KR101548311B1 - Method for producing cordycepic acid by means of liquid fermentation of cordyceps militaris - Google Patents

Abstract

The present invention relates to a method for producing chordysepinic acid using liquid fermentation of a pupa of Cordyceps sinensis, which comprises: (1) cultivating a puddied Cordyceps sinensis strain in a PD liquid fermentation medium, activating the culture, transferring it to a seed fermentation medium, Proceed to obtain seed solution; (2) The seed liquid is inoculated into a liquid fermentation medium at a volume ratio of 5 to 15%, followed by liquid fermentation for 20 to 30 hours, then expansin solution is added, and the culture is continued for 96 to 144 hours, To obtain a pupae mycelium of pupa; (3) Cordycepinic acid in the pupae of Cordyceps sinensis cells is obtained from the mycelia of pupae. The production of chondisipinic acid fermentation can be improved by using expansin for the production of liquid fermentation of cordisepinic acid in the pupa of cordyceps.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing chordysepinic acid by using liquid fermentation of Cordyceps sinensis,

The present invention pertains to the field of bio-fermentation process technology, and more particularly, to a method for producing chondiesinic acid using liquid fermentation of a pupa of Cordyceps sinensis.

Cordyceps militaris is a medicinal fungus combined with chrysanthemum. It is a species of Ascomycota, Hypocreales, Clavicipitaceae and Cordyceps. Cordyceps militaris is a type of Cordyceps militaris, And so on. Cordycepic acid is a major physiologically active substance, and its anti-free radicals (Pseudomonas aeruginosa) are the major physiologically active substances. ) And antioxidant properties. In addition, the content of cordisepinic acid is one of the main indexes for the quality of Chinese caterpillar fungus, and the medicinal value of Chinese caterpillar fungus is high, which is generally high in cordisipinic acid content. According to the test results, the cordycepsin contains about 7% of cordycepinic acid component, and the content of active ingredient of cordycepinic acid in the pupae cordyceps is remarkably higher than that of cordyceps. According to pharmacological studies, cordu- sipinic acid promotes the metabolism of the human body, improves the microcirculatory system, significantly reduces blood lipids, inhibits bacteria, strengthens the immune system against diseases, It is effective in suppressing asthma, removing sputum, and treating asthma. The immune strengthening effect of Cordyceps enhances the liver's detoxifying action, protects hepatocytes effectively by preventing hepatic fibrosis and lipid peroxidation, and can also prevent and treat diseases such as cerebral thrombosis, cerebral hemorrhage, myocardial infarction, and long-term debilitation .

As the demand for active ingredients such as chrysanthematous chrysanthematous chrysanthemum has increased, there has been an increase in the number of wild chrysanthemum collecting caterpillars, and since the process of chemically synthesizing the active ingredients is complicated, the production amount is extremely small. Therefore, There is. According to the conventional studies, the active ingredient of Cordyceps wort thrombolytic enzyme directly extracted from the pupae and the mycelia through liquid fermentation has basically the same biochemical structure and pharmacological action, has a short incubation period, is easy to control the production process, It is easy to do. In addition, the extractive content and efficiency of active substances such as chordysinic acid are much higher than those produced and extracted using artificially cultivated caterpillar fungus. However, the production of active ingredients such as pupae and cordycepinic acid in the past is still a conventional solid culture center, and the research and application of liquid culture has a short time of technological development and the development of many fermentation techniques and processes is still insufficient, Production and fermentation levels are relatively low, limiting industrial production.

Exxsin is a new type of protein found in plant cell walls. Previous studies have shown that expansin almost participates in all developmental processes of the plant, and more specifically, in addition to the function of expanding cells, expansin also induces cell growth, seed germination, myosinogenesis, myosinogenesis, leaf growth, It plays an important role in the maturation, organs and the growth of the pistil. It is generally known that expansin is present in the cell walls of various dicotyledonous and monocotyledonous plants as the expansin is found in various plants such as Arabidopsis, tomatoes, strawberries, cotton, rice, and corn. Experiments have shown that expansin is a cell wall enzyme protein that induces cell wall expansion and pressure relaxation by destroying hydrogen bonds between cell wall polymers, which can be an important regulator of physiological regulation and cell wall expansion during plant growth. However, since the mechanism of action of exshenthin has not yet been clearly defined, it has not been disclosed at present in domestic or foreign countries to produce exenphin by applying it to liquid fermentation of edible and medicinal pupae, Cordyceps, and enzymes .

