KR102536601B1 - Process for cultivating Cordyceps militaris - Google Patents

Abstract

The present invention relates to a method for cultivating Cordyceps militaris , and more particularly, to a method for increasing the production of fruiting bodies by reducing contamination sources and ensuring good germination during cultivation of Cordyceps militaris. According to the present invention, the source of contamination of the pupa Cordyceps sinensis is reduced, and after subculture, the ascospores of the pupa Cordyceps sinensis are irradiated with light, and the ascospores containing white hyphae and yellow hyphae are separated from the ascospores by the dilution plate culture method. It is possible to increase the production of fruiting bodies of the pupal Cordyceps sinensis by selecting only mycelia containing hyphae and subsequent culturing.

Description

Cultivation method of pupa Cordyceps militaris {Process for cultivating Cordyceps militaris}

The present invention relates to a method for cultivating pupa Cordyceps sinensis, and more particularly, to a method for increasing the yield of fruiting bodies by reducing pollutants during cultivation of pupa Cordyceps sinensis and ensuring good germination.

Cordyceps militaris is a mushroom belonging to the genus Cordyceps sp. It has been known as one of the three major medicines along with ginseng and deer antler since ancient times. There are 300 species, and about 20 species are distributed in Korea. Currently, three types of medicinal mushrooms are distributed: Cordyceps sinesis , Cordyceps militaris , and Isaria japonica .

This chrysalis cordyceps is an expensive herbal medicine that has been used since ancient times in China for the treatment of tuberculosis, asthma, and jaundice, as an antidote for opium addiction, as an aphrodisiac and tonic for illness, and as an immune function enhancer.

As for the artificial cultivation method of Cordyceps sinensis known so far, a method in which Cordyceps sinensis forms a fruiting body using an insect as a host is mainly known. For example, Korean Patent Publication No. 1998-023907, Patent Publication No. Patent Registration Publication No. 10-0443200, Patent Publication No. 10-1998-0033558, etc. disclose methods for producing Cordyceps sinensis fruiting bodies using insects.

However, in general farms, there are problems such as management of pollutants, so measures for increased production of fruiting bodies are still continuously required.

Korean Registration No. 10-0432472 (May 11, 2004) Korean Registration No. 10-0610983 (August 02, 2006) Korean Registration No. 10-2123594 (June 10, 2020)

The inventors of the present invention solved the problems of the prior art as described above, and while conducting intensive research to increase the production of fruiting bodies, reduced the source of contamination during cultivation of pupa Cordyceps sinensis and increased the production of fruiting bodies. When irradiated with light, in a culture solution containing yellow or yellow mycelia derived from ascospores containing white mycelium and yellow mycelia, using the dilution smear method, randomly from small pieces of hyphae separated without overlapping with adjacent mycelia Ideally, 1 colony is separated and transplanted to grow mycelia by dark culture until a certain period of time passes, and then, when light culture is performed, the mycelia that remain white and the mycelia that change to yellow (finally yellow to orange) are separated. It was discovered that the production of fruiting bodies of pupa Cordyceps sinensis could be increased by excluding white strains using the technique described above, selecting yellow (finally yellow to orange) single colonies and subsequent culturing, thereby completing the present invention.

Therefore, in one aspect, an object of the present invention is to reduce the source of contamination during cultivation of pupa Cordyceps sinensis and to increase the production of fruiting bodies by irradiating light to the ascospores of pupa Cordyceps sinensis after subculture to obtain white hyphae and yellow hyphae. Only hyphae containing yellow or yellow mycelia from ascospores are selected by dilution smearing, and white strains are excluded to select single colonies and subsequently cultured to increase the production of fruiting bodies of pupa Cordyceps. Provide a cultivation method of Cordyceps pupa. is in doing

The purpose of the present invention as above is

Subculturing the pupal Cordyceps sinensis;

After culturing, irradiating light to the ascospores of the pupa Cordyceps sinensis;

Selecting only mycelia containing yellow or yellow mycelia from ascospores containing white mycelia and yellow mycelia by dilution smearing and excluding white strains to select single colonies; and

It can be achieved by a method for increasing the unit yield of the pupal Cordyceps sinensis fruiting body comprising the step of continuing the culture in a state where the selected colony is dominant.

According to the present invention, by reducing the source of contamination of pupa Cordyceps sinensis, and irradiating light to the ascospores of pupa Cordyceps sinensis after subculture, ascospores containing white hyphae and yellow hyphae or those derived from tissue separation containing yellow or yellow mycelia It is possible to increase the production of fruiting bodies of Cordyceps pupa by selecting only mycelia by dilution smearing, excluding white strains, selecting single colonies, and subsequent culturing.

1 is a process chart of a method for increasing the unit production of a pupa cordyceps fruiting body of the present invention.
2 is a photograph showing the shape of a fruit body of a sample according to Example 1.
Figure 3 is a photograph showing the difference in the color of mycelium germinated by falling spores in a wild state.
Figure 4 is a photograph showing the hyphae in the plate medium in the state of re-implantation of the independent colony after separation from the spores.
5 is a photograph of isolated colonies.
Figure 6 is a photograph showing the state immediately before and after the pin bale.
7 is a photograph of a fruiting body on a plate medium according to growth conditions.
8 is a photograph showing the quantity (g) of live fruiting bodies that have been cultivated using Panbagu.
9 is an explanatory diagram of the number of times a microscope (objective lens 100X, oil immersion) of 1 slide glass for a microscope is used.
10 is a photograph showing contaminating bacteria.
11 is a photograph of the characteristics of the snow flower / pupa Cordyceps sinensis strain and the form of the contamination-causing bacteria generated in the manufacture.
12 is a photograph showing the hyphae of the mushroom mycelium.
13 is a photograph showing an example of the separation of two types of hyphae from the pupa Cordyceps sinensis.
14 is a photograph showing another example of separating two types of hyphae from the pupa Cordyceps sinensis.
15 is a photograph showing another example of separating two types of hyphae from the pupa Cordyceps sinensis.
16 is a photograph showing an example of separating two types of hyphae from the pupa Cordyceps sinensis.
17 is a photograph showing the shape of hyphae and ascospores under extension (transformation) according to microscope magnification in the progress of triangular culture.
Figure 18 is a photograph showing the form of specific contaminants incorporated in the liquid culture of Cordyceps pupa.
19 is a photograph showing microscopic strains involved in contamination.
Figure 20 is a picture comparing the size of the fungus causing mushroom decay and the microscopic fungus.
21 and 22 are photographs of spoilage-causing microscopic fungi.
23 shows that in this study, when two of the lines with good extraction and proven yield were selected and confirmed again by the dilution plate separation method, even after a long time after refrigerated storage, in the lot of mono spore-derived yellow mycelia, It is also a picture that explains the appearance of mostly yellow to orange hyphae.
23 is a photograph explaining that yellow mycelia are also shown in lots showing yellow mycelia derived from Mono Spore even after refrigerated storage for a long period of time.
24 is an initial photograph of cancer culture after transplanting independent colonies to a new medium.
25 is a photograph showing the progress of light culture after transplanting an independent colony to a new medium.
26 is a photograph showing the appearance of white hyphae in independent colonies.
27 is a photograph showing the state of mycelia in the process of being cultured by light irradiation (light culture) after culture on the 8th day (dark culture) after transplantation into Petri Dishes.
28 is a photograph showing the culturing process of independent colonies.
29 is a photograph confirming symptoms of primary lice.

