JP2003116522A - Medium for culturing cordyceps sinensis sacc. and method for culturing cordyceps sinensis sacc. - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a culture composition for culturing a fruit body of Cordyceps sinensis Sacc. having the same form as that of wild Cordyceps sinensis Sacc. and a pharmacological component not inferior to or superior to that of wild Cordyceps sinensis Sacc. and to provide a method for industrially and inexpensively culturing a fruit body of Cordyceps sinensis Sacc. by the medium. SOLUTION: This medium for culturing Cordyceps sinensis Sacc. comprises grain and an insect tissue body as main components to which nutrition components such as amino acids, minerals, or vitamins are added.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for cultivating Cordyceps sinensis and a culture medium used therefor.

[0002]

BACKGROUND OF THE INVENTION Cordyceps is broadly said to be a group of insect parasitic fungi belonging to the subphylum Ascomycota, Nucleococci, Bacterial order, Bacteromycetes. In this genus Cordycepas, or
In addition to complete fungi such as rubiella, Paecilomyces, Be
There are also imperfect bacteria such as auveria.

[0003] Caterpillar is literally a complex of an insect defeated by Cordyceps fungi and a grass (fruit body) produced from the worm body. It is common that two parts of the host (worm body) and the fruiting body that grows from the host (parasite), which is also infested with C. sinensis, which is a noble drug from China, is used at the same time. Is. Therefore, many researchers have conducted a great deal of research in order to form fruit bodies similar to those of wild Cordyceps sinensis, but due to the characteristic of Cordyceps fungus that parasitizes live insects, the complete fungal larvae in an artificial medium are cultivated. Cultivation of entities (sexual generation) has become extremely difficult and is not yet in practical use. As a substitute for the complete fungus of Cordyceps sinensis, most of the studies have been on the culture of mycelium (asexual generation) before the formation of fruiting bodies of the complete fungus of Cordyceps sinensis or the culture of incomplete fungi.

As a method for culturing mycelium of Cordyceps sinensis,
The mycelium of Cordyceps sinensis is transplanted into a solid medium in which saccharides, proteins, etc. are the main components and cereals are added to these main components as a substrate, and cultivated. Only those showing fruit body formation in this solid medium in the next step A method of transplanting to a liquid medium and culturing is disclosed (Japanese Patent Publication No. 61-53033). As described in this publication, this grain is used as a substrate for forming a solid medium rather than a nutrient containing grain, and the addition amount of grain and an example are not shown. Didn't. Further, since only the solid body in which fruit body formation is observed is transplanted to the next step, it is considered that this culture method is not necessarily reliable.

For the purpose of cultivating incomplete fungi of Cordyceps sinensis, in JP-A-54-80486, culturing of incomplete fungi of Cordyceps sinensis in a culture solution prepared by mixing garlic decoction, regular oil and sugar, or Alternatively, a method of adsorbing this liquid culture solution to grains such as rice and wheat or adult insects such as silkworms and cicadas, pupae, larvae and the like to carry out solid culture has been proposed. This method is also similar to the above, garlic decoction, normal oil, a culture solution in which sugar is mixed is the main component, grains or insects are used as an adsorbent of the solid medium, the amount of addition of grains and No specific example was shown regarding the kind of addition of insects and the effect of addition. In addition, 90 days are required for culturing incomplete fungi in a solid medium, and there is a problem that the culturing period is long.

On the other hand, as a method of culturing fruit bodies of complete Cordyceps sinensis fungi, Japanese Patent Application Laid-Open No. 8-75 discloses that a Cordyceps sinensis fungus inoculated into a host insect is grown at a low temperature around the growth limit temperature of the host insect to form a sclerotia. After that, a method of breeding at a suitable growth temperature to obtain fruiting bodies has been proposed. However, the formation rate of fruiting bodies is only 5
Since it is only 0% and requires a period of 2 to 3 months, it cannot be said to be an efficient culture method.

[0007] Further, after drying the dead pupa, putting it in a culture container and injecting the culture liquid so that a part of the pupa of the silkworm is exposed to the air, the pupae of insects belonging to the order Lepidoptera A method of inoculating a parasite of Cordyceps sinensis and culturing the same to form fruiting bodies on silkworm pupae has been disclosed (JP-A-9-47149). It is described in this publication that it is a method for cultivating fruiting bodies of Cordyceps sinensis that parasitize insects belonging to the order Lepidoptera, but Paecilomyces fari
Nosus) culturing an incomplete fungus is shown, and nothing is said about culturing a fruiting body of a complete fungus, which is considered difficult.

