Cordyceps militaris extract and extraction process thereof

Abstract

The present application relates to a kind of cordyceps militaris extract and its extraction process, belong to traditional Chinese medicine extraction technical field.The present application is through enzymolysis, pulse ultrasonic and supercritical extraction synergistic effect to improve extraction efficiency.Specifically including: cordyceps militaris raw materials and cellulase, pectinase mixed enzymolysis, under the protection of inert gas, adding the protective agent containing glutathione and ascorbic acid and ethanol-citrate buffer solution is treated by pulse ultrasonic;Extract liquid is concentrated after supercritical CO2 extraction removes fat-soluble impurities, freeze-drying obtains extract.The obtained extract cordycepin content is ≥0.8%, and the mass ratio of cordycepin and cordycepic acid is stably at 1:0.2-1.The process solves the problem of serious damage of active ingredient in traditional method, low efficiency.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction technology, and relates to a Cordyceps militaris extract and its extraction process. Background Technology

[0002] Cordyceps militaris, a fungus with both medicinal and edible value, is highly regarded in the health supplement, pharmaceutical, and functional food industries due to its rich content of various active ingredients such as cordycepin, cordycepic acid, and polysaccharides. Cordycepin, as its characteristic active ingredient, possesses multiple biological activities including antibacterial, anti-inflammatory, and immunomodulatory effects, while cordycepic acid plays a crucial role in improving metabolism and protecting organ function. With the increasing market demand for Cordyceps militaris-related products, how to efficiently and stably extract and retain these active ingredients from Cordyceps militaris has become a key research focus in the industry.

[0003] Currently, the extraction processes for Cordyceps militaris extract mainly include traditional solvent extraction and hot reflux extraction. Traditional solvent extraction involves soaking the raw material in solvents such as ethanol and water. While simple to operate, it has low extraction efficiency, and the active ingredients are prone to degradation due to prolonged soaking. Hot reflux extraction promotes the dissolution of components through heating, which can improve efficiency, but high temperatures can easily destroy heat-sensitive components such as cordycepin.

[0004] Existing processes generally suffer from the following shortcomings: it is difficult to balance extraction efficiency with the retention of active ingredients; high temperature and strong oxidizing environments easily lead to the loss of effective components such as cordycepin, resulting in low cordycepin content in the extract, which is insufficient to meet the requirements of high-quality products. This proposed solution, through the synergistic design of compound enzymatic hydrolysis to break down cell walls, inert gas and compound protective agents for oxidation, pulsed ultrasonic temperature-controlled extraction, and supercritical CO2 for precise impurity removal, provides a feasible path for the efficient extraction of high-quality Cordyceps militaris extract. Summary of the Invention

[0005] The purpose of this invention is to provide a Cordyceps militaris extract and its extraction process, which uses cellulase and pectinase for compound enzymatic hydrolysis, significantly improving the extraction rate and retention rate of active ingredients.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] An extraction process for Cordyceps militaris extract includes the following steps:

[0008] (1) Mix and enzymatically hydrolyze the Cordyceps militaris raw material with cellulase and pectinase solution for 1-2 hours;

[0009] (2) Place the enzymatic hydrolysis product obtained in step (1) in an inert gas environment, add a protective agent and extraction solvent, and perform pulsed ultrasonic treatment;

[0010] (3) After concentrating the extract obtained in step (2), place it in a supercritical extraction vessel and extract it for 1-2 hours with CO2 as the medium. Remove the lipid-soluble impurities and freeze-dry the resulting aqueous solution to obtain the extract.

[0011] As a preferred technical solution of the present invention, the enzyme activity ratio of cellulase and pectinase in step (1) is 1:0.5-2, the amount of enzyme added is 0.5-1% of the weight of Cordyceps militaris raw material, and the mass ratio of Cordyceps militaris raw material to enzymatic hydrolysis solution is 1:8-12.

[0012] As a preferred technical solution of the present invention, the enzymatic hydrolysis in step (1) is performed at a pH of 4.5-5.5 and a temperature of 40-45℃.

[0013] As a preferred technical solution of the present invention, the protective agent in step (2) is glutathione and ascorbic acid in a mass ratio of 1:0.4-0.8; the amount of the protective agent added is 1.2-1.8% of the mass of Cordyceps militaris raw material.

[0014] As a preferred technical solution of the present invention, the extraction solvent in step (2) comprises: 10-30% anhydrous ethanol and 0.05-0.2 mol / L citrate buffer; the pH of the extraction solvent is 4.8-5.2.

[0015] As a preferred technical solution of the present invention, the working and intermittent time of the pulsed ultrasound in step (2) is 1-3s and 0.5-2s, respectively; the pulsed ultrasound processing temperature is controlled at 35-40℃.