The present invention relates to a method for producing chordysepinic acid produced by liquid fermentation of a pupa of Cordyceps sinensis using expansin, which has overcome the disadvantages of the prior art. A concrete method of the present invention is as follows.

A method for producing chordysepinic acid using liquid fermentation of a pupa of Cordyceps sinensis,

Step (1): Pupae Cordyceps is inoculated into a PD liquid fermentation medium to activate the culture, then an inoculum amount of 10 to 15% by volume is transferred to the seed fermentation medium and the seed culture is carried out. The seed culture is carried out at 20 to 25 & Shaking for 72 hours to obtain a seed solution;

Step (2): The seed liquid obtained in the step (1) is inoculated into a liquid fermentation medium at a volume ratio of 5 to 15%, and the liquid fermentation is carried out at 20 to 25 캜 for 20 to 30 hours. The concentration of pansin is adjusted to 0.5 to 2.5 mg / mL, followed by continued culturing for 96 to 144 hours to obtain a mycelia of pupae; And

Step (3): Cordycepinic acid, i.e., chordysepinic acid, is extracted from Cordyceps sinensis cells from the pupae of Cordyceps sinensis obtained in step (2).

Preferably, in the step (1) of the present invention, the constituents per liter of the PD liquid fermentation medium are 200 g of potato, 20 g of glucose and a static capacity of distilled water of 1000 mL.

More preferably, in the step (1) of the present invention, the cultivation activation conditions are a shaking rotation speed of 100 to 160 r / min, a temperature of 20 to 25 캜, and a cancer culture activation of 24 to 72 hours.

Preferably in the above step (1) of the present invention, per liter of composition of the seed fermentation medium is glucose 3g, yeast supernatant powder 2.5g, 0.1g MgSO _4, CaCl ₂ 0.03 g, KH ₂ PO ₄ 0.1g, K ₂ 0.1 g of HPO ₄ , and 2 to 5 glass spheres having a diameter of 3 to 6 mm. After culturing in a seed fermentation medium, the amount of the fungus can be improved by more than 60%, the diameter of the fungus can be reduced by more than 40%, and the hyphae are uniformly spread.

Preferably, in the step (2) of the present invention, the constituent components per liter of the liquid fermentation medium are 3 g of lactose, 2 g of molasses, 2 g of soybean flour, 3 g of peptone, 0.2 g of MgSO ₄ , 0.03 g of CaCl ₂ and 0.3 g of KH ₂ PO ₄ , And K ₂ HPO ₄ 0.3 g.

Preferably, in step (2) of the present invention, the concentration of expansin is 1.0 to 2.5 mg / mL, more preferably the concentration of expansin is 1.8 to 2.0 mg / mL, (2), the concentration of expansin is 1.5 mg / mL.

In the above step (2), the exxanthine solution may be prepared by referring to the prior art, for example, McQueen-Mason et al., McQueen-Mason SJ, Durachko DM, Cosgrove D J. Two endogenous proteins that induce cell wall extensions in plants. Plant Cell, 1992, 4: 1425-1433, and the exxan solution can also be prepared by the following method.

The bean curd or cucumber seeds are sterilized with 0.05 to 0.15% by weight of HgCl ₂ for 4 to 6 minutes, washed with running water for 5 to 7 hours, placed in wet vermiculite and cultured at 25 to 28 ° C for 4 to 6 days ; The seedlings were cut to a size of 4 to 5 cm, pre-cooled at -20 ° C for 1 to 2 hours, homogenized by adding a homogenized buffer pre-cooled to 0 to 4 ° C, and then filtered with a nylon mesh having a diameter of 60 to 80 μm The filtrate residue is washed with a homogeneous buffer, the filtrate residue is added to the homogeneous buffer solution and left at room temperature for 1 to 3 hours to obtain a static solution; It was added to extract the static solution at 0 to 4 ℃ 24 to 30 hours, and filtered to extract and slowly added (NH _{4) 2} SO ₄ the filtrate to 0.3 to 0.5g / mL amount, (NH _{4) 2} SO stirring was continued during the addition of the ₄ (NH _{4) 2} SO ₄ is then partially preventing supersaturation in, for 5 to 10 minutes centrifugation, the precipitate was acidic buffer to 25000g at 4 ℃ allowed to stand for 24 to 30 hours And dialyzed at 4 ° C with a dialysis bag having a molecular weight of 3000 Da. The dialysis solution is centrifuged at 20,000 g for 5 to 10 minutes, and the supernatant is taken to prepare an exampan solution.

