AU2013248824A1 - Method for producing cordycepic acid by means of liquid fermentation of Cordyceps militaris - Google Patents

Description

-4 Method for cordycepic acid production of Cordyceps militaris by liquid fermentation Technical Field The present invention relates to the method of producing cordycepic acid by liquid fermentation of Cordyceps militaris, which belongs to biotechnology fermentation engineering field. Background Cordyceps militaris (Cordyceps militaris) is a kind of entomogenous fungus which can be used in drugs. It belongs to Ascomycota subphylum (Ascomycota), Hypocreales order (Hypocreales), Clavicipitaceae family (Clavicipitaceae), Cordyceps genus (Cordyceps), pattern stain, and is also named North Cordyceps militaris, North Cordyceps sinensis, etc. As a kind of rare Chinese herb, it compares favourably with Cordyceps sinensis (Cordyceps sinensis) due to its officinal components and various effects. Cordycepic acid is the primary physiological active substance with the effects of anti-free radical and anti-oxidation. Furthermore, the content of cordycepic acid is one of primacy standard to scale the quality of Cordyceps militaris. It is considered commonly that Cordyceps militaris with more cordycepic acid content will have higher drug value. The experiments have shown that the Cordycepic acid content of Cordyceps sinensis (Cordyceps sinensis) is 7%, while that of Cordyceps militaris is much higher which brings to broad attention for its value. Pharmacology researches show that cordycepic acid has effects on such as promoting body metabolism, improving body micro-circulation system, evidently reducing blood fat, restraining bacteria, and boosting up the resistibility to disease. It has also antitussive, expectorant and antiasthmatic effects. Basing on the immunity promotion effect of Cordyceps militaris, the hepatic detoxification effect is improved. Furthermore, it has effects of anti-liver tissue fibrosis and anti-lipid peroxidation, and can also protect efficiently the hepatic cells. Many diseases such as cerebral thrombus, cerebral hemorrhage, myocardial infarction and long-term exhaution, can be prevented and treated. Due to the rapid growth of Cordyceps militaris cordycepic acid demand, crazy picking of the wild Cordyceps makes the resources increasingly scarce and expensive. The chemosynthesis technics of active ingredients of Cordyceps militaris is too complex, and the yield is poor. So the resource of material constrains its application scope severely. Studies have shown that cordycepic acid and other active ingredients of Cordyceps militaris obtained by liquid fermentation have basically the same biochemical structure and physiological function as those extracted directly from cultured Cordyceps fruiting body or cultured Cordyceps militaris (Fr.) Link mycelium. And the method using liquid fermentation has also many advantages such as short training period, easily controled production process, easily extracted active substance, etc. Extract content and efficiency of Cordyceps militaris active substances, like cordycepic acid, is much higher than that of cultivated Cordyceps militaris. However, at present Cordyceps militaris and its active substances like cordycepic acid, are commonly produced by traditional solid culture methods. The research and application of liquid culture develop in the short time, so the technology and process of fermentation are still immaturity. The overall yield and fermentation level are low. These limit the development of its industrial production. Expansin is a new type of protein species found in plant cell walls. Studies have shown that expansin is involved in almost the entire development process of plants. In addition to the function Page 4 of 11 -5 of aggrandizing cell, expansin plays an important role in cell growth, seed germination, root hair formation, root growth, leaf primordial formation, leaf growth, fruit ripening, organ abscission and pollen tube growth. Expansin has been found in Arabidopsis, tomato, strawberry, cotton, rice, corn and other plants, and is considered existing in various dicotyledonous and monocotyledonous plant cell walls. Experiments have shown that expansin is a category of cell wall enzyme protein, which can induce acid-dependent cell wall extension and stress relaxation by breaking the hydrogen bonds between the cell wall polymers, and also may be an important regulatory factor for physiological regulation and cell wall extension process in the period of plant growth. However, the mechanism of expansin is not yet a clear conclusion. Currently, it is not reported at home and abroad that the expansin is applied in liquid fermentation of edible and medicinal mushroom such as Cordyceps militaris, for producing secondary metabolites such as cordycepic acid. Summary of The Invention In view of the defects of the prior art, the present invention aim to provide a method using expansin to produce cordycepic acid of Cordyceps militaris by liquid fermentation. The technical scheme of the present invention is as follows. The method for producing cordycepic acid of Cordyceps militaris by liquid fermentation, comprises the following steps of: (1) inoculating Cordyceps militaris strains into the PD liquid fermentation medium to perform activation culture, and transferring to a seed fermentation medium to a volume ratio of 10-15% to perform seed culture in the swing bed at 20-25"C for 24-72 hours so as to obtain a seed liquid; (2) inoculating the seed liquid obtained in step (1) into a liquid fermentation medium to a volume ratio of 5-15% to perform liquid fermentation culture at 20-25 C for 20-30 hours, then adding an expansin protein solution to a concentration of 0.5-2.5mg/mL to perform further culture for 96-144 hours, and separating to obtain the mycelium of Cordyceps militaris; and (3) extracting intracellular cordycepic acid of Cordyceps militaris from the mycelium of Cordyceps militaris obtained in step (2) so as to obtain cordycepic acid; Preferably, according to the invention, per litre of the PD liquid fermentation medium in the step (1) comprises the following components: 200g potato, 20g glucose, dilute to 1000 mL by distilled water. According to the invention, the preferred activation culture in the step (1) is processed in the darkroom with a shaking speed of 100 - 160 r/min at 20 - 25 0 C for 24 - 72 h. Preferably, according to the invention, per litre of the seed fermentation medium in the step (1) comprises the following components: 3g glucose, 2.5g yeast extract supernatant, 0.1g MgSO 4 , 0.03g CaCl 2 , 0.lg KH 2

