JP5850497B2 - Method for producing and purifying cordycepin - Google Patents

Description

  The present invention relates to a method for easily and efficiently producing and purifying a highly pure physiologically active substance cordycepin by culturing Cordyceps.

  Cordyceps is a fungus classified as an ascomycete fungus and is a general term for mushrooms that parasitize insects. The parasitic forms are as follows: (1) The scattered spores attach to the host insect and enter the body. (2) Spores that enter the body grow while absorbing my nutrients and extending mycelia, forming mycelium that is a mass of mycelia. (3) After maturity, the body of an insect is broken through to produce mushrooms (fruit bodies).

  In China, Cordyceps sinensis parasitizing larvae of the bat (Hepialus armoricanus) has been used as a herbal medicine since the 1700s. In recent years, Cordyceps sinensis has also been shown to have various pharmacological activity functions such as antibacterial, antitumor, immune enhancement, hypoglycemic, and vasodilatory effects. Cordycepin, ergosterol Oxides, myriocin, various polysaccharides and glycoproteins have been reported. Among these active ingredients, cordycepin, which is an analog of nucleoside (adenosine), has attracted particular attention as a substance exhibiting antibacterial and antitumor actions. As described above, cordyceps are expected to be used for pharmaceuticals and health foods, but they are extremely small and the number of inhabitants is difficult to collect, and by collecting and harvesting naturally grown cordyceps, It is not possible to meet the growing demand.

  Therefore, in recent years, several proposals have been made as a method for artificially cultivating mycelium and fruit bodies of Cordyceps sinensis and a method for extracting active ingredients therefrom. For example, as an artificial culture method for cordyceps mycelium and fruiting bodies, a carbon source, amino acids, minerals, or vitamins are added to the extract of the components of cocoon buds extracted under high temperature heating with water. A method of cultivating mycelia and fruiting bodies of Cordyceps sinensis in a medium to which at least one of the species is added (Patent Document 1). Examples thereof include a method of adding an extract of an insect of the same species as the insect (Patent Document 2). Patent Documents 3 to 5 can be cited as methods for extracting active ingredients from mycelia and fruiting bodies of cordyceps cultivated artificially. That is, Patent Document 3 discloses that a spore suspension of a Cordyceps medicinal strain such as Irisa japonica is inoculated into aseptically raised pupae or pupae to form fruiting bodies. A method for extracting active ingredients using ethanol or water is described. Patent Document 4 includes cereals such as brown rice and insect tissues (using the same insect as the parasitic insects and the same family of insects) as main components, and at least one of amino acids, minerals, and vitamins. After inoculating Cordyceps fungus on a solid medium supplemented with the above-mentioned additives and growing mycelium in 18-26 ° C and humidity of 80% or more in total darkness, irradiance is 100 Lux or more, and irradiation is performed for 6 hours or more per day. A method of culturing and pulverizing the cultured product as it is or extracting an active ingredient as necessary is described. Furthermore, Patent Document 5 discloses that Cordyceps mycelium is cultured by continuously or intermittently irradiating light having a peak in a wavelength range of 350 to 550 nm, and an active ingredient is obtained from the obtained mycelium with an organic solvent or water. Is described.

  In addition, in order to increase the productivity of cordycepin, which is one of the most important active ingredients, suitable culture media and culture conditions have been studied (Patent Documents 6 and 7, Non-Patent Documents 1 to 3, etc.). That is, Patent Document 6 includes 1 to 6% by weight of at least one selected from glucose, sucrose, molasses and maltose, 1 to 7% by weight of at least one selected from peptone, yeast, malt and milk, Inclusion of cordycepin using a cordyceps medium containing 0.1 to 0.5% by weight of at least one selected from asparagine, aspartic acid, glycine and DNA, or a trace amount of biotin and / or vitamin B A method for improving the rate is described. Patent Document 7 discloses a first step of inoculating Cordycepsin-producing Cordyceps fungus that produces cordycepin in a Cordyceps medicinal medium, and culturing at 5 to 28 ° C. for 20 to 90 days in the dark, and after the cultivation, light having an illuminance of 100 to 150,000 Lux. Under irradiation, a method is described in which the temperature is raised from the first step within a range of 35 ° C. or lower to increase the cordycepin content. Further, in Non-Patent Document 1, in deep culture using an aeration stirrer, the dissolved oxygen concentration (DO) is controlled to 60% of saturation from the start of the culture, and is reduced to 30% when the specific production rate of cordycepin decreases. Non-patent document 2 describes 42 g / L glucose as a carbon source and 15.8 g / L peptone as a nitrogen source. A method for obtaining high cordycepin productivity by culturing in a shake flask at 25 ° C., 110 rpm for 20 days using a medium containing the solution is described. Furthermore, Non-Patent Document 3 describes a method of efficiently obtaining cordycepin by using 45 g / L yeast extract as a nitrogen source and performing stationary culture for 8 days after shaking culture for 8 days. Has been.