In the exxancin solution preparation method, the homogeneous buffer solution is composed of 25 mmol / L of HEPES (N-2-hydroxyethylpiperadine-N-2-ethanesulfonic acid), 3 mmol / L of Na ₂ S ₂ L of HEPES, 1.0 mmol / L of EDTA, 3 mmol / L of Na ₂ O, ₅ mmol / L of EDTA (ethylenediaminetetraacetic acid), 0.1 wt% of Triton X-100, S ₂ O ₅ , 0.5 mol / L NaCl, pH 6.8. The preparation of the acidic buffer solution is performed by dissolving 2.05 g of sodium acetate in water, adjusting the pH to 4.0 with acetic acid, and setting the static capacity of water to 1 L.

Preferably, in the step (2) of the present invention, the separation is performed by centrifuging at 5 ° C for 12 minutes at 4 ° C for 12 minutes.

Preferably, in the step (3) of the present invention, the method for extracting Cordyceps sinensis Cordyceps pupa is as follows.

The pupae obtained in the above step (2) was dried at 65 ° C for 1 to 3 hours to grind the powder. Then, 20 mL of an ethanol solution having a volume concentration of 50% was added to 0.1 g of the pupae pupae mycelia powder, For 1 minute, and centrifuged at 12,000 r / min for 5 minutes to obtain a supernatant. 20 ml of an ethanol solution having a volume concentration of 50% is added to the precipitate, and microwave treatment and centrifugation are carried out according to the above step, and then the supernatant is combined to obtain chordysinic acid.

Compared with the prior art, the advantages of the present invention are as follows.

1. In the present invention, expansin can be utilized for production of liquid fermentation of Cordycepinic acid in the pupa of the pupa, with a yield of 2.621 g / L, which is 7.3 times higher than that of the control (Comparative Example 1). The present invention has a considerably bright industrial use prospect because it greatly improves the yield of the active ingredient of cordisepinic acid in all the pupae Cordyceps.

2. The liquid aerobic fermentation method used in the present invention generally takes 5 to 7 days, and it has a higher yield and shorter cycle time than the prior art, and does not require processes such as static culture and induction of synthetic products, great. Produced pupae Cordycepinic acid active ingredients of Cordycepsia can be used directly in the manufacture of medicines such as controlling immunity, lowering cholesterol, protecting the liver, lowering blood sugar.

3. In the present invention, the above-mentioned expansin can be extracted from a large number of dicotyledonous and unicorn plants, and thus has a wide range of sources and a relatively low cost. Since the production method of the present invention is relatively simple by optimizing the steps and can be produced by scale production because of its high production yield, the fermentation production of the active substance of the cordycepinic acid of the pupae of Cordyceps sinensis has excellent industrial promotion and improvement effect.

4. The pupa fermentation process used in the present invention is simple, eco-friendly, non-toxic, relatively low in cost of raw materials, can control the entire fermentation process, and is suitable for industrial production , Can be widely used.

5. In the present invention, since the method of combining the liquid fermentation medium and the seed fermentation medium is optimized, the production amount of the chordysepinic acid active ingredient of the pupae of Cordyceps sinensis can be greatly improved.

FIG. 1 is a graph showing the effect of different concentrations of exxanthine solution on the production of cordieresinic acid in the pupa of Cordyceps sinensis.

The following examples are intended to further illustrate the present invention and do not limit the scope of protection of the present invention.