PO

4 , 0.1g

K

2

HPO

4 , 2-5 pieces of glass beads with 3-6 mm diameter. After cultured in the seed fermentation medium, the amount of mycelium pellet may increase more than 60%, and the diameter of mycelium pellet may reduce more than 40%. The mycelium is loose and uniform. Preferably, according to the invention, per litre of the liquid fermentation medium in the step (2) comprises the following components: 3g lactose, 2g molasses, 2g soybean powder, 3g peptone, 0.2g MgSO 4 , 0.03g CaCl 2 , 0.3g

KH

2

PO

4 , 0.3g K 2

HPO

4 . Page 5 of 11 -6 Preferably, according to the invention, the expansin concentration in the step (2) is 1.0-2.5 mg/mL; further preferably, 1.8-2.0 mg/mL; most preferably, the expansin concentration in the step (2) is 1.5 mg/mL. The preparation of the expansin protein solution in the step (2) may refer to the prior technique, such as the method described in Two endogenous proteins that induce cell wall extension in plants. McQueen-Mason et al. Plant Cell, 1992, 4: 1425-1433. McQueen-Mason S J, Durachko D M, Cosgrove D J. The expansin protein solution in step (2) may also be prepared by the method which comprises the following steps of: sterilizing a broad bean or cucumber seed for 4-6 minutes with 0.05-0.15wt.% mercury chloride (HgCl 2 ), washing with running water for 5-7 hours, planting in wet vermiculite, culturing in the darkroom for 4-6 days at 15-28 C, picking up 4-5cm of seedling epicotyl or root system, precooling for 1-2 hours at -20 C, adding a homogenate buffer solution that is pre-cooled to 0-4'C, filtering with a nylon net with an aperture of 60-80 m after homogenate, washing the filter residue with a homogenate buffer solution, adding the filter residue in the homogenate buffer solution, settling for 1-3 hours to obtain a settled solution, adding an extracting solution in the settled solution, extracting for 24-30 hours at 0-4 C, slowly adding 0.3-0.5g/mL ammonium sulfate ((NH4) 2

SO

4 ) in the filtrate, meanwhile stirring to prevent a partial supersaturation of the (NH4) 2

SO

4 , settling for 24-30 hours, centrifuging for 5-10 minutes at 4 C, dissolving the sediment with an acid buffer solution, dialyzing in a dialysis bag with a molecular weight of 3000 Da at 4"C, centrifuging the dialyzate at 20000g for 5-10 minutes, and collecting the supernatant so as to obtain the expansin protein solution. In the above preparation method of the expansin protein solution, the homogenate buffer solution comprises 25 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3 mmol/L sodium pyrosulfite (Na 2

S

2 0 5 ), 1 mmol/L EDTA (ethylene diamine tetraacetic acid) and 0.1 wt% Triton X-100 at pH 7.0. The extracting solution comprises 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1.0 mmol/L EDTA, 3 mmol/L Na 2

