KR100339701B1 - Inonotus obliqua strain, method for separating it, and method for mass production of its fruitbody - Google Patents

Abstract

The present invention relates to chaga mushroom strains capable of artificial cultivation, rapid growth of mycelia and fruiting bodies, high quality and high yield, isolation methods thereof, and methods of mass production of fruiting bodies.

Description

Chaga mushroom strain, its isolation method and fruiting body mass production method of the strain {Inonotus obliqua strain, method for separating it, and method for mass production of its fruitbody}

The present invention relates to a novel chaga strain, its isolation method, and a method for mass production of fruiting bodies of the strain.

In the Kamchatka Peninsula, Russia, natural chaga mushrooms are collected and their extracts are used to treat digestive cancer and diabetes. A wild chaga mushroom in Hokkaido, Japan, developed drugs used for the treatment and prevention of AIDS virus, digestive cancer, and diabetes. However, chaga has not been artificially cultivated so far, and the artificial cultivation method using chaga mushroom strain has no technology developed at home and abroad.

Therefore, the present invention was to develop a method for isolating chaga mushroom strain capable of artificial cultivation and mass production of fruiting body with the strain. In other words, we tried to establish a mass production system by developing artificial environmental composition and propagation method of chaga mushroom which has been used for medicinal purpose by collecting natural mushroom.

Figure 1 shows the comparison of mycelial growth by Chaga mushroom medium type.

Figure 2 shows the mycelial growth by Chaga mushroom culture temperature.

Figure 3 shows the color of chaga mushroom hyphae.

The present invention provides a chaga mushroom strain capable of artificial cultivation, high mycelial growth and fruiting body formation rate, high quality and high yield, separation method and mass production method of the strain or fruiting body.

The scientific name of chaga is Inonotus obliqua (pers) pilot, Fuscoporia obliqua Aoshima. In general, chaga mushrooms are subcultured after mycelial separation, so mycelial forces have fallen sharply and are not killed or proliferated. Generally, as a method of separating mushrooms, spore separation and tissue separation methods are provided. However, chaga is characterized by a weak mycelium even when spore separation or tissue separation is weakened the vitality of the mycelia during subculture, and eventually disappear.

Therefore, the present invention performs a method consisting of the following steps to isolate the strain of chaga can be artificially cultured: after immersing the spores or tissues of chaga in sterile water for more than 24 hours in the culture chamber of 27 ~ 32 ℃ Taking out; Putting dry cotton wool in a container for 8-10 hours at 140-160 ° C. for dry heat sterilization, and immersing in 27-32 ° C. incubation room for at least 24 hours and then removing it; Chaga mushroom tissue or spores in the dry heat sterilized container step again to 25 to 30 ℃ incubation temperature for 8 to 50 hours. Mycelia of chaga isolated by the above method can recover the forces, the mycelial vitality is vigorous, even after inoculation to the logs, the degree of fruiting body formation.

Although it was not successful in the fruiting body artificial cultivation for chaga mushrooms with low mycelial activity all over the world, chaga mushroom strains isolated from the above according to the present invention successfully formed fruiting bodies in eight species.

Chaga is a fungus belonging to the family Hymenochaetaceae , forming a large fungal nucleus in the form of a tube of black birch. It is a germ nucleus of pathogens that parasitic on the trunks of birch trees, causing white swelling. Mycobacterium forms a black surface, with many cracks on both sides , and yellowish brown inside, similar to Phellinus linteus . The diameter of the bacterium is large, about 10-20 cm. The part used mainly for medicinal use is mycelia. The fruiting body has a thickness of about 2 ~ 8mm and there are holes in the front, and there are several bristle bodies. The color of spores of chaga is light brown, and the size of spores is about 7 ~ 10 (㎛) × 6 ~ 8 (㎛). Naturally occurring areas have been reported in Russia and northern Japan. Mountain has been used as an anticancer agent in Japan and Russia. In Japan and Russia, it has been reported that the cancer of the digestive system has the effect of anticancer, not an increase in immune activity, and is known to be effective against the AIDS virus.

Chaga is a mushroom that has not yet been found in Korea, and the present inventors introduced chaga mushrooms from Kamchatka Peninsula, Russia and separated the vital mycelium from the fruiting body after enhancing mycelial activity by constructing a sterile environment, and among them, chaga bacteria KNAC 3002 strain was deposited microorganisms (Accession No .: KFCC-11190).