  However, conventional artificial culture methods for Cordyceps sinensis use silkworm powder, cocoon extract, and / or insect extract as the medium, inoculate the body of insects, and finely control the temperature, pH, and light irradiation. It is difficult to perform mass production in consideration of practical use due to complicated operations such as control. Further, when trying to obtain cordycepin, the yield was 0.195 g / L in the medium by the method described in Non-Patent Document 1, 0.345 g / L by the method described in Non-Patent Document 2, and Non-Patent Document 2.2 g / L by the method described in No. 3, 0.02 to 0.1 g per 1 g of cells in the method described in Patent Literature 6, and 0.05 to 0.00 per g of cells in the method described in Patent Literature 7. 1 g is not enough.

  Moreover, there are few examination examples about the purification method of the produced cordycepin, and an efficient method is not known. For example, in Patent Document 8, Cordyceps sinensis is first powdered and extracted with ethanol, and the extract is concentrated to dryness. Next, the dried product is dissolved in distilled water, and then extracted with butanol to concentrate and dry the extract. After that, the dried product was further fractionated by silica gel chromatography and high performance liquid chromatography (HPLC) to obtain highly pure cordycepin by a very complicated method.

Japanese Patent Laid-Open No. 10-42691 Japanese Patent Laid-Open No. 10-117770 JP 2002-272267 A JP 2003-116522 A JP 2004-344027 A JP 2004-242506 A JP 2004-267004 A Special table 2010-503729

Biotechnol. Prog. 20, 1408-1413 (2004) Process Biochemistry 40, 1667-1672 (2005) Biochem. Eng. J. 33, 193-201 (2007)

  This invention is made | formed in view of the above-mentioned situation, The objective is to provide the method of manufacturing highly pure cordycepin (3'-deoxyadenosine) simply and efficiently. Another object of the present invention is to provide a method for separating and purifying cordycepin simply and at a high recovery rate.

  As a result of intensive studies on the culture conditions of Cordyceps sinensis in order to solve the above-mentioned problems, the present inventors have found that cordycepin can be produced at a high concentration in the culture solution by using a culture solution sterilized by filtration. In addition, the present inventors have found that cordycepin can be separated and purified with high purity and high recovery rate by separating the culture supernatant from the obtained culture broth and crystallizing, thereby completing the present invention.

