KR100309683B1 - A new Phellinus lintenus strain and a cultivation of Phellinus lintenus using the same - Google Patents

Abstract

The present invention relates to a novel method of planting mud mushrooms (Phellinus lintenus Andong1) and artificial mushrooms of wood mud mushrooms using the same, the wood mud mushrooms cultivated using the strains of the present invention has an excellent anticancer effect and the demand is increased. However, since natural products are very rare, there are limitations to meet the demand and problems that may be misunderstood as imported products due to differences in their natural forms, colors, and shapes. There is an advantage that can be solved.

Description

A new Phellinus lintenus strain and a cultivation of Phellinus lintenus using the same

The present invention relates to novel strains of wood mud (Phellinus lintenus Andong1) and artificial culture method of wood mud mushrooms using the same.

Sangsin (桑臣), different from (桑耳), call hand is (胡孫眼) and with two people (異名), such as, Phellinus native to mulberry mushroom woody clay belonging to the pine scales mushrooms and (Hymenochaltaclal) (Phellinus lintenus ), which has been used for the treatment of gynecological disorders such as uterine bleeding and menstrual irregularities, and has recently been recognized as a cancer treatment drug because it is known to be superior to other mushrooms such as dung mud and fir mud mushrooms. have.

However, the situation mushroom is very rare species in nature, and it is very difficult to obtain fruiting body, and also it is difficult to artificially culture mycelium, so it is not actively used as a tumor treatment agent. Situation mushrooms are sometimes collected in mountainous areas because they are limited in terms of development conditions and environments. They are wild in the shade and grow toward the ground, so they occur on stems or branches above the ground rather than on the roots of plants. It is difficult to collect because it grows into a permanent year.

Therefore, many studies are being conducted to artificially cultivate situational mushrooms. For example, Korean Patent Publication No. 1999-180097 (1999. 02. 01) discloses a method of artificially cultivating situational mushrooms by inoculating the situational mushroom spawn in the logs. It develops and produces situation mushrooms in large quantities.

However, the situation mushroom is different from the domestic natural mushrooms in its form, color, shape, etc., and can be misunderstood as imported from consumers, there is a limit to easily purchase by anyone.

Accordingly, the present inventors have studied a method for artificially cultivating situation mushrooms having the same shape, color, and shape as domestic wild mushrooms, and producing them in large quantities. As a result, a novel woody mildew strain was discovered and mulberry trees When inoculated in cultivation, the present invention was found to be able to provide the same situation mushrooms in the form, shape, color and the like with the natural wild.

Accordingly, it is an object of the present invention to provide a novel woody fungi strain.

Another object of the present invention to provide a method for artificially cultivating wood mud mushrooms using the above-mentioned strain.

1 is a PCR amplification photograph of the ITS1, 5.8S rDNA and ITS2 region of the wood mud mushroom (Phellinus lintenus ) Andong (andong) 1 strain (KCTC-8936) of the present invention.

<Description of Drawing>

1, 2: ITS portion of the phellinus sp. Strain

3, 4: ITS portion of wood mud mushroom Andong 1 strain (KCTC-8936) of the present invention

5. Molecular Weight Markers

Figure 2 is a molecular tree of the wood mud mushroom Andong 1 strain (KCTC-8936) by the nucleotide sequence of the ITS1, 5.8S rDNA and ITS2 region.

Figure 3 is put into the vinyl mulberry sterilized, cooled, and then inoculated with the wood mud mushroom Andong 1 strain of the present invention (KCTC-8936), is a photo cultured.

Figure 4 is a picture of drying after removing the biofilm formed on the vinyl and mulberry of the mulberry cultivation is complete.

5a and 5b are photographs of the upper part of the first year wood mud mushroom.

6 is a photograph of woody mud mushrooms merged with neighboring objects as they grow gradually.

Figure 7a is a planar photograph comparing the wood mud mushrooms according to the present invention and the wood mud mushrooms conventionally marketed.

Figure 7b is a front photograph comparing the wood mud mushrooms according to the present invention and the wood mud mushrooms conventionally commercially available.

Figure 7c is a bottom photograph comparing the wood mud mushrooms according to the present invention and the wood mud mushrooms conventionally marketed.

<Description of Drawing>

I: Wood Mud Mushroom II of the Present Invention

In order to achieve the above object, the cultivation method of the present invention comprises the steps of preparing a mulberry slice; Putting the prepared mulberry pieces in a plastic bag, sealing them, and then sterilizing and cooling them; Inoculating the mulberry tree stubble with the Phellinus lintenus Andong 1 strain (KCTC 8936P); Culturing the mulberry seed block inoculated with the strain at 21-23 ° C. for 50-60 days; Removing the vinyl covering the cultured mulberry slices, and the biofilm formed on the mulberry, and then drying; Planting the dried mulberry chips in soil in a cultivation facility having a water content of more than 90%; And supplying 80-85% of water to the soil, maintaining the temperature of the ground at 25-28 ° C, and maintaining the temperature of the ground at 28-31 ° C, and then growing and harvesting the mushrooms. It features.

