Membrane separation extraction method of hirsutella hepialid mycelium

Abstract

The invention discloses a membrane separation and extraction method of Hirsutella hepialid mycelium, which comprises the following steps: by taking the Hirsutella hepialid mycelium as a raw material, carrying out pretreatment, enzymolysis and extraction, then carrying out membrane separation, enriching polysaccharide and adenosine, and finally concentrating and drying to obtain the Hirsutella hepialid mycelium extract. According to the method, multiple extraction technologies are combined for use, the extraction efficiency and purity of the hirsutella hepialid mycelium are remarkably improved, the content of polysaccharide and adenosine in the hirsutella hepialid mycelium extract is far higher than that of polysaccharide and adenosine in a traditional water extraction process, and the additional value and market competitiveness of a product are improved.

Description

Technical Field

[0001] The invention belongs to the field of biological extraction, and specifically discloses a membrane separation and extraction method for the mycelium of Trichosporon bat moth. Background Art

[0002] Cordyceps sinensis is a traditional precious Chinese medicinal material. It is a fungus-insect complex, that is, an ascomycete on the body of the bat moth larvae. The main active ingredients of Cordyceps sinensis are cordycepic acid (D-mannitol), adenosine, polysaccharides, ergosterol, polypeptides and amino acids, and multiple vitamins. The bat moth Trichosporon mycelium is an artificially fermented Cordyceps powder, which is one of the artificially fermented Cordyceps mycelium approved for use by the state. It has the effects of retaining essence and replenishing qi, nourishing the lungs and kidneys, stopping bleeding and reducing phlegm, and also improves sleep.

[0003] The production process for extracting the mycelium of Trichosporon bat moth currently mostly uses a water decoction method, which has a low extraction rate and contains more impurities in the extract. Summary of the invention

[0004] In order to solve the technical problems existing in the background technology, the present invention proposes a membrane separation and extraction method for the mycelium of the bat moth hair spore. The present invention separates the mycelium extract of the bat moth hair spore by microfiltration and ultrafiltration through membrane integration technology, thereby realizing efficient and high-purity extraction of the mycelium of the bat moth hair spore. The process improves the extraction efficiency and purity of the mycelium of the bat moth hair spore.

[0005] The invention uses the mycelium of the bat moth as raw material, carries out pretreatment, enzymolysis, extraction, membrane separation, enriches the polysaccharide and adenosine content, and finally concentrates and dries to obtain the mycelium extract of the bat moth.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A membrane separation and extraction method for the mycelium of Trichosporon bat moth, the method comprising the following steps:

[0008] a) ultrafine grinding the mycelia of Hirsuta spp. to obtain Hirsuta spp. mycelia powder;

[0009] b) mixing the obtained mycelial powder of Trichosporon batriida with water;

[0010] c) hydrolyzing the obtained mixed solution with a composite enzyme, and then ultrasonically extracting;

[0011] d) separating and purifying the extract by membrane separation, concentrating under reduced pressure, and drying.

[0012] Preferably, in step a), the particle size of the mycelium powder of Trichosporon batiola is 2 to 15 μm.

[0013] Preferably, in step b), the mass ratio of the mycelium powder of Trichosporon batrix to water is 1:5-15; preferably, the mass ratio of the mycelium powder of Trichosporon batrix to water is 1:6-12.

[0014] Preferably, in step c), the complex enzyme used for enzymolysis is cellulase and pectinase in a mass ratio of 1:0.3-3; the mass ratio of the complex enzyme to the mycelium powder of Hirsutella batriida is 0.1-1.5:100.

[0015] Preferably, in step c), the mass ratio of the cellulase to the pectinase is 1:1-2; the mass ratio of the complex enzyme to the mycelium powder of Hirsutella batriida is 1:80-100.

[0016] Preferably, in step c), the pH of the enzymatic hydrolysis is 6.5-7.5; the enzymatic hydrolysis time is 0.5-3h, and the enzymatic hydrolysis temperature is 30-60°C; the pH of the enzymatic hydrolysis mixture is preferably adjusted to 6.5-7; the enzymatic hydrolysis time is 1-2h; and the enzymatic hydrolysis temperature is 40-50°C.

