / 5 of the volume of the original suspension to obtain a concentrated solution;

[0008] Step 2: adjusting the concentrated solution to alkaline, centrifuging after water bath to obtain supernatant A; then adjusting the supernatant A to acidic, standing to allow protein to precipitate, centrifuging to obtain supernatant B and precipitate A, and freeze-drying the precipitate A to obtain edible fungus protein;

[0009] Step 3: mixing the supernatant B and ethanol with a volume concentration of 50%-100% according to a water-to-alcohol volume ratio of 1:(2-10), standing at a temperature of 0-10°C and then centrifuging to obtain a precipitate B, and then washing the precipitate B, cooling and drying it to obtain edible fungus polysaccharides;

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

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

[0012] Furthermore, the edible fungus in step one is one of Pleurotus eryngii, Pleurotus ostreatus or Flammulina velutipes.

[0013] Furthermore, 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.

[0014] Furthermore, 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.

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

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

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

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

[0019] Mixed solution A was obtained by mixing Fe3O4 nanoparticles, ethanol and water at a mass ratio of 1:4000:1000, and then 28wt% ammonia solution was mixed with mixed solution A at a volume ratio of 1:(30-40) to obtain mixed solution B. Finally, tetraethyl orthosilicate was fully mixed with mixed solution B at a volume ratio of 1:(30-40), and then adsorbed and recovered by a magnet, washed, and vacuum dried to obtain Fe3O4@SiO2 nanoparticles; 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 molecular 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, and polymerizing in a water bath at 65°C for 48h to obtain magnetic surface molecular imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP is 1:(3-7):(3-7), the mass ratio of ergothioneine to ethylene glycol acrylate EDMA is 1:(26-30), the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles is 1:(4-9), the mass ratio of ergothioneine to azobisisobutyronitrile is 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran is 1:40:40:20;

[0021] (3) Removing the template molecule: using an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to elute the magnetic surface molecularly imprinted nanomaterial to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer having an imprinting cavity complementary to the target molecule ergothioneine in terms of functional group, size and shape.

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

[0023] Traditional extraction methods usually extract a single active ingredient, while the cascade extraction process of the present invention can 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. The process has significant economic and environmental advantages and contributes to the comprehensive development of multiple active ingredients in edible fungi. 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 in conjunction with specific embodiments.

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

[0026] Step 1: Mix the Pleurotus eryngii superfine powder and ultrapure water in a mass ratio of 1:20 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;

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

[0028] Step 3: Mix the supernatant B and 50% ethanol by volume in a water-to-alcohol ratio of 1:2, let stand at 10°C for 1 hour, centrifuge at 6000 rpm for 5 minutes to obtain precipitate B, wash the precipitate B three times with 80% ethanol, cool and dry to obtain Pleurotus eryngii polysaccharide;

[0029] Step 4: dissolving the Pleurotus eryngii protein obtained in step 2 in water, mixing it with the magnetic surface molecular imprinting polymer at a mass ratio of 1:1 at room temperature, eluting it 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 it, so as to achieve the enrichment of ergothioneine.

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

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

[0032] Mixing Fe3O4 nanoparticles, ethanol and water at a mass ratio of 1:4000:1000 to obtain a mixed solution A, then mixing 28wt% ammonia solution with the mixed solution A at a volume ratio of 1:30 to obtain a mixed solution B; finally, tetraethyl orthosilicate and the mixed solution B were fully mixed at a volume ratio of 1:35, and then adsorbed and recovered by a magnet, and Fe3O4@SiO2 nanoparticles were obtained after washing and vacuum drying; Fe3O4@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, and amino-functionalized Fe3O4@SiO2 nanoparticles were obtained after washing and vacuum drying;

[0033] (2) Preparation of magnetic surface molecular 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, and polymerizing in a water bath at 65°C for 48h to obtain magnetic surface molecular imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP is 1:3:4, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA is 1:26, the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles is 1:4, the mass ratio of ergothioneine to azobisisobutyronitrile is 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran is 1:40:40:20;

[0034] (3) Removing the template molecule: using an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to elute the magnetic surface molecularly imprinted nanomaterial to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer having an imprinting cavity complementary to the target molecule ergothioneine in terms of functional group, size and shape.