[0016] As a preferred technical solution of the present invention, 5%-10% ethanol is added as an entrainer during the supercritical CO2 extraction in step (3).

[0017] As a preferred technical solution of the present invention, the supercritical CO2 extraction pressure in step (3) is 15-25 MPa and the temperature is 35-45℃.

[0018] As a preferred embodiment of the present invention, the concentration in step (3) is performed by vacuum concentration to a density of 1.10-1.25 g / cm³. 3 .

[0019] In this application, cellulase specifically hydrolyzes β-1,4-glycosidic bonds, and pectinase cleaves α-1,4-galacturonic acid bonds, with the two enzymes working synergistically to depolymerize the cell wall polysaccharide network; the sulfhydryl groups of glutathione can scavenge ·OH free radicals, and ascorbic acid reduces oxidized glutathione, maintaining the circulation and protecting the active substances, while low-temperature pulsed ultrasonic dissolution is performed; ethanol solution is used as an entrainer for CO2 supercritical extraction to remove lipid impurities, and cordycepin and cordycepic acid are retained in the aqueous phase for purification.

[0020] Furthermore, the Cordyceps militaris extract prepared by the extraction process described above has a cordycepin content of ≥0.8%; the mass ratio of cordycepin to cordycepic acid is 1:0.2-1.

[0021] The beneficial effects of this invention are:

[0022] This invention first employs a combined enzymatic hydrolysis using cellulase and pectinase. By optimizing the enzyme activity ratio, dosage, and hydrolysis conditions, it efficiently disrupts the cell wall structure of Cordyceps militaris, promoting the release of intracellular active ingredients. Simultaneously, during pulsed ultrasonic treatment, an inert gas environment isolates oxygen, and a composite protective agent composed of glutathione and ascorbic acid effectively inhibits the oxidative degradation of active ingredients such as cordycepin. Furthermore, the rational control of the pulsed ultrasonic operation, intervals, and temperature avoids localized high temperatures that could damage the components, significantly improving the extraction and retention rates of active ingredients. Detailed Implementation

[0023] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0024] Example 1

[0025] An extraction process for Cordyceps militaris extract includes the following steps:

[0026] (1) Mix Cordyceps militaris raw material with cellulase and pectinase solution and enzymatically hydrolyze for 1.5 h;

[0027] (2) Place the product from step (1) in an inert gas environment, add a protective agent and an extraction solvent, and perform pulsed ultrasonic treatment;

[0028] (3) After concentrating the extract from step (2), place it in a supercritical extraction vessel and extract it for 1.5 hours with CO2 as the medium. Remove the lipid-soluble impurities and freeze-dry the resulting aqueous solution to obtain the extract.

[0029] In step (1), the enzyme activity ratio of cellulase and pectinase is 1:1.2, and the amount of enzyme added is 0.8% of the weight of Cordyceps militaris raw material; the mass ratio of Cordyceps militaris raw material to enzymatic hydrolysis solution is 1:10.

[0030] In step (1), the enzymatic hydrolysis is performed at pH 5 and temperature 42°C.

[0031] The protective agent in step (2) is glutathione and ascorbic acid in a mass ratio of 1:0.6; the amount of the protective agent added is 1.6% of the mass of Cordyceps militaris raw material.

[0032] The extraction solvent in step (2) comprises: 20% anhydrous ethanol and 0.1 mol / L citrate buffer; the pH of the extraction solvent is 5.

[0033] The working and intermittent times of the pulsed ultrasound in step (2) are 2s and 1.5s, respectively; the temperature of the pulsed ultrasound treatment is controlled at 37℃.

[0034] In step (3), 7% ethanol is added as an entrainer during supercritical CO2 extraction.

[0035] The supercritical CO2 extraction pressure in step (3) is 20 MPa and the temperature is 40 °C.

[0036] The concentration described in step (3) is a vacuum concentration to a density of 1.18 g / cm³. 3 .

[0037] Example 2

[0038] An extraction process for Cordyceps militaris extract includes the following steps:

[0039] (1) Mix Cordyceps militaris raw material with cellulase and pectinase solution and enzymatically hydrolyze for 1 hour;

[0040] (2) Place the product from step (1) in an inert gas environment, add a protective agent and an extraction solvent, and perform pulsed ultrasonic treatment;

[0041] (3) After concentrating the extract from step (2), place it in a supercritical extraction vessel and extract it for 1-2 hours with CO2 as the medium. Remove the lipid-soluble impurities and freeze-dry the resulting aqueous solution to obtain the extract.

[0042] In step (1), the enzyme activity ratio of cellulase and pectinase is 1:0.5, and the amount of enzyme added is 0.5% of the weight of Cordyceps militaris raw material; the mass ratio of Cordyceps militaris raw material to enzymatic hydrolysis solution is 1:8.