The present invention, in one aspect,

Subculturing the pupal Cordyceps sinensis;

After culturing, irradiating light to the ascospores of the pupa Cordyceps sinensis;

Selecting only mycelia containing yellow or yellow mycelia from ascospores containing white mycelia and yellow mycelia by dilution smearing and excluding white strains to select single colonies; and

It provides a method for increasing the unit yield of pupa Cordyceps sinensis fruiting bodies, comprising the step of continuing the culture in a state where the selected colonies are dominant.

The present invention, in a further aspect,

The step of subculturing provides a method of increasing the unit yield of the pupa Cordyceps sinensis fruiting body, characterized in that the subculture is minimized by using a tissue separated from the parent strain.

Hereinafter, the present invention will be described in detail. Terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain his or her invention in the best way. It should be interpreted as a meaning and concept consistent with the technical spirit of the present invention based on the principle that there is. Therefore, the embodiments described in this specification and the configurations described in the drawings are only the most preferred examples of embodiments, and do not represent all of the technical spirit of the present invention, so various equivalents and It should be understood that variations may exist.

Hereinafter, a method for increasing unit production of fruiting bodies of Cordyceps pupa according to the present invention will be described in more detail with reference to the accompanying drawings.

1 is a process chart of a method for increasing the unit production of a pupa cordyceps fruiting body of the present invention. As shown in the figure, the method for increasing the unit yield of fruiting bodies of the present invention includes a subculturing step, a light irradiation step, a selection step, a subsequent culturing step, a harvesting step, and a packaging step.

According to the present invention, the unit production of fruiting bodies can be increased by efforts to exclude white hyphae in subculture by improving the process in cultivation of pupa Cordyceps sinensis.

After subculturing the pupa Cordyceps strain by the present inventors, when dark culture is performed at the beginning of culture, white mycelia proliferate, and when the light of a fluorescent light is irradiated on the white mycelia that have grown to a certain size, the initial culture over time It was found that white mycelium has the characteristic of showing two types of mycelial color, one that remains white by light irradiation and the other that is colored yellow.

In the cultivation of pupa Cordyceps sinensis, a number of spores (Multi Spores) germinated (awakened) in a state of close proximity to spore-derived spawn or tissue isolation-derived hyphae, which progressed to mycelia. Incubation takes place in In this case, there is no problem in culturing mycelia, but there is a problem that the number of fruiting bodies is reduced because white mycelia that do not germinate are mixed.

In order to increase the number of feet of the fruiting body in the same unit, it is preferable to use yellow hyphae by separating white hyphae and yellow hyphae from each other.

When cultivated in a state where white mycelia are mixed with yellow mycelia at mass cultivation sites, the number of fruiting body buds decreases from the initial budding process, budding is delayed, and white mycelia proliferate faster to cover the upper part of the medium. I have a problem with my feet not working well.

In most farms, it is common for the pupa Cordyceps militaris to be in the process of dominating the white mycelium every time passage is performed, so subculture is avoided as much as possible in spawn management, and superior characteristics are obtained than continuous passage methods. A small amount of liquid culture is performed for the strain having the strain, and then the culture medium is filtered (sterilized) in 10% Glycerol or 10% SDS solution and stored at a low temperature, or, best of all, using a method of storing the spawn in liquid nitrogen at -196 ° C. It is good.

In a solid medium such as a conventional PDA medium, it seems to proliferate from hyphae to hyphae, but when a series of liquid cultures are performed in the next step, a large number of ascospores generated from the hyphae are clearly produced, and the generated ascospores transform into hyphae. As it grows, it gradually progresses to hyphae and mycelia.

In the spawn preparation process, liquid culture of mycelia obtained by spore separation or tissue separation and homogenization to induce an independent colony results in white mycelia and yellow mycelia when first dark culture and then light culture. At this time, you can select and use the independent colony that turns yellow.

In particular, white mycelia have a fast mycelial growth rate, but do not produce fruiting bodies. Therefore, it is good to use yellow mycelia from a colony isolated from an independent colony by the dilution smear method.

By this method, the production of fruiting bodies can be increased by excluding the white strain of Cordyceps militaris, selecting a single colony, and subsequent culturing.

By this method, some show a single yellow state, but sometimes there are mutated mycelia, such as white hyphae popping out between yellow and orange hyphae. However, when the yellow strains selected first by this method were separated again by the dilution plate method, it was confirmed that relatively most of the yellow mycelia came out. It is judged that it is a problem that it is derived from a plurality of spores (Multi Spore) when spore separation or tissue separation in the process of collection and separation in the wild.

Therefore, in the case of the primary selection, stable fruiting is expected because there is a high possibility that yellow hyphae will be stably maintained as yellow hyphae even after selection in subsequent passages.

Subculture step

Subculture of pupa Cordyceps sinensis can be carried out using a strain stored in a farmhouse or newly collected, and can be performed through Petri dishes, flasks, bottle culture, plate culture, or basket culture using a conventional medium.

Subculture is preferably used to minimize the passage of generations from the parent strain, as described below.

Other culture conditions can be preferably performed by employing known methods related to cultivation of pupa Cordyceps sinensis.

In the present invention, when liquid culture is performed in an Erlenmeyer flask, the growth of mycelia is not from mycelia to mycelia when confirmed the next day after inoculation, but ascospores are initially generated in the mycelia, and most of the mycelia from these ascospores were confirmed to proliferate. , When these hyphae were isolated and viewed, the ratio of white:yellow to orange color was about 13~16% : 87~84%.

