KR20000066822A - Method for culturing Phellinus linteus on the solid medium - Google Patents

Abstract

PURPOSE: Provided is a solid culturing method of Phellinus linteus strain which produces polysaccharide-protein showing excellent immunity in particular using a fruiting body or a spore of Phellinus linteus KCTC 0173BP. CONSTITUTION: A solid culturing method of Phellinus linteus strain(KCTC: 0173BP) is characterized by comprising the following steps of:(i)transferring a fruiting body or a spore of Phellinus linteus strain into agar medium and performing serial passage culture 2-3 times; (ii)inoculating the above thing in solid medium which comprises a mixture of one thing selected from trees of overstory, saw dust of trees of overstory and immersion liquid of trees of overstory: bran: peptone in the weight ratio of 10:0.5-1.8:0.2-0.9 and 30-50 wt.% of mulberry extract; and existing nutrients then followed by culturing at 13-36°C for 20-200 days.

Description

Method for culturing Phellinus linteus on the solid medium

The present invention relates to a solid culture method of the Pelinus linteus strain, more specifically, after passage of tissue or spores of fruiting body of the Pelinus linteus KCTC 0173BP strain in agar medium, it is a sawdust or Dipping solution of lumber: Rice bran: Solid culture method for mass production of Felinus linteus strain which produces protein polysaccharides with strong immune activity by adding mulberry extract to a mixture of peptone and inoculating into a solid medium composed of known inorganic nutrients It is about.

It is known for a long time that many kinds of mushrooms belonging to Aphyllophorales, which is a kind of Basidiomycetes, have a therapeutic effect against various diseases, especially tumors, and they have been mainly transmitted as herbal medicines or folk medicines. Among them, Pelinus linteus, belonging to Phellinus of Polyporaceae, is a very rare mushroom called circumference in oriental medicine. . The situation is also characteristic dark yellow, even in the cut section, with a ring-shaped pattern on the upper part of the fruiting body. This situation has been used for antiperspirant or gynecological diseases as a valuable herbal medicine since ancient times. In the 1960s, it was confirmed to have a very strong anticancer activity. It is known to have excellent anti-cancer effects by enhancing the function of the immune system in the human body while being very safe in toxicity.

In such rare wild mushrooms, two methods have been mainly used to industrially produce effective pharmacological ingredients and use them as food or medicine. That is, the method of collecting fruiting bodies or artificially cultivating fruiting bodies and using the fruiting bodies as a material, and separating the mycelia from the fruiting bodies and cultivating the mycelium to extract the active ingredient from the cultured myceliums. .

However, the method of collecting and using the wild fruiting body is accompanied by the problem of resource depletion and ecosystem destruction, and it is absolutely impossible to secure the required amount for rare carrier bacteria such as Felinus linteus. In addition, the method of obtaining the fruiting body through the cultivation also requires a special facility and a lot of expenses, and can be produced only for a certain period, especially the cultivation period takes 3 to 6 months, so it is not preferable.

On the other hand, the method of separating and culturing mycelium from the fruiting body can solve the above problems at one time, but this situation is not generalized due to the following technical difficulties. In other words, it is not easy to separate the mycelium purely, and it is difficult to establish facilities and culture conditions that can be cultured in a fermentation tank without contamination because of high possibility of contamination of bacteria because of difficult culture conditions and slow growth. There is a difficulty.

In this regard, the existing mushroom mycelium cultivation method has been reported to be mass-produced by culturing Felinus linteus strain in a conventional medium, for example, a medium containing yeast extract, peptone, and dibasic potassium phosphate. Patent No. 95-29192). However, it is produced in liquid medium, and the problem of culturing mycelium in liquid medium is that it is impossible to expect the pharmacologically active component of the secondary metabolite because it hardly produces various secondary metabolites that the mushroom itself has in addition to the polysaccharide. Is the point. On the other hand, in the case of a solid medium, as a variety of secondary metabolites of the mushroom itself, it is possible to obtain a fruiting body layer and a fungal layer containing high concentration of pharmacologically active ingredients.

Felinus linteus strain of the present invention is a method of using a medium containing peptone medium, pepper medium, meyer medium, malt extract medium, sawdust, glucose, which is a conventional solid medium (Korean Patent Publication No. 88-2475, 83 -1909, etc.) has not been cultivated, so that artificial culture for mass production has not been successful.