Raw materials and badges

In the examples, the Cordyceps militaris fermentation strain of the pupa was classified into CGMCC No. 5. 701 and CGMCC No. 5. 3.4655, and purchased from China General Microbiological Culture Collection Center (CGMCC).

Mannitol, Rhamnose, ammonium acetate, and acetylacetone were all purchased from Jinan Shengwei Biotech Co., Ltd. in the examples.

In the examples, the production steps of the expansin solution are as follows.

Vicia faba L (purchased from Jinan Maofeng) or cucumber (purchased from Cucumis sativus L. CV. Jinnian No. 6, Jinan Weili) seeds were weighed to 0.15 weight After 6 min. Rinsing with HgCl ₂ for 6 min., Washing in running water for 6 hrs, put in wet vermiculite and incubate for 4 days at 27 ° C. The seedling seedling axis was cut to 4 to 5 cm, that is, about 100 g of the growth zone was preliminarily cooled in a refrigerator at -20 ° C for 1 hour, homogenized at a high speed by adding a pre-cooled homogenized buffer to 4 ° C and filtered with a nylon mesh having a diameter of 70 μm The filtrate residue is washed with a homogeneous buffer solution, the filtrate residue is added to the homogeneous buffer solution, and left at room temperature for 2 hours to obtain a static solution; Was added to extract the static solution was extracted at 4 ℃ 24 hours and filtered, and added to 0.4g / mL and slowly added amount (NH _{4) 2} SO ₄ the filtrate to a, (NH _{4) 2} SO ₄ was added to the process of continue stirring at (NH _{4) 2} SO ₄ is then partially preventing supersaturation in, allowed to stand for 24 hours, remove 5 minutes centrifuged at 25000g at 4 ℃, and the precipitate and re-dissolved with the acid buffer, at 4 ℃ Dialyzed with a fluoride film (PVDF, polyvinylidene fluoride) dialysis bag (purchased from Beijing Borunlaite Science Tech Co., Ltd.) with a molecular weight cutoff of 3000 Da, centrifuged at 20000 g for 5 minutes, To prepare an expansin solution and store at 4 ° C.

The above-mentioned steps, which have not been described yet, are described in McQueen-Mason et al., McQueen-Mason SJ, Durachko DM, Cosgrove D J. Two endogenous proteins that induce cell wall extensions in plants. Plant Cell, 1992, 4: 1425-1433, and https://homes.bio.psu.edu/expansins/Protocols/Extraction.htm.

The constituents of the homogeneous buffer solution were 25 mmol / L HEPES (N-2-hydroxyethylpiperadine-N-2-ethanesulfonic acid), 3 mmol / L Na ₂ S ₂ O ₅ , 1 mmol / L EDTA, 0.1 wt% X-100, pH 7.0;

The components of the extract were 25 mmol / L HEPES, 1.0 mmol / L EDTA, 3 mmol / L Na ₂ S ₂ O ₅ , 0.5 mol / L NaCl, pH 6.8;

The preparation of the acidic buffer solution per liter is carried out by dissolving 2.05 g of sodium acetate in water and adjusting the pH to 4.0 with acetic acid to make the static capacity of water to 1 L.

Coomassie Brilliant Blue may be used as a method for measuring the exxanthine solution concentration, and more particularly, Coomassie Brilliant Blue may be used for measuring the concentration of expansin solution, and more particularly, Coomassie Brilliant Blue Brilliant Blue can be referred to, and bovine serum albumin is used as a standard curve, and the concentration of expansin in the exxan solution is 0.33 g / mL.

In the examples, the components per liter of the PD liquid fermentation medium are 200 g of potato, 20 g of glucose and a static capacity of 1000 mL of distilled water.

In the examples, the components per liter of the seed fermentation medium were 3 g glucose, 2.5 g yeast powder supernatant, 0.1 g MgSO ₄ , 0.03 g CaCl ₂ , 0.1 g KH ₂ PO _4, 0.1 g K ₂ HPO ₄ , Of glass spheres.

In the examples, the constituent components per liter of the liquid fermentation medium were 3 g glucose, 2 g molasses, 3 g soybean flour, 3 g peptone, 0.2 g MgSO ₄ , 0.03 g CaCl ₂ , 0.3 g KH ₂ PO ₄ , K ₂ HPO ₄ 0.3 g.