Therefore, it was confirmed that the number of fruiting bodies is increased when the subculture of the strain is suppressed as much as possible in the cultivation of pupa Cordyceps sinensis.

light irradiation step

When mycelial culture is carried out in the triangular fluid, the inoculum of the pupal Cordyceps sinensis is a few pieces of mycelium, and what first starts to proliferate in this inoculum is generated in the form of numerous ascospores as shown in FIG. 11, and mycelia progresses. In order to separate the large mycelial mass into small ones, the culture medium is homogenized at a speed of 10,000 to 12,000 rpm using a high-speed homogenizer, diluted 1/10 of each in sterile water by the dilution smear method, and then the diluted liquid is placed in each Petri dish. After smearing or pouring a certain amount of each dilution factor on the surface of the Petri dish, all the liquid is discarded, and the upper surface of the PDA is dried by the amount of air blowing inside the clean bench to dry the water. Thereafter, the sides of each diluted Petri dish are taped and cultured, and within 1 to 3 days, each independent small mycelium is separated from each independent colony, and each independent colony is transplanted into the Petri dish as shown in FIGS. 4 and 5 . When the transplanted colony proliferates and the diameter of the white mycelium becomes 0.5~2.0cm, light (eg, fluorescent light) is irradiated. When light is irradiated in the pupal Cordyceps mycelium culture, it has the characteristic of changing into white hyphae and yellow hyphae. At this time, a white fluorescent lamp or daylight color LED light is irradiated. As time goes by, it becomes possible to clearly distinguish white hyphae and yellow hyphae by light irradiation.

In the conventional cultivation method, when cultivation is performed in a state in which two types of ascospores (e.g., white mycelium/yellow mycelia), which undergo countless mutations, are mixed, fruiting bodies do not germinate well when cultured while irradiating light in mycelial culture. There are white hyphae and yellow hyphae that are easily pierced by fruiting bodies. This estimation has been confirmed empirically that when white mycelia increase during initial germination of fruiting bodies, the number of fruiting body shoots decreases, and white mycelia become dominant in growth. This is because it has been confirmed that when the white hyphae form a film on the upper part, the number of fruiting bodies decreases because fewer fruiting bodies come out or are completely disturbed.

screening stage

Mycelia obtained from ascospores are used by dilution smearing, and when culturing each independent colony, light is irradiated to exclude white strains, and only yellow or yellow mycelia are selected.

Subsequent incubation steps

Cultivation is continued in a state where yellow or yellow selected colonies predominate.

Harvest stage and packaging stage

As mentioned above, the selected inoculum was a liquid seed fungus cultured in a large volume of 60 to 150 L of triangular liquid [(The nutrient source of the liquid seed fungus is sugar 3% + soybean meal 1% + MgSO₄ / KH₂PO₄ 0.5% each + Antiform 204 (Sigma Aldrich A -6426) 2ml ], and inoculate 15 to 30ml of the seed germ amount to be inoculated into 1 medium bottle at high pressure to evenly inoculate the upper surface of the brown rice medium. The incubation period at ℃ is about 12 to 15 days, and when the dark red fruiting body feet show symptoms, it is moved to the growth chamber, the initial humidification maintains a relative humidity of 85 to 95% at a temperature of 16 to 18 ℃, and the lid is covered with the feet. It is opened and proceeds when the size of the fruit body is about 2 to 3 cm, and the harvest period after inoculation is stable for about 40 to 55 days. The pupal Cordyceps sinensis fruiting bodies that have been cultured are harvested and packed in the required size.

When grown in a brown rice medium (brown rice 80g + water 110ml) in a brown rice medium (brown rice 80g + water 110ml), the number of fruiting bodies is about 40-45g, and when irradiated with light while minimizing subculture In the case of using only the inoculum using the mycelia showing the maximum number of even yellow mycelia, about 60 g of fruiting bodies could be obtained when cultivated by the improved method.

Therefore, it is necessary to manage the spawn when cultivating the pupal Cordyceps sinensis according to the following criteria.

1. Must be aseptically isolated from wild-collected strains.

2. For the first spore isolation, hyphae derived from a single colony are used.

3. Contamination test is conducted for contamination of germs, and contaminants are continuously managed and controlled.

4. For genetic stability, multiple hybridizations should be performed in parallel to obtain a colony with excellent traits and should not be mutated even in subcultures in a genetically stable state.

5. Pupa Cordyceps is a strain that includes an incomplete generation, so attention should be paid to the proliferation and transformation of ascospores.

6. In several analyzes, pupa Cordyceps is proliferated by ascospores in liquid culture, unlike general mushroom hyphae, and the spores generated innumerable in the production of these ascospores are considered to be genetic defects, which is unusual. When light is given after mycelial propagation, there are white mycelia and those that change to yellow mycelia. As confirmed by these mycelial color changes, it was confirmed that the white hyphae did not become germinated, and the mycelia that turned yellow were confirmed to be germinated. In this state, as it was confirmed that abundant mushrooms can be harvested only when the number of fruiting bodies is large, the ratio of white hyphae and yellow hyphae was reviewed. In this process, the ratio of white mycelia and yellow mycelia was confirmed to be approximately 85-87%: 1 among the isolated colonies that were purely isolated. Although the growth rate is small, it was confirmed that after the initial proliferation, the growth rate of the yellow hyphae, which become germs, far exceeded. Therefore, it was confirmed that when the mycelia containing white hyphae were used by subculture, the phenomenon that the rate of emergence gradually decreased.

7. To confirm this phenomenon, ① an attempt was made from strains previously used, and ② multi spores were separated from two specimens of wild-collected strains [L-type (derived from Large pupa), M-type (derived from Middle pupa)] As an attempt from , the ratio of white hyphae and yellow mycelia in those derived from independent colonies representing white bacteria was confirmed to be approximately 15-13% : 85-87% in those derived from independent colonies representing white bacteria each time the passage proceeded. .

8. Therefore, in order to prevent mutation of the pupal Cordyceps sinensis as much as possible, an independent colony of a factor having a pure yellow color should be isolated and used, and the strain should be managed and cultivated by reducing the number of subcultures as much as possible. In this way, when first spore separation and tissue separation after wild collection, when derived from Multi Spore, when passage is performed, mycelia derived from two types of ascospores are mixed, and white mycelia multiply rapidly thereafter. Efforts to reduce the number of subcultures are required in order to ensure good germination. To this end, a sterilized aqueous solution containing 10% Glycerol, 10% SDS, and 10% PEG sterilized solution is prepared and stored in liquid nitrogen at -196 ° C. However, storing in a low-temperature storage at -70 ° C is a stable and optimal method to increase the yield. In addition, storage of ordinary mushroom strains stored in a refrigerator at 4 ~ 8 ℃ and continuous subculture should be avoided.

9. Once pure separation from those derived from Multi Spores and those derived from Mono Cells are obtained by selecting and securing a single yellow mycelial color and spawn line with good germination, from the mycelia of the flat plate stored at a low temperature for a long time, dilution plate method again Even if it is isolated again, it has been proven to have relatively single-phase hyphae thereafter.