S

2 0 5 and 0.5 mmol/L sodium chloride (NaCl) at pH 6.8. The acid buffer solution is prepared by dissolving 2.05g sodium acetate in water, adjusting pH to 4.0 with glacial acetic acid, and adding water to IL. Preferably, according to the invention, the separation in the step (2) is carried out by centrifuging at 12000 r/min, 4 0 C for 5-10 minutes. Preferably, according to the invention, the extraction method of Cordyceps militaris cordycepic acid in the step (3) comprises the following steps of: drying Cordyceps militaris mycelium obtained in the step (2) at 65 0 C for 1-3 hours and grinding to power, adding 0. 1g Cordyceps militaris mycelium powder into 20mL ethanol solution of 50% volume concentration and microwaving for 1 minute with 500W microwave power, then centrifuging at 12000 r/min for 5 minutes and separating supernatant, adding 20mL ethanol solution of 50% volume concentration to the deposit, microwaving and centrifuging as the above steps, incorporating the supernatant so as to obtain cordycepic acid. Compared with the prior art, the present invention has the following advantages: 1. The expansin is applied in the method of the invention for producing cordecepic acid of Cordyceps militaris by liquid fermentation, therefore the yield of cordycepic acid may achieve 2.621g/L, which is greater than or equal to 7.3 times of the control cordycepic acid fermentation yield (Comparative Example 1). And the cordecepic acid yield of the whole Cordyceps militaris can be increased significantly. So the method in the invention has excellent prospects for industrial Page 6 of 11 -7 application. 2. The method of the invention adopts liquid aerobic fermentation method, which has generally a fermentation time of 5 to 7 days. Compared to the prior art, it has advantages of high-yield, short-cycle, high production efficiency, and dispensing with static culture and inducing synthetic products processes. The generative Cordyceps militaris cordycepic acid can be directly used in the preparation of drugs for regulating immunity, reducing fat, protecting liver and reducing blood sugar, etc. 3. The expansin mentioned in the invention can be extracted from most dicotyledonous and monocotyledonous plants, which is widely-available and low-cost. The preferred method of the invention is relatively simple and high yield, so it can be applied for scale extraction production and also has a good effect on promoting and upgrading the active substance production of Cordyceps militaris such as cordycepic acid. 4. The liquid fermentation method of Cordyceps militaris in the invention is simple, environment-friendly, non-toxic and low raw material costs. The whole fermentation process is easily controlled and not restricted by external environmental conditions, so it is suitable for production in industrial-scale and popularization. 5. The method in the invention optimizes the formulation of the seed fermentation medium and liquid fermentation medium, and increases significantly the yeild of cordycepic acid of Cordyceps militaris. Figure Description Figure 1 is the curve about the effect of the different concentrations of expansin protein solution on the output of cordycepic acid of Cordyceps militaris. Embodiment The following is the detail description of the present invention with reference to examples, but the scope of the present invention is not limited thereof. Raw Materials and Medium Cordyceps chrysalides (Cordyceps militaris) fermentation strains described in Examples, with Culture Collection Number of CGMCC No.5.701 and CGMCC No. 3.4655, were purchased from China General Microbiological Culture Collection Center. Mannitol, rhamnose, ammonium acetate and acetylacetone were purchased from Jinan Shengwei Biotechnology Co., Ltd. The expansin protein solution of examples can be prepared by the method which comprises the following steps of: sterilizing the broad bean (Vicia faba L.; purchased from Jinan Mao Feng Seed Co.,Ltd) or cucumber seed (Cucumis sativus L. CV Jinnian No 6; purchased from Jinan Weili Seed Industry Co., Ltd.) with 0.15 wt% HgCl 2 for 6 minutes, washing with running water for 6 h, planting in wet vermiculite, dark culturing for 4 days at 27'Q picking up 4-5 cm of seedling epicotyl which is about 100g of growing area, setting at -20'C for 1 hour to pre-cool, adding homogenate buffer solution that pre-cooled to 4C filtering with a nylon net with an aperture of 70 jim after high speed dividing, washing the filter residue with homogenate buffer solution, adding the filter residue in the homogenate buffer solution, settling for 2 hours to obtain a settled solution, adding an extracting solution in the settled solution, extracting for 24 hours at 4 C, slowly adding 0.4 Page 7 of 11 -8 g/mL ammonium sulfate ((NH 4 )2sO 4 ) in the filtrate, meanwhile stirring to prevent a partial supersaturation of the (NH 4

)