That is, the present invention is as follows:
[1] A method for producing cordycepin, comprising a step of cultivating cordyceps using a culture solution sterilized by filtration and producing cordycepin at a high concentration in the culture solution.
[2] Cordyceps is a mutant of Cordyceps sinensis obtained by irradiating Cordyceps mycelium with a high energy ion beam and selecting a strain having a different mycological property from the parental mycelium. The production method according to [1] above.
[3] The production method according to [2], wherein the mutant strain of Cordyceps is a mutant strain of Cordyceps militaryis NBRC 9787 (Accession Number: FERM P-21315).
[4] The production method according to any one of [1] to [3] above, wherein the culture of Cordyceps is performed by liquid surface culture.
[5] Any of the above [1] to [4], further comprising a step of separating the culture supernatant from the culture solution obtained by cultivating Cordyceps sinensis, and separating and purifying cordycepin from the culture supernatant by crystallization. The manufacturing method as described in.
[6] The production method according to [5] above, wherein the step of separating and purifying cordycepin includes a step of pulverizing the culture supernatant and re-dissolving it in a solvent.
[7] The production method according to [6], wherein the powderization is performed by freeze-drying.
[8] The production method according to the above [6] or [7], wherein the solvent is water.
[9] After freeze-drying the culture supernatant separated from the culture solution obtained by cultivating Cordyceps sinensis using the culture solution sterilized by filtration, an aqueous solution obtained by re-dissolving in water at 30 to 90 ° C. A method for separating and purifying cordycepin, which cools to 10 ° C. and recovers the precipitated cordycepin.
[10] After freeze-drying the culture supernatant separated from the culture solution obtained by cultivating Cordyceps sinensis using the culture solution sterilized by filtration, the solution is converted into an acidic aqueous solution having a pH of less than 4 or an alkaline solution having a pH of more than 12.5. A method for separating and purifying cordycepin, which is re-dissolved and then the pH of the aqueous solution is adjusted to 4 to 12.5 to recover the precipitated cordycepin.
[11] A culture supernatant separated from a culture solution obtained by culturing Cordyceps sinensis using a culture solution sterilized by filtration is freeze-dried, and then an acidic aqueous solution having a pH of less than 4 or an alkaline aqueous solution having a pH of more than 12.5 is obtained. A method for separating and purifying cordycepin, which is redissolved at 30 to 90 ° C., then adjusted to pH 4 to 12.5, cooled to 0 to 10 ° C., and the precipitated cordycepin is recovered.
[12] Cordyceps is a mutant of Cordyceps obtained by irradiating Cordyceps mycelium with a high energy ion beam and selecting a strain having a different mycological property from the parent strain from the irradiated mycelium. The separation and purification method according to any one of [9] to [11] above.
[13] The separation and purification method according to the above [12], wherein the mutant strain of Cordyceps sinensis is a mutant strain of Cordyceps militaryis NBRC 9787 (accession number: FERM P-21315).
[14] The separation and purification method according to any of [9] to [13] above, wherein the culture of Cordyceps is performed by liquid surface culture.

According to the present invention, high-purity cordycepin can be easily and efficiently produced by culturing Cordyceps.
Further, cordycepin can be separated and purified with high purity and high recovery rate from the culture supernatant separated from the culture solution of Cordyceps sinensis.

It is the graph which compared the time-dependent change of the density | concentration of the cordycepin produced in a culture medium by the manufacturing method of Example 1-3 and Comparative Example 1-3. The open circle shows the results for Example 1-3, and the closed circle shows the results for Comparative Example 1-3. It is the HPLC chromatogram of cordycepin manufactured by the manufacturing method of Example 1-3. 3 is a HPLC chromatogram of cordycepin separated and purified by the production method of Example 2. FIG. It is a HPLC chromatogram of cordycepin separated and purified by the production method of Example 3-1.

  The present invention is described in detail below.

  The method for producing cordycepin of the present invention includes a step of cultivating cordyceps using a culture solution sterilized by filtration and producing cordycepin at a high concentration in the culture solution.

  The cordyceps used in the present invention is not particularly limited as long as it produces cordycepin, whether it is a wild-type cordyceps or artificially-grown cordyceps. Preferably, what discharge | releases the produced cordycepin out of a microbial cell is good. For example, Cordyceps militaryis NBRC 9787, Cordycesps militaryis NBRC 30377, Cordycesps militaryis ATCC 26848, Cordyceps militaryis ATCC 34164, Cordycespsmitirty 34. More preferably, from the viewpoint of the amount of cordycepin production, the mycelium of Cordyceps spp., Disclosed in JP 2009-34045 A, is irradiated with a high-energy ion beam, and the mycelium subjected to the irradiation treatment is used for parental strain and mycological properties. Cordyceps mutants obtained by selecting different strains can be used. As the “strain having a different mycological property from the parent strain”, a strain that can survive in the presence of an adenine such as 8-azaadenine and 8-azaguanine and / or an analog of guanine may have high cordycepin productivity. , Preferably selected. In the present invention, as the cordycepin high-producing strain, a cordycepin production amount that is 1.3 times or more that of the parent strain is preferably used, and a strain that is 1.5 times or more is more preferably used. As such a mutant strain, a mutant strain obtained from Cordycesps militaryis NBRC 9787 is more preferable. 1-1-1 Higashi 1-1-1, Tsukuba Center, Tsukuba City, Ibaraki Pref. Deposited at National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Accession number: FERM P-21315 strain (date of trust: July 5, 2007) is particularly preferred.