The present inventors have been studied to solve the above problems, microbiological characteristics are different from the existing Felinus linteus, and the anti-cancer effect of the situation mushroom collected in Korea is known to be very good and the culture method has not yet been established Felinus The artificial culture method using solid medium with different composition from the existing medium was established by using the strain of Linteus (KCTC 0173BP), and the addition of mulberry extract filtrate to the solid medium showed more than 20% of B cell immune activity. The present invention has been completed by demonstrating that increased protein polysaccharide can be obtained from cultured mycelium.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for solid culture of Phellinus linteus KCTC 0173BP strain, which can be usefully produced for producing polysaccharides having anti-cancer immune activity by producing an anti-cancer immunoactive substance.

Figure 1 shows the results of the search for B cell specific immune enhancing activity.

1: control group

2: PBS (phosphate-buffered saline, negative control)

3: LPS (lipopolysaccharide, positive control)

4: conventional solid medium

5: solid medium of the present invention (conventional solid medium + mulberry extract (Morus alba extract, 100g / 1.5 liters))

In order to achieve the above object, the present invention provides a solid medium of a new composition for use in the solid culture of the strain of Felinus linteus (KCTC 0173BP). More specifically, lumber, lumber sawdust or immersion liquid (hereinafter referred to as 'wood sawdust'); Chaff; It provides a solid culture medium for mass production of Felinus linteus strain, containing peptone, and other known inorganic nutrients and in addition to the mulberry extract.

The present invention also provides a method for solid culture of a Felinus linteus strain, which is characterized by culturing the tissues or spores of the fruiting body of the Felinus linteus strain and culturing them in the agar medium.

In addition, by the solid culture method of the present invention provides a protein polysaccharide showing an immuno-enhancing activity from the culture mycelium of Felinus linteus strain and its preparation method.

Hereinafter, the present invention will be described in detail.

Felinus linteus used in the present invention is a strain deposited with KCTC 0173BP in the Genetic Bank of Korea Institute of Science and Technology Biotechnology Institute, an international microorganism depositing institution, and has the following microbiological characteristics (Table 1).

Microbiological Characteristics of the Felinus Linteus KCTC 0173BP Strain of the Invention division Characteristic Fruiting body · No stipe, wood of horseshoe shape · Gad: semicircular, flat, about 10 cm wide, surface is dark brown, back surface is yellowish brown to dark brown. Spores It is globular and pale yellow.Size: about 5 ?? m Bristle body Many in size and thick film size: 20 ~ 40 x 8 ~ 12 ?? m

In the present invention, the culture of the Felinus linteus strain is characterized by using a nutrient source that can be used by ordinary microorganisms, but its composition ratio is remarkably different from the conventional one. Specifically, the weight ratio of lumber sawdust: bran: peptone is 10: 0.5-1.8: 0.2-0.9 and the composition is preferably contained so as to contain 50-70% of water. Sawdust used is better coarse, bran such as rice bran serves as a nutrient additive. Rice bran is rich in crude fat, crude protein, vitamins, etc. Particularly, skim rice bran is widely used because of its stable quality, and recently, bran is also used. The ratio of bran is preferably 0.5 to 1.5 weight per 10 weights of lumber sawdust. If the ratio of bran is high, the amount of occurrence of the bran is high, but the occurrence of various germs also increases, so it is desirable to lower the ratio in summer and increase the ratio in winter. Do. This is because if the temperature is high in the summer, the possibility of breeding of germs is high. Therefore, by reducing the amount of bran in the summer, it is possible to suppress the breeding of germs and increase the amount of bran enough to cultivate the situation in the winter when the possibility of germ contamination is low .

Inorganic nutrients to be used include potassium hydrogen phosphate; Sulfates such as magnesium sulfate, zinc sulfate, and copper sulfate; Chloride, such as calcium chloride, manganese chloride, and the like may be used, but is not limited thereto, and known components in the art may be used.

In addition to the above, the solid medium of the present invention includes a mulberry extract. Mulberry extract is preferably added at about 30 to 50% by weight. By adding the mulberry extract, a protein polysaccharide showing an immunological activity of about 20% or more higher than that by the conventional culture method can be obtained from Felinus linteus.