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

[0036] Step 1: Mix the ultrafine powder of Pleurotus ostreatus and ultrapure water in a mass ratio of 1:40 to form a suspension, let it stand and then centrifuge it at a speed of 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 is adjusted to 10, and the concentrate is centrifuged at 5000 rpm for 10 min at 60° C. for 2 h in a water bath to obtain supernatant A; then the pH value of the supernatant A is adjusted to 2.8, and the concentrate is allowed to stand for 90 min to allow protein to precipitate, and the concentrate is centrifuged at 5000 rpm for 10 min to obtain supernatant B and precipitate A, and the precipitate A is freeze-dried to obtain Pleurotus ostreatus protein;

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

[0039] Step 4: dissolving the Pleurotus ostreatus protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecular imprinting 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 eluent, thereby achieving the enrichment of ergothioneine.

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

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

[0042] Mixed solution A was obtained by mixing Fe3O4 nanoparticles, ethanol and water at a mass ratio of 1:4000:1000, and then mixed solution A with 28wt% ammonia solution at a volume ratio of 1:40 to obtain mixed solution B; finally, tetraethyl orthosilicate was fully mixed with mixed solution B at a volume ratio of 1:30, and then recovered by adsorption with a magnet, washed, and vacuum dried to obtain Fe3O4@SiO2 nanoparticles; 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;

[0043] (2) Preparation of magnetic surface molecular 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, and polymerizing in a water bath at 65°C for 48h to obtain magnetic surface molecular imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP is 1:5:7, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA is 1:30, the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles is 1:9, the mass ratio of ergothioneine to azobisisobutyronitrile is 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran is 1:40:40:20;

[0044] (3) Removing the template molecule: using an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to elute the magnetic surface molecularly imprinted nanomaterial to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer having an imprinting cavity complementary to the target molecule ergothioneine in terms of functional group, size and shape.

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

[0046] Step 1: Mix the Flammulina velutipes ultrafine powder and ultrapure water in a mass ratio of 1:30 to form a suspension, let it stand and then centrifuge it at a speed of 4000 rpm for 20 minutes to obtain the supernatant, and 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 is adjusted to 11, and the concentrate is centrifuged at 6000 rpm for 5 min in a water bath at 80°C for 6 h to obtain supernatant A; then the pH value of the supernatant A is adjusted to 3, and the concentrate is allowed to stand for 60 min to allow protein to precipitate, and the concentrate is centrifuged at 5000 rpm for 10 min to obtain supernatant B and precipitate A, and the precipitate A is freeze-dried to obtain Flammulina velutipes protein;

[0048] Step 3: Mix the supernatant B and 70% ethanol by volume in a water-to-alcohol ratio of 1:6, let stand at 8°C for 2 hours, and centrifuge at 5000 rpm for 15 minutes to obtain precipitate B, wash the precipitate B three times with 80% ethanol, and cool and dry to obtain Flammulina velutipes polysaccharide;

[0049] Step 4: dissolving the Flammulina velutipes protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecular imprinting polymer at a mass ratio of 1:1 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 eluent to achieve the enrichment of ergothioneine.

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

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

[0052] Mixed solution A was obtained by mixing Fe3O4 nanoparticles, ethanol and water in a mass ratio of 1:4000:1000, and then mixed solution A with 28wt% ammonia solution in a volume ratio of 1:32 to obtain mixed solution B; finally, tetraethyl orthosilicate was fully mixed with mixed solution B in a volume ratio of 1:40, and then recovered by adsorption with a magnet, and Fe3O4@SiO2 nanoparticles were obtained after washing and vacuum drying; Fe3O4@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane were mixed in a mass ratio of 1:2000:20, and amino-functionalized Fe3O4@SiO2 nanoparticles were obtained after washing and vacuum drying;

[0053] (2) Preparation of magnetic surface molecular 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, and polymerizing in a water bath at 65°C for 48h to obtain magnetic surface molecular imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP is 1:7:3, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA is 1:28, the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles is 1:7, the mass ratio of ergothioneine to azobisisobutyronitrile is 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran is 1:40:40:20;

[0054] (3) Removing the template molecule: using an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to elute the magnetic surface molecularly imprinted nanomaterial to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer having an imprinting cavity complementary to the target molecule ergothioneine in terms of functional group, size and shape.