Chaga strain isolated according to the present invention can be mass-produced fruiting body when inoculated in various species such as cherry, jujube, river oak, oyster oak, silver fern, acacia, white birch in addition to black birch.

The microorganisms deposited chaga ( Inonotus obliqua ) KNAC 3002 (KFCC-11190) is Potato Dextrose Agar (PDA: Potato 200g, Dextrose 20g, Agar 20g / 1ℓ), Birch Dextrose Agar (BDA: Birch sawdust 200g, Dextrose 20g, Agar 20g / 1ℓ), Oak Dextrose Agar (ODA: Oak sawdust 200g, Dextrose 20g, Agar 20g / 1ℓ) medium, the optimum pH is 6.0, sterilization conditions 121 ℃, 20 minutes (in vitro). The culture conditions are aerobic, the culture titration temperature (℃) is 30 ℃, both shaking or standing (liquid, solid) culture is possible.

Inonotus obliqua KNAC 3002 (KFCC-11190) is characterized as follows.

1. Culture Characteristics

The medium is black birch extraction medium. The titration temperature is 30 ℃ and the mycelium is killed above 35 ℃. It is not killed at low temperature.

2. Morphological features

The morphology of chaga mushroom hyphae has a clamp connection in the colonies, and especially in the malt extraction medium (MEA), the colonies are initially yellow and later black. In medium other than malt extraction medium, colonies are initially white and later black. Chaga mushrooms form basidiospores in basidium (Basidium) to reproduce sexually. In addition, basidiomycete and spore spores are formed on the hymenia of Chaga mushroom, which is the Basidiocarp. Asexual reproduction of chaga produces conidia and dividers (Oidia) in poor culture. Dividers are formed in 1-nucleus mycelium to fuse with mononuclear mycelium to form binuclear mycelia. The septum of chaga mycelium has dolipores.

The present invention will be described in detail through the following examples.

Example 1 Isolation Method of Chaga Mushroom Strains

Spores and tissues of chaga were immersed in sterile water (sterilized for 60 minutes at 121 ℃) for 24 hours in a 30 ℃ culture chamber and taken out. Dry cotton wool was put in a container with a lid, and dry heat sterilized at 140 ° C. for 10 hours, and then immersed in a 30 ° C. culture room for 24 hours in sterilized water (sterilized at 121 ° C. for 60 minutes) and taken out. Chaga tissue or spores were placed in a dry heat sterilized container and left to stand at 25 ° C. incubation temperature for 48 hours. Mycelia of chaga isolated in this way was able to recover the forces. In addition, the chaga fungi treated with these treatments had a strong mycelial activity, and even after inoculating the logs, the degree of fruiting was increased.

Example 2 Mycelial growth and density according to the type of medium of chaga bacteria (see FIG. 1)

As a result of looking at the mycelial growth according to the type of medium as shown in Table 1 among the chaga mushrooms isolated in Example 1, the culture temperature of 25 ℃, GNA medium KNAC 3001 strain is another chaga strain isolated by the present inventors Mycelial growth and density were the highest at 63mm / 10 days at. Next was PDA, CDA, ODA, BDA, YM, MCM, PODA, CHA, MEA (pH7.0), MEA (pH4.7). KNAC 3002 strain had the best mycelial growth and density at 48.0mm / 10 days in BDA medium, but mycelial growth rate was significantly slower than KNAC 3001 strain. It was followed by ODA, PODA, MCM, CDA, YM, PDA, GPA, CHA, MEA (pH 7.0) and MEA (pH 4.7). In CHA, mycelial growth was the fastest at 30.7mm / 10 days, but mycelial density was very low.