In the embodiment, the disodium hydrogenphosphate-citric acid buffer solution is prepared by dissolving 0.69 g of disodium hydrogenphosphate and 0.012 g of citric acid in distilled water and adjusting the static volume to 1 L at a pH of 8.0.

Example 1

The steps of the liquid fermentation process for improving the production of chondiesepinic acid in the pupa of Cordyceps mellifera using expansin are as follows.

(1) Cordyceps militaris strain having the strain serial number CGMCC No. 5,701 was inoculated into a PD liquid fermentation medium to activate the culture, followed by incubation at 25 ° C with a rotation speed of 150 r / min for 48 hours And a volume ratio of 15% was transferred to a seed fermentation medium to conduct seed culturing. The seed culture was shake cultured at 25 占 폚 for 48 hours to obtain a seed solution;

(2) seed liquid obtained in step (1) is inoculated into a liquid fermentation medium at a volume ratio of 15%, liquid fermentation culture is carried out at 25 ° C for 24 hours, and expansin solution is added to give a concentration of expansin of 1.0 mg / , Followed by continuous culturing for 120 hours and centrifugation at 12,000 r / min for 10 minutes to remove the fermentation supernatant to obtain a mycelia of Cordyceps;

(3) The steps of extracting the chordysepinic acid from the pupae of Cordyceps sinensis obtained in step (2) are as follows.

In step (2), the pupae mycelia of the pupa produced in 1 L of the fermentation broth were dried at 65 ° C for 1 to 3 hours, and 4.072 g of the pupa pupae were obtained. The pupae were polished with powder, 20 mL of an ethanol solution having a volumetric concentration of 50% was added thereto. The mixture was treated with microwave of 500 W for 1 minute, centrifuged at 12,000 r / min for 5 minutes to obtain a supernatant, and 20 mL of an ethanol solution , And the supernatant is combined to prepare chordysepinic acid.

Preparation of Atmospheric Samples for Measurement of Cordycepinic Acid

The chondysetinic acid prepared in the above step is taken, and the static volume is adjusted to 50 mL with an ethanol solution having a volume concentration of 50%.

Measurement of chordysephinic acid active ingredient

Comparison of the test and measurement of active ingredients of the pupae of Chinese pupae and the associated pupae of Chinese pupa, commonly used in the technical field of the present invention (refer to the general formula, Ichifan, Tang Wing, Food Science, 2004, 25 (8): 155-157).

(1) Standard curve creation

Standard curves are prepared based on mannitol. After drying and cooling at 105 ° C for 2 hours, the mannitol powder left at room temperature is taken and a standard solution of 5 mg / mL is added. The mannitol standard solution was added to a test tube in an amount of exactly 0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL and 0.9 mL, Of sodium iodate solution (3.2 g of sodium periodate dissolved in distilled water, 10.2 mL of concentrated hydrochloric acid was added to adjust the distilled water to a static volume of 1 L), and the mixture was homogeneously mixed. After being left at room temperature for 10 minutes, Add 2 mL of 0.1% by weight of rhamnose and 4 mL of Nash solution (150 g of ammonium acetate dissolved in distilled water, add 2 mL of glacial acetic acid and 2 mL of acetylacetone to adjust to a static capacity of 1 L), and shake to uniformly mix , And allowed to stand in a water bath at 53 캜 for 15 minutes at a constant temperature. The absorbance of the reaction solution is measured at 420 nm using a spectrophotometer, and a standard curve is prepared using the mannitol content (mg / mL) as the abscissa and the absorbance A420 as the ordinate.

(2) Measurement of production of active ingredient of chondispinac

Measurement of chordysepinic acid Take 500 μL of the atmospheric sample, place it in a test tube, adjust the static volume to 1.0 mL with distilled water, and shake to mix uniformly. The control group should be 1.0 mL of water. Thereafter, 1 mL of 15 mM sodium iodate solution (3.2 g of sodium iodate was dissolved in distilled water, 10.2 mL of concentrated hydrochloric acid was added, and distilled water was adjusted to a static capacity of 1 L), and the mixture was homogeneously mixed. After allowing to stand, add 2 mL of 0.1% rhamnose, add 4 mL of freshly mixed Nash solution (dissolve 150 g of ammonium acetate in distilled water, add 2 mL of glacial acetic acid and 2 mL of acetylacetone to adjust to 1 L of static solution) And allowed to stand in a water bath at 53 캜 for 15 minutes at a constant temperature. The absorbance of the reaction solution is measured at 420 nm using a spectrophotometer. According to the results calculated based on the standard curve, the content of the chondiespinic acid in the chondisipinic acid measurement sample is 237.23 μg.