2

SO

4 , settling for 24 hours, centrifuging at 25000g, 4 C for 5 minutes, dissolving the sediment with an acid buffer solution, dialyzing in a polyvinylidene fluoride (PVDF) dialysis bag (purchased from Beijing Lubrizol Wright Science and Technology Co., Ltd.) with a molecular weight of 3000 Da at 4"C, centrifuging the dialyzate at 20000g for 5-10 minutes, collecting the supernatant so as to obtain the expansin protein solution and reserving at 4 C. Other steps that are not described can be referred to the descriptions in "Two endogenous proteins that induce cell wall extension in plants. McQueen-Mason et al. Plant Cell, 1992, 4: 1425-1433. McQueen-Mason S J, Durachko D M, Cosgrove D J." and "https://homes.bio.psu.edu/expansins/Protocols/ Extraction.htm". The above extracting solution comprises 25mmol/L HEPES, 1.0mmol/L EDTA, 3mmol/L Na 2

S

2 0 5 and 0.5mol/L NaCl at pH 6.8. Per litre of the acidic buffer can be prepared by dissolving 2.05g sodium acetate in water, adjusting pH to 4.0 with glacial acetic acid, dilute to 1 L with water. The concentration determination of the expansin protein solution can employ Coomassie brilliant blue method, which may specifically refer to the operations of Coomassie blue method described in "Quintessence of Protein Science Laboratory Manual" (ISBN: 703018086, publication date: 1900-1-1), by using bovine serum albumin as the standard curve. The expansin concentration of the expansin protein solution detected by the above method and the result is 0.33g/mL. Per litre of the PD liquid fermentation medium described in examples comprises the following components: 200g potato, 20g glucose, dilute to 1000 ml with distilled water. Per litre of the seed fermentation medium described in examples comprises the following components: 3g glucose, 2.5g yeast extract supernatant, 0.1g MgSO 4 , 0.03g CaCl 2 , 0.1g KH 2

PO

4 , 0.1g K 2

HPO

4 , 2-5 pieces of glass beads with 3-6 mm diameter. Per litre of the liquid fermentation medium described in Examples comprise the following components: 3g lactose, 2g molasses, 2g soybean powder, 3g peptone, 0.2g MgSO 4 , 0.03g CaCl 2 , 0.3g KH 2

