Cordyceps militaris extract and extraction process thereof

Abstract

The invention relates to a cordyceps militaris extract and an extraction process thereof, and belongs to the technical field of traditional Chinese medicine extraction. The extraction efficiency is improved through the synergistic effect of enzymolysis, pulse ultrasound and supercritical extraction. The method specifically comprises the following steps: mixing a cordyceps militaris raw material with cellulase and pectinase for enzymolysis, and adding a protective agent containing glutathione and ascorbic acid and an ethanol-citrate buffer solution under the protection of inert gas for pulse ultrasonic treatment; and concentrating the extracting solution, performing supercritical CO2 extraction to remove fat-soluble impurities, and performing freeze drying to obtain the extract. The cordycepin content of the obtained extract is larger than or equal to 0.8%, and the mass ratio of cordycepin to cordycepic acid is stabilized to be 1: 0.2-1. The process solves the problems of serious damage to active components and low efficiency of a traditional method.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction and relates to a Cordyceps militaris extract and an extraction process thereof. Background Art

[0002] Cordyceps militaris, a fungus with both medicinal and edible properties, is attracting significant attention in the healthcare, pharmaceutical, and functional food sectors due to its rich content of active ingredients, including cordycepin, cordycepic acid, and polysaccharides. Cordycepin, its signature active ingredient, exhibits numerous biological activities, including antibacterial, anti-inflammatory, and immune-modulating properties, while cordycepic acid plays a key role in improving metabolism and protecting organ function. With growing market demand for Cordyceps militaris-related products, the efficient and stable extraction and preservation of these active ingredients from Cordyceps militaris has become a key research priority within the industry. Currently, the extraction processes for Cordyceps militaris extracts mainly include traditional solvent extraction and hot reflux extraction. Traditional solvent extraction involves soaking the raw materials in solvents such as ethanol and water. While simple to operate, it has low extraction efficiency and the active ingredients are easily degraded due to prolonged soaking. Hot reflux extraction promotes ingredient dissolution through heating, which improves efficiency. However, high temperatures can easily destroy heat-sensitive ingredients such as cordycepin. Existing processes generally suffer from the following shortcomings: It's difficult to balance extraction efficiency and active ingredient retention. High temperatures and strong oxidative environments can easily lead to the loss of active ingredients like cordycepin, resulting in low cordycepin content in the extract, making it difficult to meet high-quality product requirements. This solution, by synergizing complex enzymatic cell wall disruption, inert gas and composite protective agents for antioxidant protection, pulsed ultrasonic temperature-controlled extraction, and supercritical CO2 for precise impurity removal, provides a viable path for the efficient extraction of high-quality Cordyceps militaris extracts. Summary of the Invention

[0003] The present invention aims to provide a Cordyceps militaris extract and an extraction process thereof, which adopts cellulase and pectinase for composite enzymatic hydrolysis, thereby greatly improving the extraction rate and retention rate of active ingredients.

[0004] The purpose of the present invention can be achieved through the following technical solutions: An extraction process for a Cordyceps militaris extract comprises the following steps: (1) Mixing and enzymolyzing the Cordyceps militaris raw materials under the action of cellulase and pectinase solution for 1-2 hours; (2) placing the enzymatic hydrolysis product obtained in step (1) in an inert gas environment, adding a protective agent and an extraction solvent, and performing pulsed ultrasonic treatment; (3) The extract obtained in step (2) is concentrated and placed in a supercritical extraction kettle, extracted with CO2 as a medium for 1-2 hours, and the resulting aqueous solution is freeze-dried to obtain an extract after removing fat-soluble impurities.

[0005] As a preferred technical scheme of the present application, the enzyme activity ratio of the cellulase and the pectinase in step (1) is 1:0.5-2, and the enzyme addition amount is 0.5-1% of the weight of the cordyceps militaris raw material; the mass ratio of the cordyceps militaris raw material to the enzymolysis solution is 1:8-12.

[0006] As a preferred technical scheme of the present application, the enzymolysis pH in step (1) is 4.5-5.5, and the temperature is 40-45℃.