On the other hand, the type and amount of the base material added to the medium are not only involved in the growth of mycelia and the formation of fruiting bodies, but also have a great influence on the medicinal components of the mycelium and fruiting bodies thus formed. Is known to exist. Most of the above publications are aimed at forming mycelia or fruiting bodies, and do not mention the pharmacological activity of mycelia or fruiting bodies obtained by this culture method.

[0009]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems and has the same morphology as that of wild Cordyceps sinensis, and is a Cordyceps sinensis that has a pharmacological activity and a metabolic component equal to or higher than that of the wild Cordyceps sinensis. A medium composition for culturing an entity is provided, and a method for industrially cultivating the medium at a low cost is provided.

[0010]

[Means for Solving the Problems] Originally, Cordyceps sinensis fungi parasitize live insects, and it is said that there is a drawback that pharmacological activity decreases when subcultured in a medium without addition of insects. On the other hand, when culturing only dead insects as a medium,
There are problems that the hyphae grow slowly and the amount of fruiting bodies formed is low.

The present invention is mainly composed of a grain serving as a carbon source, an insect tissue containing a nitrogen source and other components, and the weight ratio of the insect tissue to the grain is 0.5 grain to 1 grain of the insect tissue. It is characterized in that it provides a solid medium of -10, preferably 1-5. Preferably amino acids,
By adding minerals, or nutrients such as vitamins, the growth of hyphae and the formation of fruiting bodies and ascus shells are promoted, and a medium for Cordyceps sinensis culture is obtained in which fruit bodies similar to wild Cordyceps sinensis are obtained. Characterize.

The present invention also comprises inoculating the above medium with an isolate of Cordyceps or Paecilomyces genus Cordyceps sinensis, and after growing mycelia at 18 to 26 ° C. and a humidity of 80% or more in total darkness, the illuminance is 100 Lux. As described above, a fruiting body is formed by performing irradiation for 6 hours or more per day.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Cereals and insect tissues in a solid medium will be described below.

Although brown rice is mentioned as an example of grains, rice, millet, corn, okara, germ, wheat, beans, millet and the like may also be used. Examples of insect tissues include larvae, pupae, and adults of insects such as Lepidoptera, Hemiptera, Isoptera, Orthoptera, Hymenoptera, and Diptera. Although the kind of insect as a tissue body varies depending on the type of Cordyceps, it is desirable to use the same insects or the same family as the insects that the Cordyceps have hosted. For example, in the case of cultivating Bombyx mori using a Bombyx mori parasitized by the wasp, a paper wasp or the like belonging to the same family as the wasp can be used. Further, when cultivating Usagisanagitake using the moss fungus, which is parasitic on the pupa of Kusan, it is possible to use the same pupae of the Lepidoptera Bombycidae and mosquitoes of the same species as Cusan. In particular, pupae of silkworms after reeling cocoons in the reeling industry, insects such as bees, white butterflies, stag beetles, and ants, which are easy to artificially breed, can be greatly utilized as insect tissues. In addition, in order to measure the homogeneity of the medium composition, it is preferable to dry these medium base materials in advance and crush them appropriately. The weight ratio of the insect tissue and the grain varies depending on the type of the Cordyceps sinensis, and the grain is preferably 0.5 to 10 weight, and more preferably 1 to 5 weight with respect to the insect tissue 1. Examples of nutritional components include minerals such as phosphorus, calcium, magnesium, iron, vitamins, yeast extract, peptone, sugars, etc., but the addition amount is 1 to 10 relative to 100 in total of grain and insect tissue. It is better to put it in the weight range.

These medium materials are mixed, placed in a culture vessel, water is added to the medium so that the water content is 55 to 75%, the mixture is stirred, and heat sterilization is performed. The cultivation container is heat-resistant and may be a transparent or translucent container or a glass container for mushroom cultivation. Also, the time of heat sterilization depends on the scale of culture,
Depending on the type of sterilizer, 30 minutes ~ 100 ℃ ~ 120 ℃
It should be done in 240 minutes.