[0017] Preferably, in step c), the ultrasonic extraction time is 30 to 120 min; the ultrasonic frequency is 15 to 60 KHz; preferably, the ultrasonic extraction time is 60-90 min; the ultrasonic frequency is 20-30 KHz.

[0018] Preferably, in step d), the membrane separation is sequentially performed by microfiltration and ultrafiltration, the microfiltration membrane used in microfiltration is a cellulose membrane or a ceramic membrane with a pore size of 0.1 to 10 μm, the microfiltration operating temperature is 20 to 50°C, and the pressure is 0.1 to 0.5 MPa; the feed concentration after microfiltration is 1 to 4%, the ultrafiltration membrane used in ultrafiltration is a polysulfone ultrafiltration membrane or a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 5000 to 30000D, the ultrafiltration operating temperature is 25 to 50°C, and the pressure is 0.1 to 0.6 MPa; the most preferred microfiltration membrane is a ceramic microfiltration membrane with a pore size of 0.2 μm, the microfiltration operating temperature is 40-50°C, the pressure is 0.2-0.3 MPa, the feed concentration is 1-2%, the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cutoff of 10000-20000D, the ultrafiltration operating temperature is 40°C, and the pressure is 0.3-0.4 MPa.

[0019] Preferably, in step d), the filtrate obtained by separation and purification is concentrated under reduced pressure, and dried in a vacuum, freeze-dried or sprayed manner to obtain a brown-yellow powder.

[0020] The membrane separation technology used in the present invention can maintain the original characteristics and structure of the natural product components to the greatest extent, and will not generate pollution during the separation process. It is green and environmentally friendly, has small losses, good separation effects, relatively simple processes, low costs, and can achieve automated control.

[0021] The present invention adopts microfiltration method for refining, removing impurities, clarifying and sterilizing the drug solution, which can effectively remove impurities and retain effective components, has low energy consumption, high efficiency, low pollution, simple process, convenient operation, and can carry out concentration and purification at the same time.

[0022] Freeze drying is used to freeze the extract solution below freezing point, and then the ice is converted into vapor under a higher vacuum and removed. The dried material retains its original biological activity, structural integrity and color.

[0023] The above technical solution of the present invention has the following beneficial technical effects:

[0024] The present invention combines a variety of extraction technologies to significantly improve the extraction efficiency and purity of the mycelium of the bat moth. The polysaccharide and adenosine content in the mycelium extract of the bat moth is much higher than that of the traditional water extraction process, thereby improving the added value and market competitiveness of the product. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0026] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The raw material of the following examples, the mycelium powder of the bat moth, was purchased from Jiangsu Shenhua Pharmaceutical Co., Ltd. (polysaccharide: 5g / 100g, adenosine: 120mg / 100g), and the cellulase and pectinase were purchased from Jiangsu Baiyao Biotechnology Co., Ltd.

[0027] Example 1

[0028] The mycelium powder of the bat moth hair spore was ultrafinely ground and then mixed with water, the mass ratio of the mycelium powder of the bat moth hair spore to water was 1:6; the mixed solution was enzymolyzed with a composite enzyme, the composite enzyme was cellulase and pectinase, and the mass ratio was 1:0.5; the mass ratio of the composite enzyme to the mycelium powder of the bat moth hair spore was 1:100; the pH of the enzymolysis mixed solution was adjusted to 7, the enzymolysis time was 2h, and the enzymolysis temperature was 40℃; then ultrasonic extraction was performed for 90min; the ultrasonic frequency was 20KHz; and then The membrane separation system is used for separation by microfiltration and ultrafiltration membrane in sequence. The microfiltration membrane used in the microfiltration is a ceramic microfiltration membrane with a pore size of 0.2 μm, an operating temperature of 50°C, and a pressure of 0.3 MPa. The obtained feed liquid concentration is 2%. The ultrafiltration membrane uses a polysulfone ultrafiltration membrane with a molecular weight cutoff of 20000D, an operating temperature of 40°C, and a pressure of 0.3 MPa. The obtained filtrate is concentrated by reduced pressure and vacuum dried to obtain a brown-yellow bat moth hair spore mycelium extract powder. The impurity removal rate of this method is 20%, and the adenosine loss rate is 1.0%.