[0007] As a preferred technical scheme of the present application, the protective agent in step (2) is glutathione and ascorbic acid with a mass ratio of 1:0.4-0.8; and the addition amount of the protective agent is 1.2-1.8% of the mass of the cordyceps militaris raw material.

[0008] As a preferred technical scheme of the present application, the extraction solvent in step (2) comprises 10-30% anhydrous ethanol and 0.05-0.2 mol / L citrate buffer; and the pH of the extraction solvent is 4.8-5.2.

[0009] As a preferred technical scheme of the present application, the working and intermittent time of the pulse ultrasonic is 1-3s and 0.5-2s; and the pulse ultrasonic treatment temperature is controlled at 35-40℃.

[0010] As a preferred technical scheme of the present application, 5%-10% ethanol is added as an entraining agent during the supercritical CO2 extraction in step (3).

[0011] As a preferred technical scheme of the present application, the supercritical CO2 extraction pressure in step (3) is 15-25MPa, and the temperature is 35-45℃.

[0012] As a preferred technical scheme of the present application, the concentration in step (3) is reduced pressure concentration to a density of 1.10-1.25g / cm 3 .

[0013] In the present application, the cellulase specifically hydrolyzes β-1,4-glycosidic bonds, the pectinase cleaves α-1,4-galacturonic acid bonds, and the two enzymes cooperate to depolymerize the cell wall polysaccharide network; the thiol group of glutathione can remove ·OH free radicals, ascorbic acid reduces oxidized glutathione, maintains the circulation of protective active substances, and simultaneously performs low-temperature pulse ultrasonic dissolution; ethanol solution is used as an entraining agent for CO2 supercritical extraction, lipid impurities are removed, and cordycepin and cordycepic acid are retained in the water phase for purification.

[0014] Further, a cordyceps militaris extract prepared by the extraction process has a cordycepin content of ≥0.8%; and the mass ratio of cordycepin to cordycepic acid is 1:0.2-1.

[0015] The present application has the following beneficial effects: The present invention first uses cellulase and pectinase for composite enzymolysis. By optimizing the enzyme activity ratio, addition amount, enzymolysis conditions, etc., it can efficiently destroy the cell wall structure of Cordyceps militaris and promote the release of intracellular active ingredients. At the same time, during pulsed ultrasonic treatment, oxygen is isolated by an inert gas environment, and a composite protective agent composed of glutathione and ascorbic acid is used to effectively inhibit the oxidative degradation of active ingredients such as cordycepin. In addition, the pulsed ultrasonic working and intermittent time and temperature control are reasonable, avoiding the destruction of ingredients by local high temperature and significantly improving the extraction rate and retention rate of active ingredients. DETAILED DESCRIPTION

[0016] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0017] Example 1 An extraction process for a Cordyceps militaris extract comprises the following steps: (1) Mixing the Cordyceps militaris raw material with cellulase and pectinase solution and performing enzymatic hydrolysis for 1.5 h; (2) placing the product of step (1) in an inert gas environment, adding a protective agent and an extraction solvent, and performing pulsed ultrasonic treatment; (3) After the extract in step (2) is concentrated, it is placed in a supercritical extraction kettle and extracted with CO2 as the medium for 1.5 hours. The fat-soluble impurities are removed and the resulting aqueous solution is freeze-dried to obtain an extract.

[0018] In step (1), the enzyme activity ratio of the cellulase to the pectinase is 1:1.2, and the amount of enzyme added is 0.8% of the weight of the Cordyceps militaris raw material; the mass ratio of the Cordyceps militaris raw material to the enzymatic hydrolysis solution is 1:10.

[0019] The enzymatic hydrolysis pH in step (1) is 5 and the temperature is 42°C.

[0020] The protective agent in step (2) is glutathione and ascorbic acid in a mass ratio of 1:0.6; the added amount of the protective agent is 1.6% of the mass of the Cordyceps militaris raw material.

[0021] 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.

[0022] The pulsed ultrasound working and rest time in step (2) are 2s and 1.5s respectively; the pulsed ultrasound treatment temperature is controlled at 37°C.

[0023] During the supercritical CO2 extraction in step (3), 7% ethanol is added as an entrainer.