As the Cordyceps sinensis to be cultured in the present invention,
It may be of the genus Cordyceps or the genus Paecilomyces. Bacteria are isolated from wild Cordyceps sinensis collected in advance, and a strain grown on a slope medium is prepared.

After aseptically inoculating the Cordyceps sinensis solution to a medium sterilized by heat and growing mycelia in the dark at 18 to 26 ° C. and a humidity of 80% or more for 1 week to 15 days, an illuminance of 100 Lux or more,
Irradiate for 6 hours or more a day to form fruiting bodies. In the case of incomplete fungi, when the formation of bundles (fruit bodies) and conidia is confirmed, and in the case of complete fungi, formation of fruit bodies and ascus is confirmed, it is judged to be mature, and harvest is performed before and after that.

[0018]

EXAMPLES The present invention will be described in detail below. (Example 1) Based on the composition of each test group in Table 1, a medium base material was mixed and stirred, and water was added so that the water content became 65% to prepare a medium. This was packed in a glass bottle for mayonnaise, heat sterilized at 120 ° C. for 40 minutes in an autoclave, and then naturally cooled.

Next, using a strain isolated cultured from wild Cordyceps Militaris that parasitic on pupae of Lepidoptera killer whale pike moths department, the suspension was prepared in sterile water to conidia Cordyceps Militaris bacteria is 10 ^6/1 ml, sterile Under the conditions, 2 ml was inoculated per bottle.

After that, the hyphae were grown for 10 days in an environment of about 25 ° C., a humidity of 80% or more, and a total darkness.
Irradiation was carried out for 100 to 400 Lux for 8 to 12 hours a day to form fruit bodies.

As a result, C, D, E, F, G containing a lot of cereals
In the test area, the growth of hyphae was vigorous, and on the 10th day of culture, hyphae spread over the entire medium. However, the amount of fruiting bodies in E and F wards was small. On the contrary, in the A, B, and H groups, which had many pupa tissues, the growth of hyphae was slow, and it took more than 2 weeks until the hyphae spread to the entire medium. The fruiting bodies and ascus capsules in the B, C, and G plots were well formed. The hyphae were actively grown in the pupal tissue-free F group, but the amount of fruit bodies formed was small, and no mature ascus was confirmed. In addition, since the hyphae grew slowly in the H-digestion without brown rice, it also affected the subsequent formation of fruit bodies, and the amount of fruit bodies formed on the 45th day of culture was small.
In addition, compared with the non-addition group, the addition amount of vitamin B group and KH ₂ PO ₄ tended to show a slightly higher amount of fruiting body formation, and the fruiting body showed a bright orange color, and Was almost the same. The fruit bodies in the non-added section were light orange and loquat (Table 1).

[0022]

[Table 1]

Component analysis was carried out on the fruit bodies of Pleurotus cornucopiae cultured from the B-1 test section of Example 1 and the medium after the culture. As analysis items, cordycepin, which is said to have anti-tumor activity, β-glugan, which is said to have an action to improve immunity, super enzyme that eliminates active oxygen
Oxide dismutase (SOD) and mannitol.

As Comparative Example 1 of the component analysis, a mixture of pupae of Pleurotus cornucopiae and its parasites, which was cultured from the pupae of a live silkworm, was infected with Pleurotus cornucopiae.
As Comparative Example 2, a fruiting body cultivated in a liquid medium containing a silkworm pupa extract as a main component and supplemented with nutrient components such as vitamins, amino acids, and sugars, and a culture solution thereof were used.

As a result, in the fruiting body obtained by the medium composition according to the present invention and in the medium after culturing, cordycepin,
Compared to Comparative Example 1 or 2, the pharmacological components such as β-glugan are
It was confirmed that it was contained in a large amount (Table 2). The fact that a large amount of useful metabolic components were detected in the medium after culturing is considered to be that the fruiting bodies ingested and grew nutrients in the medium and the metabolites biosynthesized from it were excreted into the medium. Moreover, the production amount seems to depend on the addition amount of cereals, insect tissues and nutrients. Although the disposal of the solid medium after culturing has been a problem so far, it has been found that the medium after culturing by the culturing method of the present invention can be used as a fine culture product like the fruit body.