Hereinafter, the present invention will be described in more detail.

Example 1 Isolation of Strains

The fruiting bodies were collected from the hardwoods of Yasan, Andong, Gyeongbuk, and the fruiting bodies were sterilized before they were dried. The fruiting bodies were removed and transplanted into agar medium. Then, the mycelia were obtained by incubating for about 10 days at about 25 ° C., and the mycelia were grown to obtain strains. This strain was added to sawdust medium and propagated to obtain seedlings for fruiting.

Example 2. Identification of Strains

After culturing the seed, the fruiting body was obtained, and the morphological characteristics and nucleotide sequence of the fruiting body were analyzed.

<Morphological characteristics>

· Holes: Square to circular, distributed 6 ~ 8 per mm, mainly dark yellowish brown, thin wall, 1mm length

Mycelium: Binary mycelium form

Spore: Size 3.8 ~ 4.8 × 2.9 ~ 4㎛

Cell wall: thick, pale yellow to pale yellow ovate to photo elliptic

Bristle: Dark brown awl to wedge shape with size of 18 ~ 28 × 8 ~ 11㎛

<Base sequence analysis>

Sequences were analyzed using Internal Transcribed Spacer 1 (ITS1), 5.8S rDNA, and ITS2 genes, which are part of the gene encoding ribosomal RNA (rDNA). Since these genes are common to all living organisms, and because the substitution rate of genes is constant, they act as very important factors for correlation and identification of evolutionarily similar microorganisms. The sequence was analyzed.

That is, the mycelium cultured in PDB (Potato Dextrose Broth (Difco)) was recovered by centrifugation. Here, DNA was extracted by the benzyl chloride method (Nucleic Acid Res. 21: 5279 ~ 5280 (1993)) of Zhu et al., And then amplified genes of the desired ITS1, 5.8S rDNA and ITS2 moieties by PCR (FIG. 1). ). PCR amplified gene was determined by nucleotide sequence using 1% agarose gel. The base sequence is shown in SEQ ID NO: 1.

The determined nucleotide sequence was multi-aligned using a clustal W program (Thompson, JD et. Al., Nuclic acid Res. 22: 4673-4680), and then the Neighbor-joing method (Saitou and Nei, Mol. Biol. Evol. 4: 406-425 (1987)) to generate a phylogenetic tree (FIG. 2).

Taken together, the strains from the wildPelinus linteusTurned out to be. Therefore, the inventors of this strainPelinus linteusAndong 1 And was deposited with the Biotechnology Research Institute on March 10, 1999 and was assigned accession number KCTC-8936P.

Example 3 Cultivation of Wood Mud Mushrooms

1) Preparation of mulberry chips

Mulberry trees are harvested from December, when the sap has completely stopped, to late March of the following spring. The thickness of the tree is preferably 5 ~ 30 ㎝, cut it to a length of 15 ~ 20 ㎝, and then dried for 5 to 10 days to prepare the ground to make the moisture of the tree 30 to 40%.

2) Sterilization and Cooling

Put the prepared mulberry pieces in a high density plastic bag of 0.03 ~ 0.05mm, seal the inlet part with a string, then pile up in a high-temperature sterilizer, sterilize with a high-pressure steam sterilizer. Sterilization time and temperature is preferably sterilized for about 12 hours at about 100 ℃.

Once sterilization is complete, the mulberry bush is cooled as quickly as possible. This is to suppress the propagation of various germs, and in particular, by cooling by using a wind such as a fan to remove water droplets generated in the steam sterilization to prevent mold in the air.

3) strain inoculation

Mycelia of woody mushrooms are very weak compared to other mushrooms, so the inoculation room must be disinfected before inoculating the strains. That is, the inoculation room can be sealed and sprayed with an air purifier installed one day before inoculation by spraying antiseptic alcohol on the front of the inoculation room, or by smoking about 0.5ℓ of green powder of 5 pyeong a day before smoking. After sterilization, the strains are inoculated.

Place a rubber band on the bottom 5cm of the top part of the sterilized mulberry tree, then unscrew the vinyl string and sterilize Andin 1 (KCTC-8936P) with Pelinus linteus. The spawn amount is appropriately selected according to the size of the mulberry, it is preferable that the inoculation of about 100g is good work efficiency and mycelial state.

On the other hand, in order to remove the cause of bacterial contamination before inoculation, it is preferable to inoculate after cutting the upper part of the entrance of the bottle containing the strain of Felinus linteus Andong 1 (KCTC-8936P) about 3 cm.

After inoculating strains on the mulberry tree, turn the vinyl 3-4 times, fold it, and tie it with a rubber band.

