JPH11103666A - Culture of cordyceps sinensis sacc. - Google Patents

Abstract

PROBLEM TO BE SOLVED: To purely culture Cordyceps sinensis Sacc., even when an insect body picked at a site is stained with other fungi, by culturing the hyphae of the Cordyceps sinensis Sacc. in the insect body and subsequently separating the cultured Cordyceps sinensis Sacc. SOLUTION: This method for culturing Cordyceps sinensis Sacc. comprises storing and maintaining an organism containing the early Cordyceps sinensis Sacc. converted from nutritive growth to reproductive growth at a low temperature to interrupt the reproductive growth, and subsequently maintaining the organism containing the Cordyceps sinensis Sacc. in a sterile room at the ordinary temperature to return to the nutritive growth, thus forming a new asexual white acicular tissue on the organism. A section of the new white acicular tissue not stained with various fungi at the tip portion of the new tissue or a conidium divided from the hypha of the new tissue as a spawn can purely be cultured on an artificial culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for cultivating Cordyceps sinensis, and particularly to Cordycep sinensis, which occurs in larvae of bat moths and is prized as a fertility agent for Chinese medicine.
s sinensis) at the asexual stage.

[0002]

2. Description of the Related Art Cordyceps sinensis, which is collected by local Tibetan people on a plateau between 3,000 meters and 5,000 meters above sea level between the Chinese side and the Nepal side across the Himalayas, is distinguished from cordyceps sinensis in other regions. It is called as a Chinese medicine and has been used as a fertilizer since ancient times.
Especially prized as a good product is Cordyceps sinensis, which forms a sclerotium in the body of bat moth larvae in the soil, ends vegetative growth, and is in the early stage when the developmental phase has been converted to reproductive growth, and Is from a slightly peeped period. On the other hand, at the time when the larva expands and releases sexual spores, the active ingredient in the worm is exhausted, and the commercial value of the drug is almost lost.

[0003] This means that the medicinal substance of Cordyceps sinensis is less in the mushroom, Locus, and more in the mycelium in the insect body, and the local Tibetan inhabitants know this by experience. The above individuals were collected exclusively at the beginning of the loci, that is, when they were slightly peeping out of the soil where the remaining snow remained,
It was used as a famous Chinese medicine. However, recently, overfishing has continued, and the locality of occurrence has been the pasture of local yaks, so the yield has dropped sharply. Of 1
/ 5. For this reason, environmental protection measures for resource conservation and pure culture at the asexual stage of mycelia have become desirable.However, the technology of artificial culture is very slow compared to other fungi, and moreover, it is collected locally. The culled individuals have been contaminated with many other fungi, and have not achieved the results to be seen.

[0004]

SUMMARY OF THE INVENTION According to the present invention, a cordyceps fungus, typified by Cordesepsin sinensis, is obtained by cultivating and separating mycelia at an asexual stage using insect bodies collected locally. Even if the insects collected at the site are contaminated with other fungi, the endemic bacteria can be cultured purely.

[0005]

According to a first aspect of the present invention, an individual containing an early Cordyceps fungus which has been converted from vegetative growth to reproductive growth is stored and managed at a low temperature to interrupt the reproductive growth, and then at room temperature. This is a method for cultivating Cordyceps sinensis, characterized in that a white needle-shaped new tissue in an asexual stage is formed on an individual by controlling the growth in a sterile room to reverse the vegetative growth.

That is, in the present invention, a larva of a bat moth or a horseshoe pond is used, which is an insect body collected locally and containing an early cordyceps fungus transformed from vegetative growth to reproductive growth. The harvest time is just after the mycelium in the worm has finished the asexual stage of vegetative growth, forms a sclerotium, and the phase of development has been switched to reproductive growth to leave offspring. It is a time when Koza peeked slightly from the soil with residual snow. If the time of collection is delayed and it is time for the larva to grow and release sexual spores, the active ingredient in the insect body is lost, which is inappropriate.

[0007] In general, in the culture of inoculum of mushrooms, it is customary to sterilize the surface of an individual, take a divided section at an early stage, and plant it in a culture medium. Instead of adopting the standard method, a method is adopted in which mushrooms that have already been converted to reproductive growth and that have a small number of loci are pulled back toward vegetative growth. That is, the collected individual is stored and managed at a low temperature to suspend reproductive growth. The temperature and control time are set so that reproductive growth can be interrupted. In the case of a bat moth of Cordesepsinensis, the control temperature is preferably 0 ° C to 5 ° C, and the control time is preferably 7 to 14 days. When the control temperature is lower than 0 ° C., the hypha may be weakened. On the other hand, when the control temperature is higher than 5 ° C., the reproductive growth is not interrupted, and the active ingredient in the individual is consumed. If the management time is less than 7 days, the reproductive growth is not interrupted. On the other hand, if the management time is more than 14 days, the hyphae are weakened and easily killed. The storage place is preferably a dark place such as a refrigerator.