<Example>

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

The materials used in this experiment and subsequent experiments are as follows: Autoclave, clean bench, torch lamp, scalpel blade, parafilm, PDA plate medium: PDA 24g/1L, Petri Dish diameter 90mm Disposable sterilization required for microbial manipulation Product, triangular broth composition: sugar 3% + peptone 0.5% + soybean meal 0.5% + MgSO ₄ /KH ₂ PO ₄ 0.5% each, 9ml/Tube of sterilized water to be used for dilution, high-speed homogenizer: rod type detachable blade (sterile condition) ), bacteria transplantation tool, pipette, 75% ethanol, crayfish, face cotton, and PDA plate medium were prepared the day before to keep the inside of the lid free of moisture, and the experiments and manipulations were in accordance with the usual microbial medium preparation method. It was performed accordingly.

Example 1: Test for ratio of white and yellow hyphae derived from ascospores

During the fruiting body growth of Cordyceps militaris, a chrysalis cultivated in Farm E under an unknown number of subcultures, the ratio of white mycelium and yellow mycelia was examined in a Petri dish of tissue separation in a Multi Spore state.

The color of the mycelium was subcultured in a Petri dish, then cultured on a plate with a diameter of 5.0 cm or more, and the experiment was conducted using the coloration characteristics of the mycelia when light was applied.

4 to 5 pieces of hyphae were put in triangular solution culture (250-300ml/500ml Erlenmeyer flask), and cultured with shaking (130 rpm) at a temperature of 24 ° C. for 4-5 days to obtain a liquid mycelial culture medium. The hyphae in the Erlenmeyer flask were crushed into small pieces using a high-speed homogenizer (10,000 ~ 12,000 rpm), and then diluted (1/10, 1/100, 1/1000, ....) by the dilution plate method, and each After the diluted solution was poured onto the prepared Petri dish plate, water was removed from the top of the medium by air volume in a clean bench while the lid of the Petri dish was frozen.

After marking each Petri dish, the upper and lower sides were combined and sealed with parafilm, and the Petri dish was inverted to continue mycelial culture in a constant temperature incubator. On the 2nd or 3rd day, randomly remove one by one of the independent fine hyphae and transplant one (or several) into each new Petri dish plate medium, and irradiate light when the bacteria proliferate in the Petri dish and reach a diameter of 50 mm or more. After dividing white and yellow mycelia, the percentage of white and yellow hyphae was reviewed.

1/10 amount of diluted smear up to 11 times, each liquid is cultured for 1 to 3 days on the plate of a Petri dish, and the colony that came out independently is randomly separated and transplanted to each Petri dish in a new medium and cultured. It was cultured in the dark until the average diameter progressed to 50 ~ 60mm, and then evaluated as follows in a discolored state by applying light.

Light was irradiated on the mycelia cultured on the plate medium of the Petri dish (or the slope medium of the Test Tube), and most of the colors were yellow mycelia, but occasionally the bacteria coated with white mycelia on the surface were confirmed.

Table 1 shows the independent colony color difference in percentage (%) after dilution of ascospores from E farm-derived pupal cordyceps ( C. militaris ) strains.

Yellow Color(Number) Y-W Color (number) White Color(number) from E2 Petri Dish 5 2 from E3 Petri Dish 4 3 from E4 Petri Dish 4 3 some disposal from E5 Petri Dish 9 0 E derived from 6 Petri Dish 8 2 from E7 Petri Dish 9 One from E8 Petri Dish 10 0 from E9 Petri Dish 9 One from E10 Petri Dish 10 One from E11 Petri Dish 11 2 from E12 Petri Dish 11 2 from E13 Petri Dish 11 2 from E14 Petri Dish 9 2 from E15 Petri Dish 9 2 from E16 Petri Dish 10 One dispose from E17 Petri Dish 8 2 Sum of the number of independent colonies 137 26 163 Mycelial color ratio 84.1% 15.9% 100

Example 2: Pupa Cordyceps sinensis from Farm E: (Mark 1208) Tissue isolated fruiting body form

Fruiting body tissues were isolated from the cultivation bottle according to Example 1, and the morphology of the separated fruiting bodies was confirmed. 2 is a photograph showing the shape of a fruiting body. a) is a picture showing the fruiting body from the cultivated bottle, b) is a picture showing the size of the fruiting body when the tissue is separated, c), d) are hyphae separated from the fruiting body of farm E, and white hyphae are mixed. indicates a state of being.

Tissue separation from those with relatively weak buds but a large number of buds, liquid culture in an Erlenmeyer flask from the separated bacteria, and independent colony isolated by the dilution plate method by the above-mentioned method. It was transplanted into petri dishes of dogs, cultivated mycelia, and confirmed by irradiating light when they reached a certain size (50-60 mm in diameter). There were yellow hyphae and white hyphae in the mycelia. Even when left, it was confirmed that white hyphae were mixed.

Example 3: Test for confirming differences in mycelial color in wild species

On July 29, 2017, two wild pupae of Cordyceps sinensis were collected from Goesan-gun, Chungcheongbuk-do, classified into L-type and M-type, and then spores were isolated and evaluated for their characteristics. Figure 3 is a photograph showing the difference in mycelial color germinated by dropping spores in a wild state. a) 2 wild pupa Cordyceps sinensis collected from Goesan-gun, b) M-type wild-collected bacteria (normal chrysalis without a cocoon), c) L-type wild-collected bacteria (large chrysalis in a cocoon), d) State with fruiting bodies attached to the outer upper part , e) inscription of spores dropped by turning

Origin of M type (Middle pupa) of wild collection

After isolating an independent colony derived from a multi spore colony, to determine the ratio of yellow cells and white cells, No. Independent clones of falling spores were counted in the M2 Petri Dish, and the results are shown in Table 2.

Tube dilution M2-5 M2-6 M2-7 M2-8 M2-9 M2-10 M2-11 Number of independent colonies (pieces) 809-1Cell Counting-PDB+A 66 11 7 3 0 0 0 151 809-1Cell Counting-PDAs 60 4 0 0 0 0 0 Total 1Cell Counting Independent colony derived from M2-2P 809-number of transplanted 1-cell colonies Shape evaluation in Petri D.: overall good mycelium/large colony size

In the case of dropping spores, since they have been collected from the wild, it is unknown at what moment the spores will fall. So, attach the fruit body to the top of the Petri dish and rotate the lower PDA medium at random intervals to disperse the falling spores. Several petri dish plates were used to germinate.

Subsequently, in order to determine the ratio of yellow cells and white cells of M-derived independent cells, light irradiation was performed from the 5th day of mycelial culture, and the number of independent colonies that could be evaluated was counted, and the results are shown in Table 3 below.