[0029] Example 2

[0030] The mycelium powder of the bat moth hair spore is ultrafinely crushed; water is added to mix, and the mass ratio of the mycelium powder of the bat moth hair spore to water is 1:10; the mixed solution is enzymatically hydrolyzed, and the composite enzyme is cellulase and pectinase, and the mass ratio is 1:1; the mass ratio of the composite enzyme to the mycelium powder of the bat moth hair spore is 1:100; the pH of the enzymatic hydrolysis mixed solution is adjusted to 7, the enzymatic hydrolysis time is 1.5h, and the enzymatic hydrolysis temperature is 50℃; then ultrasonic extraction is performed for 60min; the ultrasonic frequency is 30KHz; and then the membrane is used for The separation system performs membrane separation through microfiltration and ultrafiltration in sequence. The microfiltration membrane used in the microfiltration is a ceramic microfiltration membrane with a pore size of 0.2 μm, an operating temperature of 40°C, a pressure of 0.2 MPa, and a feed liquid concentration of 1%. The ultrafiltration membrane uses a polysulfone ultrafiltration membrane with a molecular weight cutoff of 10000D, an operating temperature of 40°C, and a pressure of 0.4 MPa. The obtained filtrate is concentrated under reduced pressure and freeze-dried to obtain a brown-yellow bat moth hair spore mycelium extract powder. The impurity removal rate of this method is about 20.3%, and the adenosine loss rate is about 1.05%.

[0031] Example 3

[0032] The mycelium powder of the bat moth hair spore is ultrafinely crushed; water is added to mix, and the mass ratio of the mycelium powder of the bat moth hair spore to water is 1:12; the mixed solution is enzymatically hydrolyzed, and the composite enzyme is cellulase and pectinase, and the mass ratio is 1:1.5; the mass ratio of the composite enzyme to the mycelium powder of the bat moth hair spore is 1:80; the pH of the enzymatic hydrolysis mixed solution is adjusted to 6.5, the enzymatic hydrolysis time is 1h, and the enzymatic hydrolysis temperature is 40℃; then ultrasonic extraction is performed for 90min; the ultrasonic frequency is 20KHz; and then the membrane separation system is used to pass through microfiltration in sequence. The method comprises the following steps: performing membrane separation by ultrafiltration, wherein the microfiltration membrane used is a cellulose membrane with a pore size of 0.2 μm, an operating temperature of 50°C, a pressure of 0.3 MPa, and a feed liquid concentration of 1%. The ultrafiltration membrane uses a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 10000D, an operating temperature of 40°C, and a pressure of 0.4 MPa. The obtained filtrate is concentrated by reduced pressure, freeze-dried, heated at 60°C, and a vacuum degree of 50 Pa to obtain a brown-yellow bat moth hair spore mycelium extract powder. The impurity removal rate of this method is about 20.6%, and the adenosine loss rate is about 1.2%.

[0033] Comparative Example: Water Extraction Method

[0034] The mycelium of the bat moth was boiled in water twice in an extraction tank, the first time with 15 times the amount of water for 2 hours, and the second time with 15 times the amount of water for 2 hours, the temperature was 100°C, the two extracts were filtered through filters respectively, and the filtrate was concentrated to 1.05-1.20 g / cm by a double-effect concentrator under reduced pressure. 3 The fluid extract of density is collected and spray-dried to obtain the dry powder of the mycelium extract of the bat moth. The impurity removal rate of this method is about 10%, and the adenosine loss rate is about 2.0%.