PO

4 , 0.3g K 2

HPO

4 . Example 1: Liquid fermentation method using expansin protein to increase the yield of cordycepic acid of Cordyceps militaris, comprises the following steps of: (1) inoculating Cordyceps chrysalis (Cordyceps militaris) strains which has a strain number of CGMCC No.5.701 into the PD liquid fermentation medium to perform activation culture in the darkroom with a shaking speed of 150 r/min at 25 'C for 48 hours, and transferring to a seed fermentation medium to a volume ratio of 15% to perform seed culture in the swing bed at 25C for 48 hours so as to obtain a seed liquid; (2) inoculating the seed liquid obtained in step (1) into a liquid fermentation medium to a volume ratio of 15% to perform liquid fermentation culture at 20-25 C for 20-30 hours, then adding an expansin protein solution to a concentration of 0.5-2.5mg/mL to perform further culture for 120 hours, centrifuging at 12000r/min for 10 minutes, separating and removing the fermentation supernatant to obtain the mycelium of Cordyceps militaris; and (3) extracting cordycepic acid from the mycelium of Cordyceps militaris obtained in step (2) by the following steps of: drying the mycelium of Cordyceps militaris obtained in the step (2) at 65'C for 1-3 hours to Page 8 of 11 -9 obtain 4.072g mycelium of Cordyceps militaris, grinding to power, adding 0. 1g mycelium powder of Cordyceps militaris into 20mL ethanol solution of 50% volume concentration and microwaving for 1 minute with 500W microwave power, then centrifuging at 12000 r/min for 5 minutes and separating supernatant, adding 20mL ethanol solution of 50% volume concentration to the deposit, microwaving and centrifuging as the above steps, incorporating the supernatant so as to obtain cordycepic acid. Preparation of Cordycepic acid Samples Dilute cordycepic acid obtained by the above steps with ethanol solution of 50% volume concentration to 50 mL. Determination of the Cordycepic acid The Cordycepic acid can be determined by the conventional sodium periodate chromatometry method in the art, which can be referred to "Compare of the detection methods of Cordycepic acid and related active components [J]. Lu Wen, Qifan Yin, Yuling Tang, et al. Food Science, 2004, 25 (8): 155- 157". (1) Establishment of Standard Curve: The standard curve basing on mannitol can be built by the method which comprises the following steps of: weighing up the mannitol powder which has been dried at 105 C for 2 hours and cooled to room temperature, then dissolving to 5 mg/mL standard solution, accurately sucking up OmL, 0.lmL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL standard solution of mannitol into tubes, separately adding distilled water to 1.OmL, then adding 1ImL 15mM sodium periodate (prepared by dissolving 3.2g sodium periodate in distilled water, adding 10.2mL concentrated hydrochloric acid, diluting to 1L with distilled water) and mixing to homogeneous, placing at room temperature for 10 minutes, adding 2 mL 0.1 wt% rhamnose, 4mL newly prepared Nash solution (prepared by weighing up 150g ammonium acetate, dissolving with distilled water, adding 2mL acetic acid and acetylacetone, and diluting to IL), shaking to homogeneous and placing at 53 C water bath to keep temperature for 15 minutes, mensurating absorbency value of reaction solution by spectrophotometer at 420nm, and setting the mannitol concentration (mg/mL) as X-coordinate and the absorbency value A 420 as Y-coordinate so as to obtain the standard curve. (2) Determination of the yield of cordycepic acid: The yield of cordycepic acid is determined by the method which comprises the following steps of: picking up 500 pL cordycepic acid sample into tubes, diluting to IL with distilled water and shaking to homogeneous, using 1.OmL water as the control group, then adding ImL 15mM sodium periodate (prepared by dissolving 3.2g sodium periodate in distilled water, adding 10.2mL concentrated hydrochloric acid, diluting to 1L with distilled water) and mixing to homogeneous, placing at room temperature for 10 minutes, and adding 2 mL 0.1% rhamnose, 4mL newly prepared Nash solution (prepared by weighing up 150g ammonium acetate, dissolving with distilled water, adding 2mL acetic acid and acetylacetone, and diluting to IL), shaking to homogeneous and placing at 53 C water bath to keep temperature for 15 minutes, mensurating absorbency value of reaction solution by spectrophotometer at 420nm. According to the standard curve, the content of cordycepic acid of the sample calculated is 237.23 gg. The yield of cordycepic acid per litre fermentation solution is: 237.23ggx 100x 1Ox 4.072 = 0.966g. The result is shown in Figure 1. Example 2: It is the same as the liquid fermentation method described in Example 1, except that in the Page 9 of 11 -10 step (2) the expansin protein solution is added to a concentration of 1.5mg/mL at 25"C, and performs further culture for 120 hours. After been detected and calculated, per gram (dry weight) of mycelium has 271.55 mg of cordycepic acid, i.e. the mycelium produced by per litre fermentation solutionhas 2.621g of cordycepic acid of Cordyceps militaris. The result is shown in Figure 1. Example 3: It is the same as the liquid fermentation method described in Example 1, except that in the step (2) the expansin protein solution is added to a concentration of 2.0mg/mL at 25"C, and performs further culture for 120 hours. After been detected and calculated, per gram (dry weight) of mycelium has 257.07 mg of cordycepic acid, i.e. the mycelium produced by per litre fermentation solutionhas 2.097g of cordycepic acid of Cordyceps militaris. The result is shown in Figure 1. Example 4: It is the same as the liquid fermentation method described in Example 1, except that in the step (2) the expansin protein solution is added to a concentration of 2.5mg/mL at 25"C, and performs further culture for 120 hours. After been detected and calculated, per gram (dry weight) of mycelium has 252.39 mg of cordycepic acid, i.e. the mycelium produced by per litre fermentation solutionhas 1.949g of cordycepic acid of Cordyceps militaris. The result is shown in Figure 1. Comparative Example 1: It is the same as the liquid fermentation method described in Example 1, except that in the step (2) the expansin-free acidic buffer is instead of the expansin protein solution added in the examples, then performs further culture at 25 C for 144 hours. After been detected and calculated, per gram (dry weight) of mycelium has 175.25 mg of cordycepic acid, i.e. the mycelium produced by per litre fermentation solutionhas 0.356g of cordycepic acid of Cordyceps militaris. The result is shown in Figure 1. Page 10 of 11

Claims (2)

1. Method for producing cordycepic acid of Cordyceps militaris by liquid fermentation, comprises the following steps of: (1) inoculating Cordyceps militaris strains into the PD liquid fermentation medium to perform activation culture, and transferring to a seed fermentation medium to a volume ratio of