M0-1 M0-2 M0-3 M0-4 M0-5 M0-6 M-1 M-2 M-3 each sum Yellow-Mono Colony = 86.7% 4 11 4 4 10 8 56 50 23 170 White-Mono Colony =13.3% 0 One 0 0 5 4 16 0 0 26 M spore origin Yellow-Mono Colony (Dogs) White-Mono Colony (Dogs) Mycelial color ratio (%) Yellow-Mono Colony 170 86.7 White-Mono Colony 26 13.3

From the results of Table 3, it was confirmed that the percentage of yellow colonies was 86.7% and the percentage of white colonies was 13.3%. Figure 4 is a photograph showing the hyphae in the plate medium in the state of re-implantation of the independent colony after separation from the spores. a) L type: isolated hyphae before light irradiation, b) L type: white and discolored hyphae after light irradiation, c) example of M/L independent colony separation.

Derived from L type (Large pupa) of wild collection

After isolation of the independent colony derived from the Multi Spore colony, independent clones of falling spore fungi were counted in the L4-2 Petri Dish to determine the ratio of yellow cells and white cells, and the results are shown in Table 4 (L4 = fourth fruiting body among several fruiting bodies). thing).

Example of independent cell confirmation in tube dilution L4--5 L4--6 L4--7 L4--8 L4--9 L4--10 L4--11 Independent colony classification availability and quantity too many
inability to separate too many
inability to separate 5 3 0 0 0 5th day of culture- Derived from L strain, 1 Cell Colony Counting & Light Irradiation 65 18 6 6 2 0 0 Number of independent colonies derived from L4-2Petri Dish 105 (pcs) Evaluation of mycelial morphology on Petri dishes mark example; 83EA/8 Petri D. :: overall hyphae/colony size is small

Subsequently, on the 9th day, the Multi Spore L4-erlenmeyer flask was diluted and smeared, and the number of independent colonies that could be evaluated was counted, and the results are shown in Table 5 below.

L spore origin L0-1 L0-2 L0-3 L0-4 L1 L2 L3 1 Colony
(dog) Yellow-Mono Colony 340 60 76 One 477 White-Mono Colony 62 5 14 8 89 100% M spore origin Yellow-Mono Colony (Dogs) White-Mono Colony (Dogs) Mycelial color ratio (%) Yellow-Mono Colony 477 84.3 White-Mono Colony 89 15.7

According to the separation and culture of 1 Cell of the pupa Cordyceps sinensis ( C. militaris ), even before this test, the majority of yellow hyphae and the small percentage of white hyphae were found. Although the number of fruiting bodies is somewhat small, it has become a foot. However, as revealed in this test, however, the white mycelium is in a state unrelated to the germination of fruiting bodies, and at the beginning there are only a small number of mycelia, so they are inferior, but in the later stage of culture or during the growth process, the proliferation rate proceeds faster, so the white hyphae Mycelia are now more visible. This phenomenon seems to be the reason why white mycelia become more and more dominant when subculture is repeated.

Considering this point, pupal Cordyceps sinensis ( C. militaris ) hyphae should be used while avoiding subcultivation as much as possible, and as an alternative to this, a method of freezing in liquid nitrogen and storing in liquid nitrogen at -196 ° C, or 10% Glycerol or 10 It is recommended to put it in %SDS sterilized solution, etc., and use it while storing it in a storage at least -70℃.

When this method is difficult, it was found that it is preferable to use only one piece of flat plate mycelium after selecting one good Petri dish and use it dozens of times, with a period of about 6 months or less.

5 is a photograph of isolated colonies. a) Example of isolating L-type independent colony, b) Example of isolating M-type independent colony, c) Example of isolating Yellow Type/White Type, d) Example of isolating Yellow Type Petri dish.

Example 4: Fruit body germination test using brown rice medium

In order to examine the effect on the number of embryos of fruiting bodies using a brown rice medium, white light from a fluorescent lamp was irradiated during cultivation to confirm the presence of germs. 6 is a photograph showing the appearance of the finbal before and immediately after. a) The appearance before the pinning, b) The appearance immediately after the pinning.

In the brown rice medium, when the hyphae cover the upper part and then germinate by light irradiation and have to penetrate to the upper part, when the white hyphae are vigorous and the mycelial layer becomes thick (because there are relatively few yellow hyphae that germinate), the number of fruiting bodies is small. It is a direct cause of losing. If the number of fruiting bodies is small at this time, the number of fruiting bodies will eventually be small even if growth is terminated.

As mentioned above, it was confirmed that uniform germination proceeded even in a large-capacity basket medium when there were no germs in the spawn and there were as many yellow-colored hyphae as possible.

Therefore, among the various variables in the cultivation of pupa Cordyceps sinensis, it can be evaluated that the management of the spawn is essential, firstly, that there is no mixing of various germs, and as the second important factor, it is essential to cultivate only mycelia with good germination. However, it should be noted that in the process of subculture or liquid culture rich in nutrients, the rate of subsequent generation of white mycelia inevitably occurs at a rate of 13.3 to 15.9%, and the growth rate of white mycelia is fast. Considering this point, spawn management is important, and other than that, pollution prevention in cultivation should be focused.

From this point of view, there is no doubt that it is a technique that can produce a higher number of fruiting bodies than other methods.

Example 5: Growth test of brown rice culture bottle

In order to confirm that the unit production of fruiting bodies is increased by using non-contaminated strains and seed fungi that reduce the number of passages and maintain a single color of yellow hyphae, about 80 g of brown rice is used in an 850 ml thin PP culture bottle (bottle inlet cap diameter 65 mm). , 110ml of water was added to compare the weights of fresh fruiting bodies using brown rice medium in culture bottles. The results are shown in Table 6.

traditional cultivation This implementation method sickness 1397 bottles 2880 bottles Fresh fruit body weight (kg) 56.3kg 179kg Average fruit body weight (g)/1 bottle 40.3g/1 bottle 62.2g/1 bottle Average fruit body weight (g)/16 bottles 644.8g/16 bottles 995.2g/16 bottles

From the results of Table 6, although there is a time difference and a slight difference in the cultivation process, productivity per bottle unit can be increased by improving the overall cultivation process.

When such spawn management was thoroughly performed, there was a clear increase in yield compared to other farms, and mold and the like were not prevalent in the fruiting body until the end of cultivation, and in the process of cultivation using brown rice medium, good results were obtained until the 55th day after inoculation. status was indicated.

Example 6: Growth test using basket type (plate) medium

The growth status of the fruiting bodies using brown rice + pupa powder was observed on plate medium.

7 is a photograph of a fruiting body on a plate medium according to growth conditions. a) the early stage of fruiting body growth on a plate b) the middle stage of growth of a fruiting body on a plate medium, c) the late stage of growth of a fruiting body on a plate medium, d) the final stage of fruiting body growth on a plate medium.