[0043] The enzymatic hydrolysis in step (1) is performed at a pH of 4.5 and a temperature of 40°C.

[0044] The protective agent in step (2) is glutathione and ascorbic acid in a mass ratio of 1:0.4; the amount of the protective agent added is 1.2% of the mass of Cordyceps militaris raw material.

[0045] The extraction solvent in step (2) comprises: 10% anhydrous ethanol and 0.05 mol / L citrate buffer; the pH of the extraction solvent is 4.8.

[0046] The working and intermittent times of the pulsed ultrasound in step (2) are 1s and 0.5s, respectively; the temperature of the pulsed ultrasound treatment is controlled at 35℃.

[0047] In step (3), 5% ethanol is added as an entrainer during supercritical CO2 extraction.

[0048] The supercritical CO2 extraction pressure in step (3) is 15 MPa and the temperature is 35 °C.

[0049] The concentration described in step (3) is a vacuum concentration to a density of 1.1 g / cm³. 3 .

[0050] Example 3

[0051] An extraction process for Cordyceps militaris extract includes the following steps:

[0052] (1) Mix Cordyceps militaris raw material with cellulase and pectinase solution and enzymatically hydrolyze for 2 hours;

[0053] (2) Place the product from step (1) in an inert gas environment, add a protective agent and an extraction solvent, and perform pulsed ultrasonic treatment;

[0054] (3) After concentrating the extract from step (2), place it in a supercritical extraction vessel and extract it for 2 hours with CO2 as the medium. Remove the lipid-soluble impurities and freeze-dry the resulting aqueous solution to obtain the extract.

[0055] In step (1), the enzyme activity ratio of cellulase and pectinase is 1:2, and the amount of enzyme added is 1% of the weight of Cordyceps militaris raw material; the mass ratio of Cordyceps militaris raw material to enzymatic hydrolysis solution is 1:12.

[0056] The enzymatic hydrolysis in step (1) is performed at a pH of 5.5 and a temperature of 45°C.

[0057] The protective agent in step (2) is glutathione and ascorbic acid in a mass ratio of 1:0.8; the amount of the protective agent added is 1.8% of the mass of Cordyceps militaris raw material.

[0058] The extraction solvent in step (2) comprises: 30% anhydrous ethanol and 0.2 mol / L citrate buffer; the pH of the extraction solvent is 5.2.

[0059] The working and intermittent times of the pulsed ultrasound in step (2) are 3s and 2s respectively; the temperature of the pulsed ultrasound treatment is controlled at 40℃.

[0060] In step (3), 10% ethanol is added as an entrainer during supercritical CO2 extraction.

[0061] The supercritical CO2 extraction pressure in step (3) is 25 MPa and the temperature is 45 °C.

[0062] The concentration described in step (3) is a vacuum concentration to a density of 1.25 g / cm³. 3 .

[0063] Comparative Example 1

[0064] Based on Example 1, only cellulase was added to the enzymatic hydrolysis step, while the rest remained the same as in Example 1.

[0065] Comparative Example 2

[0066] Based on Example 1, only pectinase was added to the enzymatic hydrolysis step, while the rest remained the same as in Example 1.

[0067] Comparative Example 3

[0068] Based on Example 1, the protective agent in step (2) is glutathione, and the rest is the same as in Example 1.

[0069] Comparative Example 4

[0070] Based on Example 1, the protective agent in step (2) is ascorbic acid, and the rest is the same as in Example 1.

[0071] Comparative Example 5

[0072] Based on Example 1, step (2) uses 20% ethanol aqueous solution as the extraction solvent and adjusts the pH to 5 with dilute hydrochloric acid, while the rest remains the same as in Example 1.

[0073] Comparative Example 6

[0074] Based on Example 1, step (3) is changed from pulsed ultrasound to continuous ultrasound and the total action time of continuous ultrasound is the same as that of pulsed ultrasound, while the rest is the same as in Example 1.

[0075] Comparative Example 7

[0076] Based on Example 1, step (3) supercritical CO2 extraction was replaced with petroleum ether extraction three times (material-liquid ratio 1:1) for degreasing, while the rest remained the same as in Example 1.