The culture temperature is slightly different depending on the conditions when incubated in the above conditions, but it is usually preferred to incubate at 15 ~ 35 ℃, the incubation period is 20 to 200 days by culturing the desired mass culture is possible.

In order to obtain the initial culture by subculture of the Pellinus linteus strain, PSA medium (potato sucrose agar), PDA medium (potato dextrose agar), MEA medium (malt extract agar), QEA medium (quercus extract agar) ), PEA media (rice extract), RBEA media (rice bran extract agar), WBEA media (wheat bran extract agar), MCM media (mushroom complete medium), CDA media (Czapedox agar), SCA media (Spawn complex agar) , OMA medium (oatmeal agar) and the like can be used, especially in MCM medium shows excellent growth ability.

Looking at the solid culture process of the Felinus linteus strain as follows.

First, for solid culture of the Felinus linteus strain, the tissues or spores of the fruiting body of the strain are agar medium containing potato, malt extract, mulberry sawdust, poplar, rice bran, wheat bran and oatmeal as main ingredients. Incubate at 15-35 ℃ for several weeks. This culture operation is repeated two to three times, subcultured, and after confirming that there are no incorporation of various bacteria, it is transferred to a solid medium prepared as described above and incubated at a temperature of 13 to 36 ° C. for 20 to 200 days.

At this time, the sawdust medium is decomposed and absorbed by most of the inoculated mycelium, which is converted into the situation mycelium, and in the process of ripening for a sufficient period, the unique and diverse components of the situation are produced, and the fruit has the unique dark yellow color of the situation. Layers and fungal layers are produced. Therefore, extracting the active ingredient using the fruiting layer and the mycelium layer can obtain a large amount of effective pharmacological components of the situation mushroom itself, unlike the normal sawdust cultured mycelium or liquid cultured mycelium.

Next, an appropriate amount of water is added to the cultured mycelium as described above, followed by hot water extraction and filtration with a centrifuge. The extract is subjected to ethanol precipitation, dialysis, and the like to obtain a polymer extract containing polysaccharide. The obtained extract was added to the leukocytes of the experimental animal cells and cultured, and then B lymphocyte formation was measured by flow cytometry to measure the effect of enhancing immune activity.

Polysaccharides having B cell specific immunological activity produced from Felinus linteus solid-cultured according to the present invention can be formulated in a conventional manner as a medicament using one or more pharmaceutically acceptable carriers, oral, rectal, transdermal Formulated in a form suitable for administration via a variety of routes including, subcutaneously, intravenously and intramuscularly, such as tablets, hard and soft gelatin capsules, liquids, emulsions, suspensions, suppositories or injections. have.

For the preparation of pharmaceutical preparations, the above-mentioned substances may be combined with pharmaceutically inert inorganic or organic carriers, and lactose, corn starch or derivatives thereof, talc, stearic acid or salts thereof may be used as a carrier. The pharmaceutical preparations may also contain preservatives, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for controlling osmotic pressure, buffers, coatings or antioxidants.

Dosages for cancer treatment or immunopotentiation can vary widely from a daily dosage of about 1 mg to about 5000 mg per kg of body weight and are adjusted according to each individual requirement.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited to the examples.

Example 1. Incubation of Felinus linteus KCTC 0173BP strain

(1) Manufacture of Agar Media

Various agar medium containing Potato, malt extract, mulcus sawdust, Poplar sawdust, rice bran, wheat bran or oatmeal was prepared (Table 2).

Creation of Agar Badges badge Composition (g / l) PSA badge (Potato sucrose agar) 200 potatoes, 20 per book, 20 baby PDA badge (Potato dextrose agar) Potato 200, Dextrose 20, Baby 20 MEA medium (Malt extract agar) Malt extract 20, dextrose 20, peptone 1, MgSO ₄ 7H ₂ O 0.5, agar 20 QEA medium (Quercus extract agar) Quercus Sawdust 100, Dextrose 20, Baby 20 PEA medium (Poplar extract agar) Poplar Sawdust 100, Dextrose 20, Baby 20 RBEA medium (Rice bran extract agar) Rice bran 30, baby 20 WBEA medium (wheat bran extract agar) Push 30, baby 20 MCM badge (Mushroom complete medium) Dextrose 20, Peptone 2, Yeast Extract 2, MgSO ₄ 7H ₂ O 0.5, KH ₂ PO ₄ 0.46, Agar 20 CDA badge (Czapedox agar) Dextrose 20, MgSO ₄ 7H ₂ O 0.5, KH ₂ PO ₄ 1, Agar 20 SCA medium (Spawn complex agar) Quercus Sawdust 60, Poplar Sawdust 60, Rice Bran 20, Baby 20 OMA Badge (Oatmeal agar) Oatmeal 30, Baby 20