PDA: Potato Dextrose Agar; Potato 200g, Dextrose 20g, Agar 20g / ℓ

MEA: Malt Extract Agar

CDA: Compost Dextrose Agar

BDA: Birch Dextrose Agar; Birch sawdust 200g, Dextrose 20g, Agar 20g / ℓ

ODA: Oak Dextrose Agar; Oak sawdust 200g, Dextrose 20g, Agar 20g / ℓ

PODA: Popla Dextrose Agar

MCM: Mushroom Complete Media

YM: Yeast Extract Media

CHA: Chapecks Agar

GPA: Grucose Peptone Agar

* Mycelial density: +; Weak ++; Usually +++; Good

* 9cm Petri dish

Example 3 Mycelial growth and density according to the temperature of chaga bacteria (see FIG. 2)

Chaga is a fungus that grows in cold regions above 45 degrees north latitude, but mycelial growth is unique at 30 ℃. The KNAC 3001 strain was 30 mm / 10 days in PDA medium at pH 6.0 and had the best mycelial growth and density. By temperature, mycelial growth rate and density were good in order of 25, 20, 35, 15, 10, 5 ℃. KNAC 3002 strains showed slower mycelial growth than KNAC 3001 strains, but showed the same trend. At 40 ℃, chaga did not grow and cultured again at 25 ℃, but did not grow and died. It grows at a low temperature of 5 ℃ but is very weak.

* Mycelial density: +; Weak ++; Usually +++; Good

* 9cm Petri dish

Example 4 Mycelial Growth and Density According to Acidity (pH) of Chaga

The pH was adjusted by addition of 1.0N NaOH, HCl, the preferred acidity of mycelial growth of Chaga mushroom at incubation temperature of 30 ℃ is 5-9 and the appropriate acidity is 6.0. The pH peaked at 80.7 and 40.3 mm / 10 days, respectively. The growth and density of Chaga fungi were significantly reduced at or below pH 6, and the strain KNAC 3002 showed half the low growth rate in synthetic medium compared to KNAC 3001.

Mycelial growth and density (mm / 10 days) according to acidity (pH) of chaga Acidity 5.0 6.0 7.0 8.0 9.0 10.0 Mycelial Growth Mycelial density Mycelial Growth Mycelial density Mycelial Growth Mycelial density Mycelial Growth Mycelial density Mycelial Growth Mycelial density Mycelial Growth Mycelial density KNAC3001 59.7 ++ 80.7 +++ 75.3 +++ 69.3 ++ 66.0 ++ 58.0 + KNAC3002 24.0 +++ 40.3 +++ 32.7 +++ 31.3 ++ 26.7 ++ 23.3 ++

* Mycelial density: +; Weak ++; Usually +++; Good

* 9cm Petri dish

Example 5 Mycelial Growth According to Sawdust Types of Chaga Bacteria

In order to prepare the chaga fruit body mass production system, 8 kinds of sawdust were inoculated with mycelium, and the rate of mycelial growth was measured every 5 days.The results of cherry tree, jujube tree, river oak, oyster oak, silver ash tree, akash tree The mycelial growth rate was higher in the order of wood and black birch, but in Sawdust medium, KNAC 3002 strains showed significantly more mycelial growth than KNAC 3001 strains. The original host of chaga bacteria is black birch. Black birch grows in some parts of Gangwon-do in Korea, but it is very rare. The mycelial growth was the slowest in black birch, the host, but the fruiting body formation was the best.

Example 6 Log Selection of Chaga Mushroom Cultivation

Black birch trees have the highest rate of mycelial growth, but they are best for fruit body formation and yield. The diameter of wood is about 15cm and the length is 20cm. The moisture content of wood is 42 ~ 45%, so if you log wood in winter and dry it for 3 ~ 4 months, you will get proper water content.

Example 7 Sterilization for Chaga Spawn Inoculation

To inoculate the spawn, prepare 8 to 12 holes per piece of wood with a diameter of 12 mm and a depth of 2 cm. Then, place a piece of wood in a high-density heat-resistant plastic bag and block it with a stopper containing a sponge or cotton in the upper layer. Sterilize by autoclaving or autoclaving. Atmospheric pressure sterilization is carried out for about 13 to 15 hours (based on log size 15 X 20cm) after raising to 100 ℃ by steam and heat. High pressure sterilization is 97 ~ 100 ℃ after 110 ~ 130 minutes by opening the exhaust valve and adding boiler steam. At this time, if the exhaust valve is completely opened to remove the air bag in the sterilizer and the boiler is continuously operated for 50 to 60 minutes, the temperature becomes 118 to 120 ° C. From this time, with the exhaust valve slightly open, sterilize the logs for 9 to 10 hours and soften it for good mycelial growth. After sterilization, when the temperature drops to about 90 ℃, take out the wood immediately to prevent the surface of the wood.