[0024] The supercritical CO2 extraction pressure in step (3) is 20 MPa, and the temperature is 40℃.

[0025] The concentration in step (3) is reduced pressure concentration to a density of 1.18 g / cm 3 .

[0026] Example 2 An extraction process of a Cordyceps militaris extract includes the following steps: (1) mixing Cordyceps militaris raw materials with a cellulase and pectinase solution for enzymolysis for 1 h; (2) placing the product of step (1) in an inert gas environment, adding a protective agent and an extraction solvent, and performing pulse ultrasonic treatment; (3) concentrating the extraction liquid of step (2), placing it in a supercritical extraction kettle, extracting it for 1-2 h with CO2 as the medium, removing the fat-soluble impurities, and freeze-drying the water-soluble solution to obtain the extract.

[0027] The enzyme activity ratio of the cellulase and pectinase in step (1) is 1:0.5, and the enzyme addition amount is 0.5% of the weight of the Cordyceps militaris raw materials; the mass ratio of the Cordyceps militaris raw materials to the enzymolysis solution is 1:8.

[0028] The enzymolysis pH in step (1) is 4.5, and the temperature is 40℃.

[0029] The protective agent in step (2) is glutathione and ascorbic acid with a mass ratio of 1:0.4; the addition amount of the protective agent is 1.2% of the mass of the Cordyceps militaris raw materials.

[0030] The extraction solvent in step (2) includes 10% anhydrous ethanol and 0.05 mol / L citrate buffer; the pH of the extraction solvent is 4.8.

[0031] The working and intermittent time of the pulse ultrasonic in step (2) is 1 s and 0.5 s; the pulse ultrasonic treatment temperature is controlled at 35℃.

[0032] 5% ethanol is added as a entraining agent during the supercritical CO2 extraction in step (3).

[0033] The supercritical CO2 extraction pressure in step (3) is 15 MPa, and the temperature is 35℃.

[0034] The concentration in step (3) is reduced pressure concentration to a density of 1.1 g / cm 3 .

[0035] Example 3 An extraction process of a Cordyceps militaris extract includes the following steps: (1) mixing Cordyceps militaris raw materials with a cellulase and pectinase solution for enzymolysis for 2 h; (2) The product of step (1) is placed in an inert gas environment, a protective agent and an extraction solvent are added, and pulse ultrasonic treatment is performed; (3) After the extract of step (2) is concentrated, it is placed in a supercritical extraction kettle, CO2 is used as the medium for extraction for 2 h, and the water solution obtained by removing the fat-soluble impurities is freeze-dried to obtain the extract.

[0036] In step (1), the ratio of the enzyme activity of cellulase to pectinase is 1:2, and the enzyme addition amount is 1% of the weight of the cordyceps militaris raw material; the mass ratio of the cordyceps militaris raw material to the enzymolysis solution is 1:12.

[0037] In step (1), the pH of the enzymolysis is 5.5, and the temperature is 45°C.

[0038] In step (2), the protective agent is glutathione and ascorbic acid with a mass ratio of 1:0.8; the addition amount of the protective agent is 1.8% of the mass of the cordyceps militaris raw material.

[0039] In step (2), the extraction solvent comprises 30% anhydrous ethanol and 0.2 mol / L citrate buffer; the pH of the extraction solvent is 5.2.

[0040] In step (2), the working and intermittent time of the pulse ultrasonic is 3s and 2s; the pulse ultrasonic treatment temperature is controlled at 40°C.

[0041] In step (3), 10% ethanol is added as an entraining agent during the supercritical CO2 extraction.

[0042] In step (3), the supercritical CO2 extraction pressure is 25MPa, and the temperature is 45°C.

[0043] In step (3), the concentration is reduced pressure concentration to a density of 1.25 g / cm 3 .

[0044] Comparative Example 1 Based on Example 1, only cellulase is added in the enzymolysis step, and the rest is consistent with Example 1.

[0045] Comparative Example 2 Based on Example 1, only pectinase is added in the enzymolysis step, and the rest is consistent with Example 1.

[0046] Comparative Example 3 Based on Example 1, the protective agent in step (2) is glutathione, and the rest is consistent with Example 1.