[0035] The results of the polysaccharide and adenosine content tests in the comparative example and Example 1 are shown in Table 1:

[0036] Table 1

[0037]

Claims

1. A membrane separation and extraction method for the mycelium of Trichosporon bat moth, characterized in that The method comprises the following steps: a) ultrafine grinding the mycelia of Hirsuta spp. to obtain Hirsuta spp. mycelia powder; b) mixing the obtained mycelial powder of Trichosporon batriida with water; c) hydrolyzing the obtained mixed solution with a composite enzyme, and then ultrasonically extracting; d) separating and purifying the extract by membrane separation, concentrating under reduced pressure, and drying.

2. The membrane separation and extraction method of the mycelium of Trichosporon bat moth according to claim 1, characterized in that In the step a), the particle size of the mycelium powder of Trichosporon batriida is 2 to 15 μm.

3. The membrane separation and extraction method of the mycelium of Trichosporon bat moth according to claim 1, characterized in that In the step b), the mass ratio of the mycelium powder of Trichosporon batrix to water is 1:5-15; preferably, the mass ratio of the mycelium powder of Trichosporon batrix to water is 1:6-12.

4. The membrane separation and extraction method of the mycelium of Trichosporon bat moth according to claim 1, characterized in that In the step c), the complex enzyme used for enzymolysis is cellulase and pectinase in a mass ratio of 1:0.3-3; the mass ratio of the complex enzyme to the mycelium powder of Hirsutella batria is 0.1-1.5:

100.

5. The membrane separation and extraction method of the mycelium of Trichosporon bat moth according to claim 4, characterized in that The mass ratio of the cellulase to the pectinase is 1:1-2; the mass ratio of the complex enzyme to the mycelium powder of the Hirsutella bat moth is 1:80-100.

6. The membrane separation and extraction method of the mycelium of Trichosporon bat moth according to claim 1, characterized in that In the step c), the pH of the enzymatic hydrolysis is 6.5-7.5; the enzymatic hydrolysis time is 0.5-3h, and the enzymatic hydrolysis temperature is 30-60°C; preferably, the pH of the enzymatic hydrolysis mixture is adjusted to 6.5-7; the enzymatic hydrolysis time is 1-2h; and the enzymatic hydrolysis temperature is 40-50°C.

7. The membrane separation and extraction method of the mycelium of Trichosporon batriida according to claim 1, characterized in that In the step c), the ultrasonic extraction time is 30 to 120 minutes, and the ultrasonic frequency is 15 to 60 KHz; preferably, the ultrasonic extraction time is 60-90 minutes, and the ultrasonic frequency is 20-30 KHz.

8. The membrane separation and extraction method of the mycelium of Trichosporon bat moth according to claim 1, characterized in that In the step d), the membrane separation is performed sequentially through microfiltration and ultrafiltration, the microfiltration membrane used in the microfiltration is a cellulose membrane or a ceramic membrane with a pore size of 0.1 to 10 μm, the microfiltration operation temperature is 20 to 50° C., and the pressure is 0.1 to 0.5 MPa, and the ultrafiltration membrane used in the ultrafiltration is a polysulfone ultrafiltration membrane or a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 5000 to 30000D, and the ultrafiltration operation temperature is 25 to 50° C. and the pressure is 0.1 to 0.6 MPa.

9. The membrane separation and extraction method of the mycelium of Trichosporon bat moth according to claim 8, characterized in that In the step d), the microfiltration membrane is a ceramic microfiltration membrane with a pore size of 0.2 μm, the microfiltration operating temperature is 40-50° C., the pressure is 0.2-0.3 MPa, the feed concentration is 1-2%, and the ultrafiltration membrane is a polysulfone ultrafiltration membrane with a molecular weight cutoff of 10000-20000D, the ultrafiltration operating temperature is 40° C., and the pressure is 0.3-0.4 MPa.

10. The membrane separation and extraction method of the mycelium of Trichosporon batriida according to claim 1, characterized in that In the step d), the filtrate obtained by separation and purification is concentrated under reduced pressure, vacuum dried, freeze dried or spray dried to obtain a brown-yellow powder.

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