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

[0056] Step 1: Mix the Flammulina velutipes 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 is adjusted to 13, and the concentrate is centrifuged at 5000 rpm for 10 min at 70° C. for 4 h in a water bath to obtain supernatant A; then the pH value of the supernatant A is adjusted to 4, and the concentrate is allowed to stand for 45 min to allow protein to precipitate, and the concentrate is centrifuged at 5000 rpm for 10 min to obtain supernatant B and precipitate A, and the precipitate A is freeze-dried to obtain Flammulina velutipes protein;

[0058] Step 3: Mix the supernatant B and ethanol with a volume concentration of 80% according to a water-to-alcohol volume ratio of 1:8, let it stand at 0°C for 3 hours, and then centrifuge it at a speed of 5000 rpm for 10 minutes to obtain a precipitate B, wash the precipitate B three times with 80% ethanol, and cool and dry it to obtain Flammulina velutipes polysaccharide;

[0059] Step 4: dissolving the Flammulina velutipes protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecular imprinting polymer at a mass ratio of 1:1 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 eluent to achieve the enrichment of ergothioneine.

[0060] It should be noted that the magnetic surface molecularly imprinted polymer of the imprinted cavity complementary to the target molecule ergothioneine in functional group, 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 mixed solution A, and then a 28wt% ammonia solution was mixed with the mixed solution A at a volume ratio of 1:38 to obtain a mixed solution B; finally, tetraethyl orthosilicate was fully mixed with the mixed solution B at a volume ratio of 1:31, and then adsorbed and recovered by a magnet, and Fe3O4@SiO2 nanoparticles were obtained after washing and vacuum drying; Fe3O4@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, and amino-functionalized Fe3O4@SiO2 nanoparticles were obtained after washing and vacuum drying;

[0063] (2) Preparation of magnetic surface molecular 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, and polymerizing in a water bath at 65°C for 48h to obtain magnetic surface molecular imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP is 1:4:6, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA is 1:27, the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles is 1:5, the mass ratio of ergothioneine to azobisisobutyronitrile is 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran is 1:40:40:20;

[0064] (3) Removing the template molecule: using an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to elute the magnetic surface molecularly imprinted nanomaterial to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer having an imprinting cavity complementary to the target molecule ergothioneine in terms of functional group, size and shape.

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

[0066] Step 1: Mix the Flammulina velutipes ultrafine powder and ultrapure water at 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 is adjusted to 12, and the concentrate is centrifuged at 5000 rpm for 10 min at 70° C. for 4 h in a water bath to obtain supernatant A; then the pH value of the supernatant A is adjusted to 3.6, and the concentrate is allowed to stand for 60 min to allow protein to precipitate, and the concentrate is centrifuged at 5000 rpm for 10 min to obtain supernatant B and precipitate A, and the precipitate A is freeze-dried to obtain Flammulina velutipes protein;

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

[0069] Step 4: dissolving the Flammulina velutipes protein obtained in step 2 in water, mixing the mixture with the magnetic surface molecular imprinting polymer at a mass ratio of 1:1 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 eluent to achieve the enrichment of ergothioneine.

[0070] It should be noted that the magnetic surface molecularly imprinted polymer of the imprinted cavity complementary to the target molecule ergothioneine in functional group, 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 mixed solution A, and then a 28wt% ammonia solution was mixed with the mixed solution A at a volume ratio of 1:36 to obtain a mixed solution B; finally, tetraethyl orthosilicate was fully mixed with the mixed solution B at a volume ratio of 1:30, and then adsorbed and recovered by a magnet, and Fe3O4@SiO2 nanoparticles were obtained after washing and vacuum drying; Fe3O4@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane were mixed at a mass ratio of 1:2000:20, and amino-functionalized Fe3O4@SiO2 nanoparticles were obtained after washing and vacuum drying;

[0073] (2) Preparation of magnetic surface molecular 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, and polymerizing in a water bath at 65°C for 48h to obtain magnetic surface molecular imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP is 1:5:4, the mass ratio of ergothioneine to ethylene glycol acrylate EDMA is (1:29), the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles is (1:6), the mass ratio of ergothioneine to azobisisobutyronitrile is 1:2, and the mass ratio of ergothioneine to water, ethanol and tetrahydrofuran is 1:40:40:20;

[0074] (3) Removing the template molecule: using an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to elute the magnetic surface molecularly imprinted nanomaterial to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer having an imprinting cavity complementary to the target molecule ergothioneine in terms of functional group, size and shape.