In the present invention, the growth is suspended once in the early stage of reproductive growth as described above, and then the growth is reverted to vegetative growth by managing in a sterile room at room temperature. The condition may be such that vegetative growth is resumed. In the case of the above bat moth, the temperature is 17 ° C to 24 ° C.
C., humidity 90% to 95%, illuminance 50 to 100 lx, and time are preferably 10 to 20 days, respectively, whereby the vegetative growth in the asexual stage is resumed, and a new white needle-like tissue is formed on the surface of the individual. Is done. However, when the temperature is lower than 17 ° C., the growth is too slow, and when it is higher than 24 ° C., the growth is further slowed. If the humidity is less than 90%, the growth will be slow and 9
If it exceeds 5%, the growth will be slower. Also, if the illuminance is less than 50 lx or the time is less than 10 days, the growth is delayed, and if the illuminance is more than 100 lx or the time exceeds 20 days, it is easy to shift to reproductive growth. .

[0009] By the resumption of the vegetative growth, white needle-like new tissue is formed on the surface of the individual. This new tissue is not a reproductive organ for producing sexual ascospores, but a hairy hypha. It is a new organization of the asexual stage gathered in a bundle. When producing sterile seedlings in horticultural crops, the division of new cells that are not contaminated with other fungi is concentrated, just as virus-free sterile seedlings are obtained by tissue culture at the growing point where new cell division is active. It is an aggregate of mycelia endemic to Cordyceps. Therefore, a section obtained by cutting off the tip of the new tissue or a conidium split from the hypha of the new tissue can be used as a seed for the next artificial culture.

[0010] A second aspect of the present invention is a section of the white needle-shaped new tissue which is not contaminated with various bacteria at the tip of the new tissue obtained according to the first aspect of the present invention, or a portion separated from the hypha of the new tissue. This is a method for cultivating Cordyceps sinensis, wherein live spores are purely cultured as an inoculum on an artificial medium.

[0011] In the method of the pure culture, as described in claim 3, after inoculating the inoculum into a sterile artificial medium containing potato extract and glucose as main components, it is controlled at room temperature to complete vegetative growth, Next, a method is preferred in which the growth is interrupted by preservation and management at a low temperature, and then a new needle-like tissue at the asexual stage is formed by controlling again at room temperature. The formula of the above-mentioned artificial medium is such that the standard is 4.0 g of potato extract and 20.0 g of glucose per 1000 cc of water.
This PDA medium is extremely excellent as an artificial medium for purely cultivating protozoa from a collected individual. The pH of the medium is preferably 5 to 7, preferably 5 to 7.
If it is less than 7, the growth of the hypha is slow, and if it is more than 7, the incidence of various bacteria increases.

[0012] As a medium for industrially expanding Cordesepsinensis at an asexual stage for medicinal or edible purposes, a medium used for cultivating shiitake mushroom described in JP-B-46-40169 is disclosed. And a mixture of brown rice, onion and regular oil used in a medium of shiitake mushroom described in JP-B-55-50668. In particular, a mixture of brown rice, onion and pure oil is preferable in that the medium containing mycelia can be used for food or medicine. The pH of the medium is preferably 5 to 7 as described above.

The normal temperature control immediately after inoculation is to complete the vegetative growth, the temperature is 17 to 24 ° C., and the illuminance is 50.
-100 lx, and the time is preferably 15-25 days. If the temperature is less than 17 ° C., the illuminance is less than 50 lx, and the time is less than 15 days, the growth will be insufficient.
If the temperature exceeds 4 ° C., the illuminance exceeds 100 lx, and the time exceeds 25 days, it is easy to convert to reproductive growth, and the growth may be stopped. In addition, by this vegetative growth, a white mycelial flora is formed on the artificial medium, the thickness of the mycelial layer is increased, and the color tone changes from white to pale yellow through yellowish brown, so when the yellowish brown color is reached Can be determined as the completion time of the vegetative growth.