The yield of mycelial chondiesinic acid production per liter of fermentation broth was 237.23 μg × 100 × 10 × 4.072 = 0.966 g, the results of which are shown in FIG.

Example 2

The liquid fermentation method of the first embodiment differs from the liquid fermentation method of the first embodiment in that the expansin solution is added at a temperature of 25 ° C in step (2) to adjust the concentration of expansin to 1.5 mg / ml, followed by continued culturing for 120 hours.

According to the results of the test, the content of chordysephinic acid contained in the mycelium (dry weight) per milliliter is 271.55 mg, that is, the mycelium produced in the fermentation solution per liter contains 2.621 g of the chrysalis Cordycepinic acid. The results are shown in Fig.

Example 3

The liquid fermentation method of the first embodiment differs from the liquid fermentation method of the first embodiment in that the expansin solution is added at a temperature of 25 ° C in step (2) to adjust the concentration of expansin to 2.0 mg / ml, followed by continued cultivation for 120 hours.

According to the results of the test, the content of chordysephinic acid contained in the mycelium (dry weight) per milliliter is 257.07 mg, that is, the mycelium produced in the fermentation solution per liter contains 2.097 g of the chrysalis cordatechodipine acid. The results are shown in Fig.

Example 4

The liquid fermentation method of the present invention is different from the liquid fermentation method of the first embodiment in that the expansin solution is added at a temperature of 25 ° C in step (2) to make the concentration of expenchin to 2.5 mg / ml and then continued culturing for 120 hours.

According to the results of the test, the content of chordysephinic acid contained in the mycelium (dry weight) per milliliter is 252.39 mg, that is, the mycelium produced in the fermentation solution per liter contains 1.949 g of the chrysalis cordatechodipine acid. The results are shown in Fig.

Comparative Example 1

The liquid fermentation method of Example 1 was different from that of Example 1 except that the exosphan solution added in the Example was replaced with an acid buffer solution not containing expansin in the step (2) Fermentation.

According to the results of the test, the content of chordysephinic acid contained in the mycelium (dry weight) per milliliter is 175.25 mg, that is, the mycelium produced in the fermentation solution per liter contains 0.356 g of chrysanthemum chordatephinic acid. The results are shown in Fig.

Claims (10)