  In the culture of Cordyceps sinensis of the present invention, a culture solution that can be usually used for culture of mycelium can be used, and the culture solution contains a carbon source and a nitrogen source as components and / or other additives. Examples of carbon sources that can be used include glucose, sucrose, maltose, lactose, molasses, and examples of nitrogen sources that can be used include peptone, yeast extract, casamino acid, corn steep, and wheat germ. Is preferred. Moreover, you may add any 1 type, or multiple types of nucleic acid related substances, such as amino acids, such as glycine, glutamine, aspartic acid, and cysteine, adenine, guanine, and adenosine.

  As a compounding amount of the medium component, for example, the carbon source is 1 to 20% by weight, preferably 2 to 15% by weight of the total weight of the medium including water, and the nitrogen source is 0.25 to 20% by weight of the total weight of the medium. %, Preferably 0.5 to 15% by weight. When an amino acid is added, the blending amount is 0.5 to 5% by weight, preferably 1 to 3% by weight, based on the total weight of the medium. When a nucleic acid-related substance is added, the blending amount is the total weight of the medium. 0.05 to 0.6% by weight, preferably 0.1 to 0.4% by weight.

  Specifically, for example, a platin dextrose agar plate or an agar plate containing glucose 20 g / L, peptone 2.5 g / L, and yeast extract 7.5 g / L is inoculated with one platinum loop mycelium. What was statically cultured at 15 to 35 ° C., preferably 20 to 30 ° C. for 7 to 21 days, preferably 10 to 16 days can be used as the inoculum. A liquid surface culture for producing cordycepin can be performed by cutting out the mycelium at the periphery of the mycelium grown on the plate and floating it on the surface of the culture solution in the culture bottle. The culture temperature is 15 to 35 ° C, preferably 20 to 30 ° C. The amount of the culture solution is set such that the liquid depth is 1 to 10 cm, preferably 2 to 5 cm, thereby increasing the amount of cordycepin production. As the sterilization method of the culture solution, various methods such as high-pressure steam sterilization (autoclave), low-temperature sterilization, and filter sterilization can be adopted. In some cases, autoclaving is not preferred. In particular, in the case of a culture solution containing a medium component at a high concentration, a fermentation inhibitor may be generated. Therefore, in the production of cordycepin by culture, filtration sterilization is preferable in order to improve the amount of cordycepin produced. As for filtration sterilization, a germ filter is usually removed using a membrane filter having a pore size of 0.20 μm. However, other materials and pore sizes may be used as long as no contamination such as germs is observed.

  The mycelium of Cordyceps grows on the surface of the culture solution using the components in the culture solution as nutrients to produce cordycepin. Here, 90% or more of the produced cordycepin is secreted into the culture solution. Therefore, in order to recover the produced cordycepin, it is necessary to first separate the mycelium from the culture solution to obtain the culture supernatant. As a method for separating mycelium, various methods such as centrifugation and filtration can be used, and are not particularly limited.

  The separated culture supernatant contains cordycepin at a high concentration, and cordycepin can be recovered by various crystallization methods using the solubility difference of cordycepin. For example, by concentrating the culture supernatant under reduced pressure, cordycepin that exceeds the solubility and precipitates in the supernatant can be recovered as a precipitate. It is also possible to pulverize the culture supernatant containing cordycepin in advance by freeze-drying, vacuum drying, etc., and then dissolve it in a solvent so as to obtain a high concentration, followed by crystallization. As a method for crystallization of cordycepin dissolved in a high concentration, a method of changing the temperature of the solution, a method of changing the pH, a method of adding a poor solvent for cordycepin to the cordycepin solution, and the like can be used. Examples of the poor solvent for cordycepin that can be used in the present invention include acetone, ethanol, propanol and the like.

  The solvent for dissolving cordycepin is not particularly limited as long as it does not react with cordycepin and has low toxicity and has a low boiling point, but water is preferable. The solubility of cordycepin in water is high at high temperatures and low at low temperatures. Therefore, crystallization of cordycepin can be performed by cooling after dissolving cordycepin at high temperature. The temperature for dissolving cordycepin is not particularly limited as long as hydrolysis of cordycepin or the like does not occur. However, when water is used as a solvent, 30 to 90 ° C is suitable, and preferably 40 to 80 ° C. Further, the temperature for precipitating dissolved cordycepin is not particularly limited as long as the solution does not freeze, but 20 ° C. or less is suitable, and preferably 0 to 10 ° C.