Example 8 Chaga Inoculation

In the aseptic inoculation room, the logs are inoculated at the same time as the temperature of the logs drops to around 20 ℃. The inoculation amount of sawdust spawn is 80 ~ 100g per bag, so it is important to inoculate enough to cover the surface of logs.

Example 9 Chaga Mushroom Log Culture

Chaga mushroom log culture room temperature is maintained at 25 ± 2 ℃. 30 days after incubation, the mycelial activity becomes vigorous. Pollution occurs when the stopper is wet or in poor contact. It takes 61 ~ 72 days to complete mycelial culture, but mushrooming is better if cultured for 20 days more after completion of culture.

Example 10 Chaga Mushroom Log Generation

The growers for chaga logs are to be insulated like the existing simple oyster mushroom growers, but the bottom should be well-drained soil with good drainage, clean soil free from pathogens, and three ventilation windows per building. Chaga mushroom alleys are erected and buried, but the depth is buried 70% of the total length and 30% exposed to the ground. The exposed cut surface is easy to dry, so to prevent this, clean Masato is placed 2cm thick to maintain humidity. During this period, the temperature is 30 ± 2 ℃, the humidity is over 90%, and the carbon dioxide content is not ventilated at the time of chaga generation.

Example 11 Chaga Mushroom Growth

Chaga mushroom growth environment management is important water management and ventilation management. The water management is sprayed once every two days in spring, once a day in summer, once every three days in autumn, and without water in winter. Ventilation management should be managed not to exceed 3,000ppm, but only necessary in summer.

Example 12 Chaga Mushroom Harvesting and Drying

The timely harvesting time for chaga mushrooms, which are full of fruiting bodies, is 2 years after mushroom development. However, it is incomplete but can be harvested after 6 months. Drying method is hot air drying and it keeps at 40 ± 5 ℃ for 20 hours to become a dry product.

Example 13 Fruiting Body Yield According to Logical Species of Chaga Bacteria

The growth rate according to the wood species of chaga was consistent with the test of sawdust species, and the incubation period was 61 days because it was the fastest in jujube tree, and 72 days in black and white birch trees. It was the longest. The initial number of days required was 30 to 40 days, and the fruiting period was 18 to 22 days. Yield can be 43 ~ 65g (dry weight) per piece of wood (10x20cm), the highest in black birch.

* Solid Wood: Diameter 10 × 20cm

Chaga mushroom strain isolated according to the present invention is capable of artificial cultivation, mycelial growth and fruiting body formation rate is fast, high-quality large-capacity is possible. In addition, it is possible to produce a substance with high pharmacological functionality by building mass production of chaga fruiting body according to the present invention. In particular, chaga fungus KNAC 3002 strain is a mycelial growth and fruiting body formation rate is fast and can be mass-produced in high quality, high yielding chaga superior strain.

Claims (12)

delete delete Accession No .: Chaga mushroom strain, characterized in that KFCC-11190. delete delete delete Removing the spores or tissue of chaga in sterile water for more than 24 hours in a culture room at 27 ~ 32 ℃; Putting dry cotton wool in a container for 8-10 hours at 140-160 ° C. for dry heat sterilization, and immersing in 27-32 ° C. incubation room for at least 24 hours and then removing it; Chaga mushroom tissue or spores in the dry heat sterilized container comprising the step of standing again for 25 to 30 ℃ incubation temperature for 8 to 50 hours, characterized in that the method of separating chaga mushroom according to claim 3. Chaga mushroom strains according to claim 3 are cultured in a synthetic medium or sawdust medium to produce mycelium, and the produced chaga mycelium is cherry, jujube, river oak, oyster oak, silver fern, akash wood, white birch, black The method of producing chaga fruiting body, characterized in that inoculated and grown on the selected wood from the group consisting of birch. The method of claim 8, wherein the synthetic medium is a medium selected from the group consisting of BDA, ODA, PODA, and MCM, and the method of producing chaga fruiting body is cultured at a culture temperature of 20 to 30 ° C and pH 5 to 9. . The chaga mushroom according to claim 8, wherein the sawdust badge is a sawdust badge of solid wood selected from the group consisting of cherry, jujube, river oak, oyster oak, silverfin, akash, white birch and black birch. Method of production of fruiting bodies. 9. The method of producing chaga fruiting body according to claim 8, wherein the water content of the wood is 42 to 45%. The method of claim 8, wherein the log is sterilized by autoclaving or autoclaving before inoculation.