[0075] Experimental part:

[0076] The freeze-dried edible fungus protein, edible fungus polysaccharide and ergothioneine in Example 1-5 were weighed, and the yield was calculated according to the formula: Calculate the yield of the corresponding component, where W 1 W is the weight of the polysaccharide (protein, ergothioneine) after freeze drying, in g; 2 is the weight of edible fungus powder added during water bathing, in g. The specific yield is shown in Table 1:

[0077] Table 1

[0078]

[0079] It can be seen that the present invention simultaneously enriches edible fungus protein, edible fungus polysaccharide and ergothioneine from edible fungi, wherein the polysaccharide yield is 2.21%-5.32%, the protein yield is 8.02%-13.59%, and the highest ergothioneine yield is 0.302%, which is no significant difference compared with the single extraction method yield. The yield of edible fungus protein in Example 2 (water bath temperature is 60 ° C, water-alcohol volume ratio 1: 4) is the highest. When the temperature is relatively low, the cell wall cannot be completely destroyed, and intracellular protein and polysaccharide cannot be precipitated, and the yield is relatively low. At 60 ° C, the yield reaches the highest, and then with the increase of temperature, it is easy to cause the denaturation and inactivation of protein, reducing the yield; when the water bath temperature exceeds 80 ° C, most of the protein is denatured and inactivated, and it is difficult to extract. At the same time, the water-alcohol volume ratio is too low to make the polysaccharide precipitation incomplete, and the subsequent operation of the polysaccharide, such as rotary evaporation, which is too high, will affect more time and resources, causing waste, and if ethanol is not removed, it will have a greater impact on subsequent freeze drying. 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 a supernatant, and concentrate the supernatant to 1 / 5 of the volume of the original suspension to obtain a concentrated solution; Step 2: adjusting the concentrated solution to alkaline, centrifuging after water bath to obtain supernatant A; then adjusting the supernatant A to acidic, standing to allow protein to precipitate, centrifuging to obtain supernatant B and precipitate A, and freeze-drying the precipitate A to obtain edible fungus protein; Step 3: mixing the supernatant B and ethanol with a volume concentration of 50%-100% according to a water-to-alcohol volume ratio of 1:(2-10), standing at a temperature of 0-10°C and then centrifuging to obtain a precipitate B, and then washing the precipitate B, cooling and drying it to obtain edible fungus polysaccharides; Step 4: dissolving the edible fungus protein obtained in step 2 in water, mixing it with the magnetic surface molecular imprinting polymer at a mass ratio of 1:1 at room temperature, eluting it with an eluent prepared by mixing methanol and acetic acid at a volume ratio of 1:9, and then concentrating the eluent by rotary evaporation and freeze-drying it to achieve the enrichment of ergothioneine.

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

3. The cascade extraction process of active ingredients of edible fungi according to claim 1, characterized in that: The edible fungus described in step 1 is one of Pleurotus eryngii, Pleurotus ostreatus or Flammulina velutipes.

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

8. The cascade extraction process of active ingredients of edible fungi according to claim 1, characterized in that: The preparation process of the magnetic surface molecular imprinted polymer in step 4 comprises the following steps: (1) Preparation of amino-functionalized Fe3O4@SiO2 nanoparticles: Fe3O4 nanoparticles, ethanol and water are mixed at a mass ratio of 1:4000:1000 to obtain a mixed solution A, and then a 28wt% ammonia solution is mixed with the mixed solution A at a volume ratio of 1:(30-40) to obtain a mixed solution B. Finally, tetraethyl orthosilicate is fully mixed with the mixed solution B at a volume ratio of 1:(30-40), and then adsorbed and recovered by a magnet, and Fe3O4@SiO2 nanoparticles are obtained after washing and vacuum drying; Fe3O4@SiO2 nanoparticles, ethanol and 3-aminopropyltriethoxysilane are mixed at a mass ratio of 1:2000:20, and amino-functionalized Fe3O4@SiO2 nanoparticles are obtained after washing and vacuum drying; (2) Preparation of magnetic surface molecular imprinted nanomaterials: Using ergothioneine as a template molecule, methyl methacrylate 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 carried out at 65°C for 48 hours to obtain magnetic surface molecular imprinted nanomaterials, wherein the mass ratio of ergothioneine to MAA and 4-VP is 1:(3-7):(3-7), the mass ratio of ergothioneine to ethylene glycol acrylate EDMA is 1:(26-30), the mass ratio of ergothioneine to Fe3O4@SiO2 nanoparticles is 1:(4-9), and the mass ratio of ergothioneine to azobisisobutyronitrile is 1:2 ， The mass ratio of ergothioneine to water, ethanol and tetrahydrofuran is 1:40:40:20; (3) Removing the template molecule: using an eluent prepared by mixing methanol and acetic acid in a volume ratio of 8:2 to elute the magnetic surface molecularly imprinted nanomaterial to remove ergothioneine, thereby obtaining a magnetic surface molecularly imprinted polymer having an imprinting cavity complementary to the target molecule ergothioneine in terms of functional group, size and shape.

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