  Further, the solubility of cordycepin in water is low in the vicinity of neutrality and high in the acidic region and alkaline region. Accordingly, cordycepin can be crystallized by dissolving cordycepin in an acidic or alkaline aqueous solution at a high concentration and then changing the pH of the aqueous solution to near neutral. The pH at which cordycepin is dissolved is preferably less than 4 or more than 12.5, more preferably less than 3 or more than 13. Moreover, as pH which precipitates melt | dissolved cordycepin, 4-12.5 are preferable and 5-10 are more preferable.

Further, in the present invention, cordycepin can be crystallized by combining the above temperature change and pH change.
That is, the lyophilized powder of the culture supernatant is converted into an acidic aqueous solution having a pH of preferably less than 4, more preferably less than 3, or an alkaline aqueous solution having a pH of preferably 12.5, more preferably more than 13. Redissolve at 90 ° C., preferably 40-80 ° C., then adjust the pH of the aqueous solution to preferably 4-12.5, more preferably 5-10, cool to 20 ° C. or less, preferably 0-10 ° C., The precipitated cordycepin is collected.

The precipitated cordycepin can be recovered by centrifugation or filtration. In addition, centrifugation and filtration can be performed by the method, conditions, etc. which are generally performed when fractionating the precipitated crystals. For example, centrifugation is performed at 9000 to 15000 × g for about 15 to 30 minutes using a small centrifuge, tabletop centrifuge, industrial centrifuge, or the like, depending on the production scale. Filtration can be performed using a filter paper suitable for precipitating the precipitate, but ADVANTEC No. It is preferable to use 5A filter paper or the like, such as filter paper corresponding to Type 5A defined in JIS standard P 3801 [filter paper (for chemical analysis)].
Furthermore, the cordycepin purity can be improved by washing the recovered cordycepin with low-temperature water.

  The collected cordycepin is dried by air drying, freeze drying or the like. From the viewpoint of storage stability and the like, lyophilization is preferred.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[Example 1] Production of cordycepin by liquid surface culture of Cordyceps sinensis mutants Cordyceps militaryis NBRC 9787 mutant G81-3 (AIST Patent Biological Deposit Center accession number: FERM P-21315) on a potato dextrose agar plate And pre-cultured at 25 ° C. for 13 days to obtain seeds. 100 mL of the main culture medium having the composition shown in Table 1 was sterilized by filtration using a membrane filter (“A020A047A”, ADVANTEC, pore size: 0.2 μm), and then placed in a container for static culture (capacity: 500 mL). In addition, the inoculum of the inoculum grown to 3.5 to 4.5 cm by pre-culture was cut out with a sterilized cylindrical cutter (1 cm), floated on the main culture medium, and subjected to stationary culture.

  The culture solution was sampled over time, filtered through a membrane filter (“SLHPX13NK”, manufactured by Millipore) having a pore size of 0.45 μm, and the cordycepin concentration of the filtrate was measured by HPLC. After confirming that the cordycepin concentration reached the maximum value, the main culture was terminated, and the cells were removed from the medium by centrifugation (15000 × g, 20 minutes). The culture supernatant was separated and recovered by filtration through 5A filter paper.

Here, the HPLC measurement conditions are as follows.
Column: Tosoh TSK-GEL ODS-80Ts (4.6 mm ID × 25.0 cm)
Mobile phase: methanol: 0.1% by volume phosphoric acid = 2: 98 (volume ratio)
Column temperature: 40 ° C
Flow rate: 1.0 mL / min
Injection volume: 20 μL
Peak detection: UV detector (260 nm)

  In Example 1, instead of filtration sterilization using a membrane filter, culture was performed in the same manner using an autoclave-sterilized medium for main culture to obtain a culture supernatant (Comparative Example 1). Table 2 shows the maximum cordycepin production amount (g / L-medium) and the culture period until reaching the maximum production amount in each production of Example 1 and Comparative Example 1.

  Moreover, the time-dependent change of the cordycepin density | concentration in the manufacturing method of Example 1-3 of Table 2 and Comparative Example 1-3 is shown in FIG. 1 by HPLC about the culture supernatant obtained in the manufacturing method of Example 1-3. An example of a typical analysis result is shown in FIG. From Table 2, when the culture medium sterilized by filtration was used (Examples 1-1 to 1-4), 1.3 to 3 in the case of using the autoclaved culture medium (Comparative Examples 1-1 to 1-4). It was shown that a maximum cordycepin production of 2.1 times was obtained, and that the culture period until reaching the maximum production was shortened by up to 27%. Thus, production efficiency could be improved by using filtration sterilization as a medium sterilization method. In the production method of Comparative Example 1-5 in which a medium containing glucose 120.7 g / L and yeast extract 131.3 g / L was used after autoclaving, growth of bacterial cells was not observed. Further, FIG. 2 shows that the main impurity contained in the culture supernatant is a peak (hereinafter referred to as “Main impurity”) that appears immediately after cordycepin in the retention time.