[0026]

[Table 2]

Example 2 Milled rice 12 g, dried wasp 6 g, wheat germ 1 g, rice bran 1 g, peptone 0.2 g, MgSO ₄
· 7H ₂ O 0.2 g were mixed, water was added so that the moisture content became 68%, followed by stirring. Then, it was bottled, sterilized and cooled in the same manner as in Example 1. Honeytake (Cordyc
A suspension of eps spbecocepbala) was prepared, inoculated into the medium, and cultured at a temperature of around 18 to 25 ° C. Two months later,
A pale orange-yellow fruiting body having a length of 5 to 7 cm was formed. Ascomycetes were obliquely buried and secondary spores were also confirmed. (Example 3) Rice 5 g, corn 2 g, soybean powder 1
g and 3 g of pupa of Saturniidae were mixed, water was added so that the water content was 60%, and the mixture was stirred. Then, it was bottled, sterilized and cooled in the same manner as in Example 1. A suspension of Paecilomyces farinosa was prepared and inoculated with the fungus. When culturing was performed at a temperature of around 20 to 25 ° C., 40 days later, a pale yellow bundle (fruit body) with a length of 4 to 6 cm was formed and conidia were also confirmed. (Example 4) 5 g of brown rice and 2 larva powders of Batidae
g, dried pupa of Saturniidae 2 g, soybean powder 0.5 g, millet 0.5
Mix g, peptone, vitamin B, glucose, MgS
15 g of nutrient water containing O ₄ and K ₂ HPo ₄ was added and stirred. Then, in the same manner as in Example 1, bottled, sterilized,
Cooled. After that, Cordyceps
unnii) was prepared and the medium was inoculated. twenty three
After culturing mycelium for 15 days in the total darkness at ℃,
The temperature was lowered to 20 ° C., and 200 to 500 Lux was irradiated for 10 hours a day to form fruit bodies. After 50 days from the start of culture, white fruiting bodies 3 to 4 cm in length and 0.4 to 0.5 cm in thickness and embedded ascidian shells were formed.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to culture a fruiting body having a morphology similar to that of wild Cordyceps sinensis. In addition, it is possible to efficiently and inexpensively obtain the Cordyceps sinensis fruiting bodies and cultured metabolites having a pharmacological component equal to or higher than that of the wild Cordyceps sinensis. These cultures can be crushed and processed as they are, or, if necessary, the active ingredient can be extracted and used for medicines, health foods and the like.

Claims (9)

[Claims] 1. A main component is a grain and an insect tissue, and the weight ratio of the insect tissue and the grain is 0.
A medium for cultivating Cordyceps sinensis, characterized by comprising a solid medium of 5 to 10. 2. The medium for cultivating Cordyceps sinensis according to claim 1, wherein the weight ratio of insect tissue to cereal is 1 to 5 to 1 to insect tissue. 3. The medium for culture of Cordyceps sinensis according to claim 1 or 2, wherein brown rice is used as the grain. 4. The medium for cultivating Cordyceps sinensis according to claim 1, 2 or 3, wherein as the insect tissue, an insect of the same order or in the same family as the insect hosting the Cordyceps sinensis is used. 5. The medium for cultivating Cordyceps sinensis according to any one of claims 1 to 4, wherein at least one additive is added from amino acids, minerals, or vitamins. 6. The medium for cultivating the Cordyceps sinensis plant according to claim 5, wherein the amount of the additive to be added is 1 to 10 weight ratio when the total value of the grains and the insect tissue is 100. . 7. The Cordyceps sinensis culture medium according to any one of claims 1 to 6 is inoculated with Cordyceps sinensis and the hyphae are grown at 18 to 26 ° C. and a humidity of 80% or more in total darkness, and an illuminance of 100 Lux or more, A method for cultivating Cordyceps sinensis, which comprises cultivating Cordyceps sinensis fruit bodies by irradiation for 6 hours or more per day. 8. The method for cultivating Cordyceps sinensis according to claim 7, wherein the Cordyceps genus is a bacterium belonging to the genus Cordyceps. 9. The method of culturing Cordyceps sinensis according to claim 7, wherein the Cordyceps sinensis is a bacterium belonging to the genus Paecilomyces.