When using a plate culture basket, if there is a burden of contamination, you should never use it. This is the best cultivation technique as it must be done under the conditions of perfect cultivation without contamination.

Cultivation in a plate medium not only increases the space other than the bottom area of the bottle by 30% compared to that in the bottle, but also increases the number of fruiting bodies, and it is useful to reduce the cost of cutting the bottle to take out the fruiting body at harvest. As a method, spawn management as in this study is a very useful method.

Example 7: Growth test using basket type (plate) medium

The average weight (g) of net fruiting bodies of one basket was evaluated in a medium containing pupa Cordyceps sinensis brown rice + pupa powder, and the results are shown in FIG. 8 and Table 7. 8 is a photograph showing the quantity (g) of live fruiting bodies that have been cultivated using Panbagu.

Plate basket fresh fruit body weight (g) Plate basket cultivation No. 6 Plate basket cultivation No. 7 1 basket (g) 1366.5g 1351.0g Weight per bottle (g) 85.4g/ 1 bottle 84.4g/1 bottle

The number of fruiting bodies grown in 6 baskets was almost the same, with No. 6 and 7 are average quantities, and cultivation was possible without contamination during the growth process, and the average fresh weight of the fruiting body also had a water increase effect of about 30% compared to the state of containing 16 bottles.

Example 8: Contamination test of liquid spawn and spawn screen work

The following strains of the pupal cordyceps strain were used.

* PP strains:

No. 1 to No. 39: Select M2 / M4 / M17 / M123 as the first / second speculum

* E strain:

Petri dish passaging: presence of 7-8 types of severely contaminated bacteria, 2 Petri dishes in the 1st, 1 Petri dish in the 2nd

Tissues were isolated from self-grown fruiting bodies after self-cultivation derived from the stomach.

*Y strain:

Petri dish passage: presence of 7 to 8 types of severely contaminated bacteria, tissues derived from 2 (middle) fruiting bodies grown and brought were isolated.

* S strain: Derived from fruiting body

Tissue Separation 1st: 4 vials (medium) S11/S12/S16/S20/S21/S24/S25/S27 grown and brought were selected. Tissue Separation 2nd: It was separated from the fruiting body of the fractionated culture medium.

* G strain: After bringing one mycelia culture bottle, tissue derived from the fruiting body was separated (first) by self-growth, and about 10 others were selected.

* New strains: Pure strains isolated in 2020.

Regarding liquid spawn, reference materials [Sim Kyu-gwang et al. (2012, Korea Mushroom Society: Pre-inoculation inspection of enoki mushroom liquid spawn; Journal of the Korean Mushroom Society, 2012, Vol 1, p44-48, Shim Kyu-gwang, Yoo Young-jin, Koo Chang-deok, Kim Myung-gon] Contamination of the liquid spawn was tested by applying the microscopic method according to the described method.

The test tube culture solution was smeared on a slide glass and Giemsa Stain Sol. It was examined through the staining process, and when stained with Giemsa Stain Solution, Bacillus spp. or coccal fungi can be easily distinguished. As in this application example, when using liquid spawn before inoculation, contamination before inoculation can be inspected by evaluating only the presence or absence of contaminating fungi.

In the liquid spawn test, in order to evaluate the number of contaminants by microscopy before inoculation of the liquid spawn, the relative number of contaminants was defined as follows to set the criteria for overall evaluation.

As a method of inspecting liquid spawn in a short time by microscopy before inoculation, as shown in the example shown in Figure 9 below, 22,000 observations should be made to see the entire area of 1 slide glass, but in the case of pupal cordyceps, incomplete fungi It is difficult to observe because it produces ascospores, and in particular, the frequency of contamination of spawn conditions, which have been managed by previous growers without a case of applying quantitative techniques to most spawn management, is very serious.

It takes too much time to view the entire area of the slide during the screening process for pure separation of contaminated spawn cells. Therefore, 220 horizontal lines are observed among the entire area of 1 slide glass, and the number of suspected bacteria is counted for relative evaluation. That would be one way too. 9 is an explanatory diagram of the number of times of a microscope (objective lens 100X, oil immersion) of 1 slide glass for a microscope.

As shown in FIG. 9, at the time when the liquid spawn is cultured in a large amount and the density of the spawn is high, only 0.1 ml of this culture medium is smeared on a slide glass, and it can be used as relatively quantified data. Among the existing liquid spawn contamination test methods, various methods may be performed, but the most accurate among them may be a method of confirming the contamination on a 100X objective lens under a microscope.

After collecting the pupa Cordyceps sinensis, it is common to see that there are many cases in which finely infected strains have already been established in general farmhouses due to lack of awareness of the work of pure separation, lack of technique, and lack of observation. In particular, wild pupa Cordyceps fungi collected from high-protein larvae or pupae (e.g., snow flower Cordyceps sinensis, pupa Cordyceps cultivars) are cultivated without proper isolation work. It is confirmed that 7 to 8 types of various contaminating fungi are present.

In the case of general bacteria ( Bacillus spp. ), which are usually highly infective, they are expressed in various forms (odor, turbidity, foam, rapid increase of CO₂ during aeration culture, etc.), but contamination by microscopic numbers, In the case of various forms of contamination, where the size of microorganisms is much smaller than that of common contamination causative bacteria (bacillus), it is impossible to confirm other than microscopic examination.

Example 8-1: Examination of Pupae Cordyceps strains

The strains of Petri dishes stored in Puri Mulsan Co., Ltd. (PP company) were assigned serial numbers by date (No.1 to No. 39). In addition, after classifying the petri dishes in refrigerated storage by passage date, subculture was performed on a flat plate Petri dish for each date, subculture for a second plate Petri dish, and test tube liquid passage for a third time.

After inoculating the pupal cordyceps in the liquid culture medium of the Erlenmeyer flask, the mycelia proliferated while shaking at 130 rpm, fractionated a certain amount of liquid, spread it on a slide glass, dried it, and gave Giemsa Stain Sol as a concentrated solution. The solution was dyed for 40Sec to 60Sec, washed with clean water, and then the slide glass was dried again.

In the optical microscopy, first focus was performed with an objective lens of 10X to 40X, then mineral oil was dropped, and an accurate image was focused with an objective lens of 100X, and then the slide glass as shown in FIG. 10 was moved and observed. 10 is a photograph showing contaminating bacteria. a) Hyphae of the pupal Cordyceps fungus b) Ascospores and double septa hyphae of the pupal Cordyceps fungus.

At this time, the fungi that mainly cause contamination are bacillus ( Bacillus spp.), cocci ( Coccus spp.), and occasional mold fungi.