[Example 2] Purification of cordycepin using crystallization by temperature change The purification of cordycepin was carried out mainly for the purpose of reducing the content of Main impurity. The culture supernatant obtained by the production method of Example 1-3 was freeze-dried, and a dried product containing 9.6 g of cordycepin was added to 480 mL of distilled water so that the cordycepin concentration was about 20 g / L. Dissolved. In a heated state, a small amount of insoluble matter was removed from ADVANTEC No. After filtration using 5A filter paper (the collected amount of the filtrate was 420 mL), it was transferred to an aluminum vat and left in a refrigerator at 5 ° C. The temperature of the solution, the amount of precipitated cordycepin, and the ratio of the amount of main impurity in the precipitate were monitored over time, and the precipitate was collected by filtration 36 hours after reaching a nearly steady state. ADVANTEC No. as filter paper After filtration using 5A, the precipitate was washed with ice-cold water. The washed cordycepin was lyophilized to obtain 6.54 g of purified cordycepin (purification yield: 66%). The analysis result by HPLC of the obtained cordycepin is shown in FIG. The main impurity content calculated from the peak area detected by 260 nm ultraviolet light was 3.8 to 4.0% in the culture supernatant, but decreased to 1.0% after purification according to this example. . That is, the purity of cordycepin increased from about 96% to 99%.

[Example 3] Re-purification of cordycepin using crystallization by temperature change The cordycepin obtained by the purification method of Example 2 was slightly colored. Therefore, in order to obtain even more pure cordycepin, purification using crystallization by temperature change was performed again. First, 400 mL, 300 mL, and 200 mL of distilled water were added to and dissolved in cordycepin 2.00 g, 1.50 g, and 1.00 g obtained by the purification method of Example 2, respectively (Examples 3-1, 3-2, 3-3). This solution is referred to as ADVANTEC No. By filtering with 5A filter paper, filter paper fibers and the like mixed in the previous step were removed. After each filtrate was freeze-dried, 50 mL each of distilled water was added to the dried product and heated to 60 ° C. to completely dissolve it. The solution was allowed to stand in a refrigerator at 5 ° C. for 24 hours, and then the precipitate was advantec no. It filtered and collected using 5A filter paper. Table 3 shows the final recovered amount of cordycepin and the purity of the purified product. The content of Main impurity in cordycepin obtained by the repurification method of Example 3-1 was reduced to about 0.3%. That is, the purity of cordycepin could be increased to about 99.7%. The analysis result by HPLC of cordycepin obtained by the repurification method of Example 3-1 is shown in FIG.

[Example 4] Purification of cordycepin using crystallization by pH change The culture supernatant obtained by the production method of Example 1-3 was freeze-dried, and 8.68 g of the obtained dried product (cordisepin content 1. 91 g) was suspended in 20 mL of distilled water, 5N hydrochloric acid aqueous solution was added little by little, and a 69 g / L cordycepin solution was prepared while keeping the temperature at 25 ° C. The pH of this solution was 2.7. This solution was centrifuged at 9100 × g for 20 minutes to remove some insoluble components. While maintaining the temperature of the obtained supernatant at 25 ° C., a 5N aqueous sodium hydroxide solution was added to adjust the pH to 5.3, and a large amount of precipitate was produced. This precipitate was adsorbed with ADVANTEC No. The product was collected using 5A filter paper, washed with ice-cold water, and freeze-dried to obtain 1.36 g of a purified product. Table 4 shows the final purity and recovery rate of cordycepin. It was confirmed that cordycepin having a purity of 99% or more can be recovered in a high yield of 70% or more by crystallization by pH change.