[0047] Comparative Example 4 Based on Example 1, the protective agent in step (2) is ascorbic acid, and the rest is consistent with Example 1.

[0048] Comparative Example 5 On the basis of Example 1, the extraction solvent in step (2) was 20% ethanol aqueous solution, and the pH was adjusted to 5 with dilute hydrochloric acid. The rest was consistent with Example 1.

[0049] Comparative Example 6 On the basis of Example 1, the pulsed ultrasound in step (3) was changed to continuous ultrasound and the total action time of the continuous ultrasound was kept the same as that of the pulsed ultrasound. The rest was the same as that of Example 1.

[0050] Comparative Example 7 On the basis of Example 1, step (3) supercritical CO2 extraction was replaced by extraction with petroleum ether three times (material-liquid ratio 1:1) for fat removal, and the rest was consistent with Example 1.

[0051] Performance testing: Cordycepin content: 0.1 g of the extract powder from the examples and comparative examples was added to 10 mL of pure water preheated at 80°C, vortexed for 5 minutes, cooled to room temperature in an ice bath, and centrifuged at 12,000 rpm for 10 minutes. The supernatant was filtered through a 0.45 μm filter. The mixture was separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and quantitatively detected by a UV detector (260 nm). Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid aqueous solution (15:85, v / v); flow rate: 1.0 mL / min; column temperature: 30°C. Cordycepic acid content: 1 g of the extract powder from the examples and comparative examples was added to 10 mL of deionized water and heated to dissolve. After cooling, anhydrous ethanol (final concentration 70%) was added, the mixture was allowed to stand for 1 h, centrifuged, the precipitate was redissolved in water, and filtered through a 0.45 μm filter. The cordycepic acid content was determined 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. Polysaccharide content: Take 1g of extract powder, add 10mL of 80% ethanol and reflux extraction for 2h (to remove monosaccharides and oligosaccharides). After filtering, add 10mL of deionized water to the residue and boil for 30min. Centrifuge to obtain the supernatant and adjust the volume to 50mL. Polysaccharides are dehydrated in the presence of concentrated sulfuric acid to form furfural, which condenses with phenol to form a colored substance, which is quantified colorimetrically at 490nm.

[0052] The above test results show that the cordycepin content of the extracts of the examples is 0.83-0.88%, which is 2-3 times higher than that of the traditional process, and the cordycepin / cordycepic acid ratio is about 1:0.37, which is close to the ratio of natural Cordyceps. Comparative Examples 1 and 2 use only a single enzyme for enzymatic hydrolysis, resulting in incomplete depolymerization of the cell wall and a significant decrease in the 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 reduces the solubility and the content of each active substance decreases. Comparative Example 6 continuous ultrasound leads to a 3.8% loss of polysaccharide active components. Comparative Example 7 uses petroleum ether extraction and has solvent residues.

[0053] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modifications to equivalent embodiments of equivalent changes using the technical content disclosed above without departing from the scope of the technical solution of the present invention. However, any indirect modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A process for extracting Cordyceps militaris extract, characterized in that: The following steps are involved: (1) Mixing and enzymolyzing the Cordyceps militaris raw materials under the action of cellulase and pectinase solution for 1-2 hours; (2) placing the enzymatic hydrolysis product obtained in step (1) in an inert gas environment, adding a protective agent and an extraction solvent, and performing pulsed ultrasonic treatment; (3) The extract obtained in step (2) is concentrated and placed in a supercritical extraction kettle, extracted with CO2 as a medium for 1-2 hours, and the resulting aqueous solution is freeze-dried to obtain an extract after removing fat-soluble impurities.

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

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

4. The extraction process of a Cordyceps militaris extract according to claim 1, characterized in that: The protective agent in step (2) is glutathione and ascorbic acid in a mass ratio of 1:0.4-0.8; the added amount of the protective agent is 1.2-1.8% of the mass of the Cordyceps militaris raw material.

5. The extraction process of the Cordyceps militaris extract according to claim 1, characterized in that: 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.

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

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

8. The extraction process of a 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°C.

9. The extraction process of a 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 / cm 3 .

10. A Cordyceps militaris extract obtained by the extraction process according to any one of claims 1 to 9, 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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