10-15% to perform seed culture in the swing bed at 20-25"C for 24-72 hours so as to obtain a seed liquid; (2) inoculating the seed liquid obtained in step (1) into a liquid fermentation medium to a volume ratio of 5-15% to perform liquid fermentation culture at 20-25 C for 20-30 hours, then adding an expansin protein solution to a concentration of 0.5-2.5mg/mL to perform further culture for 96-144 hours, and separating to obtain the mycelium of Cordyceps militaris; and (3) extracting intracellular cordycepic acid of Cordyceps militaris from the mycelium of Cordyceps militaris obtained in step (2) so as to obtain cordycepic acid; the expansin protein solution in step (2) is prepared by the method which comprises the following steps of: sterilizing a broad bean or cucumber seed for 4-6 minutes with 0.05-0.15wt.% mercury chloride (HgCl 2 ), washing with running water for 5-7 hours, planting in wet vermiculite, culturing in the darkroom for 4-6 days at 15-28 C, picking up 4-5cm of seedling epicotyl or root system, precooling for 1-2 hours at -20 C, adding a homogenate buffer solution that is pre-cooled to 0-4C, filtering with a nylon net with an aperture of 60~80m after homogenate, washing the filter residue with a homogenate buffer solution, adding the filter residue in the homogenate buffer solution, settling for 1-3 hours to obtain a settled solution, adding an extracting solution in the settled solution, extracting for 24-30 hours at 0-4C, slowly adding 0.3-0.5g/mL ammonium sulfate ((NH4) 2 SO 4 ) in the filtrate, meanwhile stirring to prevent a partial supersaturation of the (NH4) 2 SO 4 , settling for 24-30 hours, centrifuging for 5-10 minutes at 4 C, dissolving the sediment with an acid buffer solution, dialyzing in a dialysis bag with a molecular weight of 3000 Da at 4"C, centrifuging the dialyzate at 20000g for 5-10 minutes, and collecting the supernatant so as to obtain the expansin protein solution. 2. The method according to claim 1, wherein activation culture in the step (1) is processed in the darkroom with a shaking speed of 100-160 r/min at 20-25 C for 24-72 h. 3. The method according to claim 1, wherein per liter of the seed fermentation medium in the step (1) comprises the following components: 3g glucose, 2.5g yeast extract supernatant, 0.lg MgSO 4 , 0.03g CaCl 2 , 0.lg KH 2 PO 4 , 0.lg K 2 HPO 4 , 2-5 pieces of glass beads with 3-6 mm diameter. 4. The method according to claim 1, wherein per liter of the liquid fermentation medium in the step (2) comprises the following components: 3g lactose, 2g molasses, 2g soybean powder, 3g peptone, 0.2g MgSO 4 , 0.03g CaCl 2 , 0.3g KH 2 PO 4 , 0.3g K 2 HPO 4 . 5. The method according to claim 1, wherein the expansin concentration in the step (2) is Page 2 of 11 -3 1.0-2.5 mg/mL. 6. The method according to claim 1, wherein the expansin concentration in the step (2) is 1.8-2.0 mg/mL. 7. The method according to claim 6, wherein the expansin concentration in the step (2) is 1.5 mg/mL. 8. The method according to claim 7, wherein the homogenate buffer solution comprises 25 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3 mmol/L Na 2 S 2 0 5 , 1 mmol/L ethylene diamine tetraacetic acid and 0.1 wt.% Triton X-100 at pH 7.0. The extracting solution comprises 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 3 mmol/L Na 2 S 2 0 5 , 1 mmol/L EDTA and 0.5 mmol/L NaCl at pH 6.8. The acid buffer solution is prepared by dissolving 2.05g of sodium acetate in water, adjusting pH to 4.0 with glacial acetic acid, and adding water to IL. 9. The method according to claim 1, wherein the separation in the step (2) is carried out by centrifuging at 12000 r/min, 4 'C for 10 - 15 minutes. 10. The method according to claim 1, wherein the method of extracting Cordyceps militaris cordycepic acid in the step (3) comprises the following steps of: drying Cordyceps militaris mycelium obtained in the step (2) at 65'C for 1-3 hours and grinding to power, adding 0. 1g Cordyceps militaris mycelium powder into 20mL ethanol solution of 50% volume concentration and microwaving for 1 minute with 500W microwave power, then centrifuging at 12000 r/min for 5 minutes and separating supernatant, adding 20mL ethanol solution of 50% volume concentration to the deposit, microwaving and centrifuging as the above steps, incorporating the supernatant so as to obtain cordycepic acid. Page 3 of 11