5) Culture

After the inoculation of the strain was completed, the inoculated mulberry tree inoculated in a temperature facility that can maintain 21 ~ 23 ℃ was incubated. Particularly important at the time of cultivation is to maintain the constant temperature as the temperature. This is because it takes 3 to 5 days for the mycelial growth to stop and grow again when a change in temperature occurs, thereby increasing the incubation period and increasing the incidence rate of bacteria.

As a result, it was observed that mycelia bloomed about 3 days later, and 7 days later, mycelia grew out of the original target blood. In other words, the growth rate of wood-attached mycelium at 1 ℃ in 3 days at the culture room temperature of 22 ℃, the culture is completed in about 50 days, about 10 days to further culture for maturation. How to determine the completion of the cultivation with the naked eye is that the mycelia grows while the mycelium wraps around the entire tree, forming a solid wood biofilm and culturing is complete (Fig. 3).

When the cultivation is completed, the vinyl of the spawned seedlings is peeled off, the biofilm formed on the logs is removed, and then dried in a well-ventilated place for 2 to 3 days without rain (FIG. 4). This is because the mycelial growth stops at the same time when the logs are dried, so that the mycelia are cut off inside the logs. On the other hand, if you pile up too much or not ventilated during drying, it does not dry well, especially the blue fungus that kills the woody fungus bacteria, because it hinders the generation of mushrooms during the transplantation.

6) Mushrooming, growing and harvesting

Bury mulberry cultivated at intervals of 20 cm on the ground. At this time, the length of the buried is preferably buried about half of the mulberry on the ground, the top of the soil to put about 1 to 2 cm to prevent moisture from evaporating.

Next, irrigation is done for hydration. For watering, ground watering (spring cooler) and heat and ground watering (drip irrigation) are carried out to keep the water saturated at the bottom of the timber basement for 7 days. After that, the water in the ground is 90% or more, Water is maintained to maintain 80 to 85% of water.

Humidity should be maintained about 90%, and make a separate ventilation window to ensure sufficient ventilation.

The temperature is maintained at 25 to 28 ° C. on the ground and 28 to 31 ° C. at the ground level.

After burying the mulberry in the ground under the above conditions, 20 days later, woody mud mushrooms occur, and the shape comes out as a round bean stalk, and gradually grows to form a round horseshoe-shaped lampshade. It grows thickly to the ground, and the color during growth is golden at the bottom, and the upper part of the fruiting body is changed to a dark brown color. 5b). In addition, many (30 ~ 200) mushrooms grow on one log and merge with neighbors, and the strong ones continue to grow, and those who do not grow are stopped, and those who stop after a certain period of time There is a growing feature (Fig. 6).

On the other hand, the medicinal effect of the situation mushroom can be said that the efficacy is remarkable as it is perennial, so that until the end of the life of the mulberry can continue to grow the situation mushroom (wood mud mushroom), it is good to harvest in the second or third year.

At this time, in the case of the second year mushrooms, the color and shape of the growth stop state is brown at the upper side, forming a concentric circular layer, split horizontally and vertically, and the bottom part is yellowish brown.

Example 4

Using mulberry stub of 20 cm in length, 10 cm in diameter, and 1.5 kg in weight, wood mud mushrooms were grown in the same manner as in Example 1, and harvested in the first and second years. Yield is as follows.

1st year: 35g (yellow at bottom, maroon at top)

2nd year: 80g (yellow at bottom, maroon at top)

If you leave it without harvesting in the first year and harvest it at the same time in the second year, you can harvest about 155g.

Comparative Example 1

Wood mud mushrooms were grown in the same manner as in Example 4, except that oak chips were used, and harvested in the first and second years. Yield is as follows.

1st year: 5g (yellow at bottom, maroon at top)

2nd year: 30g (yellow on bottom, maroon on top)

As described above, wood mud mushrooms cultivated using the strain of the present invention have an excellent anticancer effect, but the demand is increasing. However, since natural products are very rare, there are limitations in meeting the demand and present. Commercially available woody mud mushrooms can solve problems that may be misunderstood as imported products due to differences in natural forms, colors, and shapes.

Claims (2)

Wood mud mushroom (Phellinus lintenus ) Andong 1 strain (KCTC-8936) 1) preparing mulberry chips; 2) putting the prepared mulberry pieces in a plastic bag, sealing, sterilizing and cooling; 3) inoculating the mulberry mulberry chopped wood chips with Phellinus lintenus Andong 1 strain (KCTC 8936P); 4) culturing the mulberry seed block inoculated with the strain at 21-23 ° C. for 50-60 days; 5) removing the vinyl covering the cultured mulberry stub, and the biofilm formed on the mulberry, and then drying; 6) planting the dried mulberry stub in the soil in a cultivation facility containing more than 90% moisture; And 7) After supplying 80 ~ 85% of moisture to the soil, keeping the temperature of the ground at 25 ~ 28 ℃ and the ground temperature at 28 ~ 31 ℃, after mushrooming, growing and harvesting mushrooms. Cultivating method of wood mud mushrooms, characterized in that.