[Example 5] Purification of cordycepin using crystallization by pH change and temperature change 7.14 g (cordisepin content 1.57 g) of the dried culture supernatant obtained in the same manner as in Example 4 was added to 30 mL of distilled water. 5N hydrochloric acid aqueous solution was added little by little to prepare a 42.4 g / L cordycepin solution while keeping the temperature at 25 ° C. The pH of this solution was 2.7. This solution was centrifuged at 9100 × g for 20 minutes to remove some insoluble components. Subsequently, 5N sodium hydroxide aqueous solution was added and pH was adjusted to 6.6, keeping the temperature of the obtained supernatant at 25 degreeC. Furthermore, when it cooled in a 4 degreeC refrigerator for 3 hours, a lot of precipitation produced | generated. This precipitate was adsorbed with ADVANTEC No. The product was collected using 5A filter paper, washed with ice-cold water, and then freeze-dried to obtain 1.17 g of a purified product. Table 4 shows the final purity and recovery rate of cordycepin. It has been confirmed that cordycepin having a purity of 98% or more can be recovered with a high yield of 75% or more by crystallization combining pH change and temperature change.

  The specific parts of the contents of the present invention have been described in detail above. However, such a specific technique is only a preferred embodiment for those having ordinary knowledge in the art, and the present embodiment is not limited to these embodiments. It will be apparent that the scope of the invention is not limited. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method which can obtain highly purified cordycepin simply and efficiently can be provided.
In addition, it is possible to provide a method for separating and purifying cordycepin with high purity and high recovery rate from the culture supernatant separated from the culture solution of Cordyceps sinensis.

Claims (11)

Cordyceps militaris NBRC 9787 mutants (accession number: FERM P-21315) of the, and culture with a sterile-filtered culture broth, and producing co Rujisepin in the culture, the culture supernatant is separated from the culture broth A method of producing cordycepin, comprising: pulverizing the culture supernatant and re-dissolving in water such that the cordycepin concentration is 20 g / L or more; and separating and purifying cordycepin from the aqueous solution by crystallization. . Culture is carried out by liquid surface culture method according to claim 1. The production method according to claim 1 or 2 , wherein the powderization is performed by freeze-drying. The manufacturing method of any one of Claims 1-3 whose cordycepin density | concentration in the process of re-dissolving in water is 20 g / L-69 g / L. The process according to any one of claims 1 to 4, wherein the step of separating and purifying cordycepin by crystallization includes the step of precipitating cordycepin at 0 to 10 ° C after dissolving cordycepin at 30 to 90 ° C. . The step of separating and purifying cordycepin by crystallization includes the step of precipitating cordycepin at pH 4 to 12.5 after dissolving cordycepin at a pH of less than 4 or more than 12.5. The manufacturing method of any one of these. Cordyceps militaris NBRC 9787 mutants (accession number: FERM P-21315) of the, the culture supernatant from the culture broth obtained by culture using a sterile-filtered culture broth were separated and the culture supernatant was lyophilized Thereafter, the cordycepin separation method for recovering the cordycepin precipitated by cooling the aqueous solution obtained by redissolving in water at 30 to 90 ° C. to 0 to 10 ° C. so that the cordycepin concentration is 20 g / L or more is recovered. . Cordyceps militaris NBRC 9787 mutants (accession number: FERM P-21315) of the, the culture supernatant from the culture broth obtained by culture using a sterile-filtered culture broth were separated and the culture supernatant was lyophilized Thereafter, the solution is redissolved in an acidic aqueous solution having a pH of less than 4 or an alkaline solution having a pH of more than 12.5 so that the cordycepin concentration is 20 g / L or more, and then the pH of the aqueous solution is adjusted to 4 to 12.5. A method for separating and purifying cordycepin, which recovers cordycepin. Cordyceps militaris NBRC 9787 mutants (accession number: FERM P-21315) of the, the culture supernatant from the culture broth obtained by culture using a sterile-filtered culture broth were separated and the culture supernatant was lyophilized Thereafter, the solution is redissolved in an acidic aqueous solution having a pH of less than 4 or an alkaline solution having a pH of more than 12.5 at 30 to 90 ° C. so that the cordycepin concentration is 20 g / L or more . 5. A method for separating and purifying cordycepin, which is cooled to 0 to 10 ° C. and recovers the precipitated cordycepin. Culture is carried out by liquid surface culture, separation and purification process according to any one of claims 7-9. The separation and purification method according to any one of claims 7 to 10, wherein the cordycepin concentration at the time of redissolving is 20 g / L to 69 g / L.

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