After the vegetative growth is completed under the normal temperature control, the growth is interrupted by the low-temperature storage control. The low-temperature storage control is performed in the same manner as the low-temperature storage control of the collected individual. That is, it is preferably controlled using a refrigerator at 0 ° C to 5 ° C for 7 to 14 days. And the next room temperature control is
As in the case of the normal temperature control of the collected individual, the temperature is 17 ° C to 24 ° C.
C. and 50 to 100 lx at 10 to 20 days, whereby a white needle-like tissue similar to the new tissue on the collected individual is formed on the surface of the artificial medium. This needle-shaped tissue can be cut out and used for medicinal and edible purposes and for the next culture. When brown rice medium is used as the artificial medium, the entire medium containing the mycelium can be used for food.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Cordyceps sinensis-containing Cordyceps sinensis (bat moth larvae) individuals are collected in the Himalayas in a state where a part of their loci protrude from the soil, and this individual is placed in a thermos bottle. And controlled at a low temperature of 0 ° C. to 5 ° C. to interrupt the growth of mycelium, and in this state, control for 7 to 14 days. Thereafter, the individual larvae were taken out of the thermos bottle and placed in a culture room (temperature 17 ° C. to 24 ° C., humidity 90% to 95%).
%, Illuminance 50 to 100 lx), and normal temperature control is performed for 10 to 20 days to restart vegetative growth in the asexual stage, and a new white needle-like tissue is formed on the surface of the individual.

Next, the tip of the above-mentioned new tissue is cut off, and the obtained section or the conidiospores split from the hypha of the above-mentioned new tissue is used as an inoculum, and a sterile preparation such as a PDA medium or a rice bran medium or a brown rice medium prepared in advance is used. Artificial medium (pH: 5
~ 7), temperature 17 ~ 24 ℃, illuminance 50 ~ 100
The vegetative growth is completed by controlling in a lx culture room for 15 to 25 days, and then the above mycelium is transferred to a refrigerator together with the artificial medium, and controlled at a low temperature of 0 to 5 ° C. for about 7 to 14 days on the artificial medium. Suspend the growth of mycelium. After that, return to the above-mentioned culture room again and perform the same ordinary temperature control for 10 days or more.
The treatment is carried out for about 20 days to form a white needle-like tissue similar to the above on the surface of the artificial medium, and further grow as necessary to form a swab-like tissue.

[0017]

[Example] 10:00 am to 11:00 am on May 23, 1997,
4500m above sea level in Himalayan (Surface temperature 5 ℃ ～ 8
C.), a cordyceps cordyceps (bat moth larva) was collected from the remaining snow with its loci protruding 2.0 cm. An individual in a state in which the development phase of Cordesepsin sinensis has changed from vegetative growth to reproductive growth. After digging the soil of the individual, wrap it in water moss, put it in a portable thermos bottle, descend at a low temperature of 0 ° C to 5 ° C while controlling at low temperature, and transfer the individual to a laboratory at 20 ° C two weeks after collection. The illuminance was adjusted to 100 lx by wrapping it in cotton that had been sufficiently absorbed to give scattered light. If naked, 50 lx is fine, but wrapped in cotton, illuminance is 10
Increased to 0lx.

Two weeks later, the wrapped cotton was carefully removed, and the individual was taken out. As shown in FIG. 1, pure white needle-like tissue 11 appeared on the surface of the individual 10. This new needle-like tissue 11 was a new tissue at the asexual stage in which hairy hyphae were collected in a bundle, and had a height of about 5 mm to 15 mm. A sterile portion having a length of about 3.0 mm was cut from the tip of the needle-shaped tissue 11, and the obtained section was used as a seed fungus for culture.
In addition, conidiospores split from the hypha of the tissue were also used as cultures for inoculum.

For culture of the above inoculum, 50 test tubes and 50 Petri dishes were prepared, and a PDA medium (formulation:
4 g of potato extract, 20 g of glucose, 15 g of agar, pH: 5 to 7) in 1000 cc of water, put a piece of the new tissue in a petri dish medium, and put the conidia of the new tissue in a test tube medium. It was inoculated by attaching it to a platinum loop and placed in a test room at a temperature of 20 ° C. and an illuminance of 100 lx and maintained for 20 days to complete vegetative growth.