[0077] Performance testing:

[0078] Cordycepin content: Take 0.1g of the extract powder from the examples and comparative examples, add 10mL of preheated pure water at 80℃, vortex for 5min, cool to room temperature in an ice bath, centrifuge at 12000rpm for 10min, and filter the supernatant through a 0.45μm filter membrane; separate by reversed-phase high-performance liquid chromatography, and quantitatively detect by ultraviolet detector (260nm); chromatographic conditions: C18 column (4.6mm×250mm, 5μm); mobile phase: methanol-0.1% phosphoric acid aqueous solution (15:85, v / v); flow rate 1.0mL / min; column temperature 30℃;

[0079] Cordycepic acid content: 1 g of the extract powder from the examples and comparative examples was dissolved in 10 mL of deionized water by heating. After cooling, anhydrous ethanol (final concentration 70%) was added, and the mixture was allowed to stand for 1 h. After centrifugation, the precipitate was reconstituted with water and filtered through a 0.45 μm filter membrane. The content of cordycepic acid was detected using an evaporative light scattering detector (ELSD). Chromatographic conditions: Alltima NH2 column (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile-water (75:25, v / v); flow rate 1.0 mL / min; column temperature 35 °C; drift tube temperature 60 °C; carrier gas (nitrogen) flow rate 2.0 L / min.

[0080] Polysaccharide content: Take 1g of extract powder, add 10mL of 80% ethanol and reflux for 2h (to remove monosaccharides and oligosaccharides), filter and add 10mL of deionized water to the residue and boil for 30min, centrifuge and take the supernatant, and make up to 50mL; the polysaccharide is dehydrated under the action of concentrated sulfuric acid to produce furfural, which condenses with phenol to produce a colored substance, and is quantified by colorimetric measurement at 490nm.

[0081]

[0082] The test results above show that the cordycepin content of the extracts from the examples is 0.83-0.88%, which is 2-3 times higher than that of traditional processes. The cordycepin / cordycepic acid ratio is about 1:0.37, which is close to the ratio of natural cordyceps. Comparative Examples 1 and 2 were enzymatically hydrolyzed with only a single enzyme, resulting in incomplete depolymerization of the cell wall and a significant decrease in cordycepin yield. The decrease in cordycepin in Comparative Examples 3 and 4 reveals that the synergistic effect of glutathione (free radical scavenging) and ascorbic acid (redox cycle) is indispensable. The lack of citrate buffer in the extraction solvent of Comparative Example 5 reduced the solubility, and the content of each active substance decreased. Continuous sonication in Comparative Example 6 resulted in a 3.8% loss of polysaccharide active components. Petroleum ether extraction in Comparative Example 7 left solvent residue.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any indirect modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An extraction process for Cordyceps militaris extract, characterized in that: Includes the following steps: (1) Mix and enzymatically hydrolyze the Cordyceps militaris raw material with cellulase and pectinase solution for 1-2 hours; (2) Place the enzymatic hydrolysis product obtained in step (1) in an inert gas environment, add a protective agent and extraction solvent, and perform pulsed ultrasonic treatment; (3) After concentrating the extract obtained in step (2), place it in a supercritical extraction vessel and extract it with CO2 as the medium for 1-2 hours. Remove the lipid-soluble impurities and freeze-dry the resulting aqueous solution to obtain the extract. The protective agent is glutathione and ascorbic acid in a mass ratio of 1:0.4-0.8; the amount of the protective agent added is 1.2-1.8% of the mass of Cordyceps militaris raw material. The extraction solvent comprises: 10-30% anhydrous ethanol and 0.05-0.2 mol / L citrate buffer; the pH of the extraction solvent is 4.8-5.

2.

2. The extraction process of Cordyceps militaris extract according to claim 1, characterized in that: In step (1), the enzyme activity ratio of cellulase and pectinase is 1:0.5-2, and the amount of enzyme added is 0.5-1% of the weight of Cordyceps militaris raw material; the mass ratio of Cordyceps militaris raw material to enzymatic hydrolysis solution is 1:8-12.

3. The extraction process of Cordyceps militaris extract according to claim 1, characterized in that: The enzymatic hydrolysis in step (1) is performed at a pH of 4.5-5.5 and a temperature of 40-45℃.

4. The extraction process of Cordyceps militaris extract according to claim 1, characterized in that: The working and intermittent time of the pulsed ultrasound in step (2) is 1-3s and 0.5-2s respectively; the temperature of the pulsed ultrasound treatment is controlled at 35-40℃.

5. The extraction process of Cordyceps militaris extract according to claim 1, characterized in that: In step (3), 5%-10% ethanol is added as an entrainer during supercritical CO2 extraction.

6. The extraction process of Cordyceps militaris extract according to claim 1, characterized in that: The supercritical CO2 extraction pressure in step (3) is 15-25 MPa and the temperature is 35-45℃.

7. The extraction process of Cordyceps militaris extract according to claim 1, characterized in that: The concentration in step (3) is to concentrate under reduced pressure to a density of 1.10-1.25 g / cm3.

8. A Cordyceps militaris extract prepared by the extraction process according to any one of claims 1-7, characterized in that: The cordycepin content is ≥0.8%; the mass ratio of cordycepin to cordycepic acid is 1:0.2-1.

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