Step (1): Pupae Cordyceps is inoculated into a PD liquid fermentation medium to activate the culture, then an inoculum amount of 10 to 15% by volume is transferred to the seed fermentation medium and the seed culture is carried out. The seed culture is carried out at 20 to 25 & Shaking for 72 hours to obtain a seed solution;
Step (2): The seed liquid obtained in the above step (1) is inoculated into a liquid fermentation medium at a volume ratio of 5 to 15%, followed by liquid fermentation culture at 20 to 25 캜 for 20 to 30 hours, The concentration of expansin is adjusted to 0.5 to 2.5 mg / mL, and the cultivation is continued for 96 to 144 hours to obtain a pupae mycelium of pupae; And
Step (3): extracting cordycepinic acid, i.e., chordysepinic acid, from Cordyceps sinensis cells in the pupae of the pupae obtained from the step (2);
In the step (2), the expansin solution is prepared by disinfecting the bean curd or cucumber seed with 0.05 to 0.15% by weight of HgCl ₂ for 4 to 6 minutes, washing it in flowing water for 5 to 7 hours, (Wet vermiculite) at a temperature of 25 to 28 DEG C for 4 to 6 days; The seedlings are cut to a size of 4 to 5 cm and preliminarily cooled at -20 ° C for 1 to 2 hours to homogenize by adding a homogenized buffer precooled to 0 to 4 ° C and then filtered with a nylon mesh having a diameter of 60 to 80 μm The filtrate residue is washed with a homogeneous buffer, the filtrate residue is added to the homogeneous buffer solution and left at room temperature for 1 to 3 hours to obtain a static solution; The extract was added to the static solution to 0 to 24 to 30 hours at 4 ℃ extracted and filtered, and slowly added (NH _{4) 2} SO ₄ the filtrate with the addition amount of 0.3 to _{0.5g / mL, (NH 4)} 2 Continue stirring in the course of adding SO ₄ and prevent (NH ₄ ) ₂ SO _{4 from} being partially supersaturated. Then, it is left for 24 to 30 hours and centrifuged at 25000 g for 5 to 10 minutes at 4 ° C., , Dialyzed at 4 ° C with a dialysis bag having a molecular weight of 3000 Da, centrifuged at 20000 g for 5 to 10 minutes, and the supernatant was taken to prepare the exsansan solution. ≪ / RTI > to produce chondisipinic acid. The method according to claim 1,
Wherein the cultivation activation conditions in step (1) are that the cultivation is carried out at a shaking rotation speed of 100 to 160 r / min, a temperature of 20 to 25 캜, and a cancer culture activation time of 24 to 72 hours. ≪ / RTI > The method according to claim 1,
3 g of glucose, 2.5 g of yeast powder, 0.1 g of MgSO ₄ , 0.03 g of CaCl ₂ , 0.1 g of KH ₂ PO _4, 0.1 g of K ₂ HPO ₄ , A method for producing chordysepinic acid by using liquid fermentation of pupa chrysanthemum phloem, characterized by having 2 to 5 glass spheres of 6 mm. The method according to claim 1,
The constituent components per liter of the liquid fermentation medium in step (2) were 3 g of lactose, 2 g of molasses, 2 g of soybean flour, 3 g of peptone, 0.2 g of MgSO ₄ , 0.03 g of CaCl ₂ , 0.3 g of KH ₂ PO _{4 and} 0.3 g of K ₂ HPO ₄ ≪ / RTI > wherein the process comprises the steps of: The method according to claim 1,
Wherein the concentration of expenchin in step (2) is 1.0 to 2.5 mg / mL. The method according to claim 1,
Wherein the concentration of expansin in step (2) is in the range of 1.8 to 2.0 mg / mL. ≪ RTI ID = 0.0 > 11. < / RTI > The method according to claim 6,
Wherein the concentration of expenchin in step (2) is 1.5 mg / mL. 8. The method of claim 7,
The constituents of the homogeneous buffer solution were 25 mmol / L HEPES (N-2-hydroxyethylpiperadine-N-2-ethanesulfonic acid), 3 mmol / L Na ₂ S ₂ O ₅ , 1 mmol / L EDTA Ethylenediaminetetraacetic acid), 0.1% by weight of Triton X-100, pH 7.0; The components of the extract were 25 mmol / L HEPES, 1.0 mmol / L EDTA, 3 mmol / L Na ₂ S ₂ O ₅ , 0.5 mol / L NaCl, pH 6.8; The preparation of the acidic buffer solution is carried out by dissolving 2.05 g of sodium acetate in water and adjusting the pH to 4.0 with acetic acid to make a static capacity of water to 1 L. The puddings of Cordyceps sinensis Way. The method according to claim 1,
Wherein the separation in step (2) is carried out by centrifuging at 5 DEG C for 12 minutes at 4 DEG C for 5 to 10 minutes. The method according to claim 1,
In the step (3), the step of extracting Cordyceps sinensis Cordyceps pseudo-chrysanthemum is performed by drying the pupae of Cordyceps sinensis obtained in step (2) at 65 ° C for 1 to 3 hours to grind powder, 20 mL of an ethanol solution having a volume concentration of 50% was added to 0.1 g of the mycelial powder, microwaved for 1 minute at an output of 500 W, centrifuged at 12000 r / min for 5 minutes to obtain a supernatant, Ethanol solution is added to the solution, followed by microwave treatment and centrifugation according to the above step, and then the supernatant is added to obtain the chordysepinic acid, thereby producing the chordysephinic acid.

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