On the tenth day from the start of the culture,
As shown in FIG. 2, the bacterial layer thickly rises in the central portion of the white flora 13 radially extending from the center of the PDA medium 12, and the bacterial layer spreads to the periphery in the latter 10 days, and Pigments specific to Cordesepsinensis bacteria appeared on the surface of the white flora. That is, a pale red color appeared at first, which gradually changed to orange or tan. The time at which the swelling of the bacteria spread to the periphery and the yellow-brown pigment settled on the surface of the bacterial flora was used as a visual criterion for the completion of vegetative growth.

Next, the above-mentioned test tube and petri dish were
A total of 10 pieces were taken out and transferred to a refrigerator at a temperature of 0 ° C. to 5 ° C. to prevent the above mycelium from converting the growth phase into reproductive growth, and preserved as it was for 10 days. Remove the tube and petri dish and re-heat to 20
After returning to the test room at 100 ° C. and an illuminance of 100 lx, the growth of the mycelium resumed. On the 7th day, the center of the mycelium rose and needle-like tissues began to appear. It grew into a white needle-like new tissue. Then, a part of the new needle-shaped tissue grows into the swab-shaped tissue 14 as shown in FIG.
As shown in FIG. 4, the spores began to split and produce conidia 14c.

On the other hand, the remaining 45 test tubes and the 45 petri dishes were kept in a test room at a temperature of 20 ° C. and an illuminance of 100 lx for 30 days without being transferred to a refrigerator even after the completion of the vegetative growth, and cultured for 30 days. As a result, out of 45 petri dishes inoculated with a section of the new tissue, one petri dish in which various bacteria had occurred and one petri dish in which no hyphae were generated. In addition, out of 45 test tubes inoculated with conidia, one test tube in which various bacteria were generated and two test tubes in which no hypha was generated. That is, although the hypha elongation was slow and flat, the elongation was uniform, and both the occurrence of various bacteria and the absence of mycelia were small, so the sections and conidia of the new tissue used for the inoculum were:
Both were confirmed to be pure asexual stages of Cordesepsinensis itself.

[0023]

According to the first aspect of the present invention, a needle-shaped tissue consisting of a new cordyceps fungus which is not contaminated with various bacteria and which is asexual at a new stage is obtained from a cordyceps individual collected locally. The obtained new tissue can be used as a seed for the next artificial culture. Claim 2
According to the invention described in (1), a cordyceps fungus similar to the invention described in (1) is obtained by utilizing, as a seed fungus, a portion of the apical portion of the new tissue obtained by the invention described in claim 1, which is not contaminated with various bacteria. Can be produced. Further, according to the invention described in claim 3, the invention described in claim 2 can be implemented more efficiently and reliably. According to the fourth aspect of the present invention, Cordesepsin sinensis, which is the most effective Cordyceps sinensis, can be cultured as a tonic of Chinese herbal medicine.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a main part of a larval individual in which a new tissue of Cordyceps fungi has developed.

FIG. 2 is a cross-sectional view showing hyphae that have completed vegetative growth on a PDA medium.

FIG. 3 is a cross-sectional view showing a swab-like tissue expressed on a PDA medium.

FIG. 4 is an enlarged sectional view showing the tip of the swab-like tissue of FIG. 3;

[Explanation of symbols]

 10: Individual (larva) 11: Needle-like tissue 12: PDA medium 13: Flora 14: Swab-like tissue 14a: Head 14b: Mycelium 14c: Conidia

Claims (4)

[Claims] Claims: 1. An individual containing Cordyceps sinensis, which has been converted from vegetative growth to reproductive growth, is preserved at a low temperature and maintained at a low temperature to interrupt the reproductive growth. Thereafter, the individual is controlled in a sterile room at room temperature to control vegetative growth. A method for cultivating Cordyceps sinensis, which comprises retrogradely forming a white needle-like new tissue in an asexual stage on an individual. 2. A seed of said white needle-shaped new tissue section or a conidia spore split from a hypha of said new tissue which is not contaminated with bacteria at the tip of said new tissue obtained by the invention of claim 1. A method for culturing Cordyceps sinensis, wherein the culture is purely cultured on an artificial medium. 3. The pure culture according to claim 2, wherein the pure culture is composed of a potato extract and glucose as main components.
After inoculating the inoculum into a sterile artificial medium of H5-7, control at room temperature to complete vegetative growth, and then store at a low temperature to interrupt the growth, and then control again at room temperature to maintain the asexual stage. A method for cultivating Cordyceps sinensis to form a white needle-like new tissue as described above. 4. The method of cultivating Cordyceps sinensis according to any one of claims 1 to 3, wherein the Cordyceps sinensis is Cordyceps sinensis.