Example 8-2: Experiments of pupal cordyceps strains

It can be seen that as the number of passages progressed toward the latter half of the year, the number of suspected contamination bacteria increased relatively more. This suggests that the number of suspected microorganisms overall increased every time the number of passages increased in the Petri dish plate medium.

In this way, in the process of selecting a lot with a small number of suspect bacteria and good fruiting body development, a lot with a well-developed mycelial color was selected.

About 39 lots stored by the PP company were stained with Giemsa Stain Sol. and examined for microscopic examination. Contamination was confirmed in all strains, and the number of passages tended to increase as the number of passages increased.

Pupal cordyceps strains are fastidious strains in which, in addition to normal (basic) hyphae, another reproductive process of ascospore-derived hyphae (flowering) exists. As a rule of thumb, with the addition of the characteristics of colored strains, strains with better color change have better germination characteristics. As such, the color characteristics of hyphae are closely related to light irradiation, and abundant yields of fruiting bodies are induced. Confirmed.

Example 8-3: Confirmation by microscopy

Figure 11 is a photograph of the form of contamination-causing bacteria generated in the characteristics and production of the snow flower cordyceps / pupa cordyceps strains. The hyphae of Cordyceps sinensis are rarely found in the form of ascospores mentioned in this study. However, in the pupa of Cordyceps sinensis, numerous ascospores can be identified at the beginning of culture when liquid culture is performed.

In this way, the degree of proliferation of cordyceps into ascospores varies depending on the change in the environment, and fungi derived from ascospores eventually become a force and appear to show strong power during the vegetative growth period, but most fundamentally During spore separation/tissue separation, there may be cases where it is started in an incorporated state that is not derived from 1 cell. Hyphae derived from ascospores may form a white mycelial film later due to various problems such as other activities, and may appear as a phenomenon that interferes with pinning. is genetically defective, but it is presumed to have the characteristics of better proliferation of mycelia, so it seems necessary to separate their hyphae from pure.

Example 8-4: Confirmation test for the presence of mixed mycelia in mycelia

Antrodia cinnamomea is a fungus unique to Taiwan, also called Antrodia cinnamomea. It grows within the rotten heartwood of Cinnamomum camphora, which mainly inhabits the mountainous regions of Taiwan at an altitude of about 450m to 1500m above sea level. It was confirmed that other hyphae could be incorporated using Ujangji mushrooms.

As for the above variety, the separation was attempted by the dilution plate method and the independent mycelia were separated. There were no germs affecting mycelial culture, and they were separated into white mycelia and yellow mycelia as follows. Figure 12 is a photograph showing the mycelia of the mushroom mycelium [a) mixed state of mycelium of mycelium (mushroom), b) separation of two types of mycelia from mycelia of mycelium].

As in the experience of completely separating two types (white and colored mycelia) into two from the mushroom mycelium, it may be necessary to increase the ratio of yellow hyphae in the chrysalis Cordyceps sinensis mycelia and yellow mycelia in white mycelia. Since it is the same, there is a need to separately separate the two mycelia of different species.

Therefore, the presence of white mycelia in different lots of pupa Cordyceps sinensis was confirmed as follows by collecting varieties stored on its own or obtained from nearby cultivation farms.

It was also confirmed that white mycelia and yellow mycelia were mixed in the pupa of Cordyceps sinensis. 13 is a photograph showing an example of the separation of two types of mycelia from the pupa Cordyceps sinensis [a) different color aspects in the liquid culture fraction, b) incorporation of white mycelia in a Petri dish]. 14 is a photograph showing another example of separating two types of hyphae from the pupa Cordyceps sinensis. 15 is a photograph showing another example of separating two types of hyphae from the pupa Cordyceps sinensis.

Therefore, when hyphae coexist, it is important which hyphae develop into fruiting bodies. If the mixed mycelia are separated and the symmetrical culture test is performed, the growth rate of white mycelia proceeds very quickly, preventing the proliferation of yellow mycelia.

16 is a photograph showing an example of separating two types of hyphae from the pupa Cordyceps sinensis. a) E isolated strain-white mycelium growth is faster, and if subculture is continued, strong strains eventually dominate, b) Competitive culture after isolation from E strain: Measure the same amount of yellow mycelium + white mycelium After transplantation, only the white mycelium proliferated strongly and a dominant state was observed.

As shown in FIG. 16, it was found that white hyphae coexist with yellow hyphae in most lots. The growth rate of yellow mycelia and white mycelia cultured on the plate of the same Petri dish was rapidly progressing, and when these mycelia were cultured in the center of one place, in the end, white mycelia proliferated more strongly. Since there is a phenomenon, as the number of times in the passage of spawn is repeated, the yellow mycelium, which finally germinates well, degenerates, and only the white mycelium growth tends to prosper better.

These results confirm that white mycelia appear more and more vigorously after they are actually cultured and germinated.

Until now, no academic document published on this principle has been reported, and no discussion on this mechanism has been made. However, since it can be recognized empirically, for this reason, the production of fruiting bodies decreases as the passage progresses.

Even though intensive time is given to spawn management when passages are performed and mycelia culture is carried out again, it is not easy to completely separate white mycelia and yellow mycelia as long as there are ascospores like pupa Cordyceps sinensis.

17 is a photograph showing the elongation (proliferation) of hyphae and ascospores according to microscope magnification.

It was confirmed that microscopic fungi exist in the form of contamination causative bacteria (classes) isolated from fruiting bodies grown by culturing petri dish strains and hyphae of pupa cultivars of Cordyceps sinensis cultivated in nearby farms. As mentioned, even if there is a pollutant intervening between the mycelia in such fine detail, in mushroom cultivation, there are farmhouses that grow it close to the end of growth. However, it is possible to increase the number of fruiting bodies per bottle by establishing perfect aseptic strains in these varieties and making efforts to use a single yellow mycelium derived from an independent colony.

Figure 18 is a photograph showing the form of specific contaminants incorporated in the liquid culture of Cordyceps pupa. It is a bacillus of the Bacillus (general bacterium: Bacillus spp.) type and belongs to a relatively large class of 1-cell size among bacteria that cause contamination. Bacillus is one of the most powerful proliferative bacteria, and shows a relatively large size with an objective lens (100X, oil immersion). It should be noted that human visceral symptoms (e.g. odor, clouding, air bubbles) may not be present.

Example 8-5: Microscopic method by optical microscopy by staining with Giemsa Stain Sol.

There are a variety of specific fungi related to contamination, and these fungi could not be identified. 19 is a photograph showing microscopic strains involved in contamination. As shown in this picture, special attention is required for contaminants that form a colony. As there is no document mentioned so far about the type of contaminating bacteria that is labeled as putrefying bacteria, the present developer developed a large-capacity medium for the above types of causative bacteria after harvesting wild wine from morel mushrooms or during the separation process of champignon mushrooms. It has also been verified in the medium and spawn manufacturing process of So far, when cultured in small amounts, the form of putrefactive bacteria cannot be detected, or when 1 unit of champignon (shiitake) medium grows to 12 ~ 13.5kg, the mycelium is weakened due to deterioration. Contamination is more serious It is a fungus that causes decay that has been confirmed to appear.

Figure 20 is a picture comparing the size of the fungus causing mushroom decay and the microscopic fungus. 21 and 22 are photographs of spoilage-causing microscopic fungi. (Super) microscopic fungi, causative fungi that cause decay, fungi that cause decay, and fungi that cause ultra-microscopic fungi.

The most basic of these is one of the most difficult tasks when isolating an independent colony of strains collected outdoors, but the biggest cause is the result of poor meticulous work, and there is a need to clarify the passage management technique of strains afterwards.

As described above, there may be differences in the varieties of pupa cordyceps ( C. militaris ), but when managing the petri dish by applying the optimal method, the mycelial color (yellow -> dark yellow) by irradiating light from the PDA flat medium of the petri dish As a technique for obtaining a single strain that changes rapidly and uniformly, the number of fruiting bodies can be increased by homogenizing the strain and using the diluted smear method.

Example 9: Verification of separation by dilution plate method

In this example, two of the high-quality lines (Lot E-No. E28 & E-No. 33) with good extraction and proven quantity were selected and confirmed again by the dilution plate method. 23 is a photograph explaining that yellow mycelia are also shown in lots showing yellow mycelia derived from Mono Spore even after refrigerated storage for a long period of time.

White mycelia and yellow mycelia were separated from the E-series pupa Cordyceps sinensis, and from those in the flat Petri Dish state left at 4 ° C for a long time, two lot numbers were selected from those that were classified as yellow mycelia and recorded as having good germination. . As shown in FIG. 23, Lot E-No. E28 & E-No. 33 was performed by the dilution plate method described above. The E-28 Petri Dish was stored at low temperature for a long period of time with a good Lot No. selected for the first separation, and then separated and verified again by the dilution plate method. 24 is an initial photograph of cancer culture after transplanting an independent colony to a new medium. 25 is a photograph showing the progress of light culture after transplanting an independent colony to a new medium (8th day after transplantation / 3rd day of light irradiation). 26 is a photograph showing the appearance of white hyphae in independent colonies. White mycelia appeared at a frequency of 1/494 in independent colonies (top: 3rd day of light irradiation, bottom: 6th day of light irradiation). After storage, triangular culture was performed from the old E-28 Petri Dish, and after homogenization, one White mycelium of the E-28-17 Petri Dish obtained by the dilution plate method was confirmed (1 white / 494 yellow = 0.2% ). 27 is a photograph showing the state of mycelia in the process of being cultured by light irradiation (light culture) after culture on the 8th day (dark culture) after transplantation into Petri Dishes.

Triangular cultures were performed from the above Petri dishes, which were noted to have good germination. The triangular solution is about 200ml, and a magnetic magnet is placed inside and cultured for 2 to 3 days while rotating. E-No. E28 two days later, E-No. 33 is homogenized at 10,000 rpm with a high-speed homogenizer after 3 days, and when independent mycelial colonies are seen by the aforementioned dilution plate method (diluted 10 to 11 times), each is separated with mescal so that they are not adjacent to each other. Incubated and then irradiated with fluorescent light. Looking at the ratio of discoloration by irradiation of light, a total of 494 were transplanted and confirmed. The ratio of white hyphae to yellow hyphae was 1/494=0.2%, and good hyphae in a very stable state were obtained.

By this method, a total of 494 independent colonies were transplanted from the pure yellow hyphae to each new plate by the dilution plate culture method in the two openings of the Petri dish of E-28 & E-33. As a result of culturing the mycelia of the transplanted independent colony on a petri dish plate and then culturing the mycelium to check the color of the mycelia, it was confirmed that only 1 out of 494 appeared white (99.8% of the hyphae rate of yellow that could be easily spread was 99.8%). ) became Therefore, it has been proven that when producing strains after collection, selecting those derived from mono spores rather than multi spores and using them as spawn strains affects the increase in the number of fruiting bodies.

Example 10: Isolated culture of independent colonies

E-28 Petri Dish Yellow-derived independent colony was inoculated into triangular fluid using one hyphae, shaken culture at 24 ° C and 130 rpm, and high-speed homogenized mycelia were aerated cultured in a 9L glass bottle, and on the 4th day of aeration culture After fractionation, add 80g of oats + 110 to 120ml of water, sterilize, and inoculate the aerated cultured liquid mycelia in a cooled container, and inoculate the oat medium and irradiate the mycelia from the beginning to cultivate the mycelia. Primary symptoms on the 12th day of cultivation in yellow mycelia This was confirmed and evaluated in an almost pure isolated state.

28 is a photograph showing the culturing process of independent colonies. 29 is a photograph confirming symptoms of primary lice.

As shown in FIG. 29, even if there is only yellow mycelium showing yellow mycelium, there is no problem in producing fruiting bodies, and white mycelium forms a white film on the upper part of the medium to interfere with (second) germination of mushrooms This has been confirmed during the cultivation process.

As described above, the present invention has been described by the limited embodiments and drawings, but the present invention is not limited to the above embodiments, and those skilled in the art in the field to which the present invention belongs can make various modifications and transformation is possible Therefore, the spirit of the present invention should be grasped only by the claims described below, and all equivalent or equivalent modifications thereof will be said to belong to the scope of the present invention.

Claims (4)

a) subculturing the pupal Cordyceps sinensis;
b) irradiating light to mycelia derived from ascospores of the pupa Cordyceps sinensis after culturing;
c) screening only mycelia containing yellow or yellow mycelia from ascospores containing white mycelia and yellow mycelia by dilution smearing and excluding white strains to select single colonies;
d) continuing the culture in a state where the selected colonies dominate; and
e) harvesting the cultivated pupa Cordyceps sinensis fruit bodies and then packaging them in the required size;
The step of a) subculture is performed by liquid culture in an Erlenmeyer flask using the tissue separated from the parent strain to minimize subculture,
In the step b), the mycelia are grown by transplantation and cancer culture, and when the diameter of the white hyphae becomes 0.5 to 2.0 cm, light is irradiated to proceed with light culture,
The culture in step d) is cultured by light culture from the beginning, and the incubation period is 12 to 15 days at a temperature of 24 ° C. Humidification maintains a relative humidity of 85-95%, and when the size of the fruit body is 2-3 cm, the lid is opened and the harvest time is 40-55 days after inoculation. The culture medium is silkworm, pupa, and brown rice and oat. delete delete delete

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