(2) Preparation of solid medium

After adding a known inorganic nutrient for the medium to the mixture of mulberry tree, coarse sawdust of its branches, or immersion liquid of mulberry tree (hereinafter abbreviated as "wood sawdust"), bran and peptone, it contains 50-70% of water. More specifically, mulberry extract was added to a mixture of lumber sawdust: rice bran: peptone = 10: 1: 0.5, and KH ₂ PO ₄ 0.87 g: MgSO ₄ · 7H ₂ O 0.5 g as an inorganic nutrient. : CaCl ₂ 0.3 g: FeSO ₄ · 7H ₂ O 10 mg: MnCl ₂ · 4H ₂ O 7 mg: ZnSO ₄ · 7H ₂ O 40 g: CuSO ₄ · 5H ₂ O 1 mg is added, and the moisture content is 65 ±. To 5%.

(3) cultivation of strains

Tissues or spores of the fruiting bodies of the Plinus linteus KCTC 0173BP strain were transplanted into agar medium prepared according to the method of (1) and incubated at 20 ± 2 ° C. for 30 days. After repeating this culture operation three times, it was confirmed that there were no mixed bacteria, and then transferred to a solid medium prepared according to the method of (2), followed by culturing at 25 ± 2 ° C. for 100 days to obtain 20 kg of mycelia.

Example 2 Extraction of Protein Polysaccharides

The mycelium obtained in Example 1 was extracted by the following method. That is, 100 g of the obtained mycelium was added to 500 ml of distilled water at 90 to 100 ° C. and extracted by boiling water for 2 hours. The mycelium cake was removed and only the hot water extract was recovered. The hot water extract is concentrated in vacuo to 100 ml, left to stand overnight with 4 times the volume of 95% ethanol, and then centrifuged at 3000 rpm for 30 minutes. The extract thus obtained was dialyzed for about 3 days using a dialysis membrane capable of dissolving in 100 ml of water and dialysis of a substance having a molecular weight of 3000 or less, and then lyophilized at -70 ° C. to about 3 g of a polymer extract containing polysaccharide. Got it.

Example 3 Screening for B Cell Specific Immunostimulatory Activity

The control group, the negative control PBS, the positive control LPS, the protein polysaccharide of the mycelium cultured in a conventional solid medium and the polymer extract obtained in Example 2 were added to splenic leukocytes of BALB / c mouse and B cell immunity. The activity was investigated. In other words, 1 ml of ValvC mouse spleen leukocytes were added to RPMI medium to which 10% of streptomycin (100 ?? g / ml), penicillin (100 U / ml), and fetal bovine serum were added. The cells were suspended to 1 × 10 ⁶ cells and the samples sterilized through the bacterial filtration membrane were added to 100 μg / ml. After culturing for 3 days in a CO ₂ incubator in a 100% humidity state containing 5% CO ₂ at 37 ° C, a method of already reporting whether or not B lymphocytes were produced was developed. According to the Autumn Symposium Abstract, 7-12, 1993). As a result, the protein polysaccharide of the mycelium cultured in the solid medium of the present invention was found to have about 20% or more of B cell-specific immune enhancing activity compared to the strain cultured in the conventional manner, which is the positive control by LPS It was higher than that.

According to the method provided in the present invention, after subcultured with Fellinus linteus in agar medium, the main ingredient is lumber sawdust, and inoculated and cultured in a solid medium to which a peptone and mulberry extract are added at a predetermined ratio. In addition to culturing Felinus linteus, which produces protein polysaccharides with a strong immunostimulatory activity of more than 20%, in addition to a large amount of unique secondary metabolites, Felinus linteus rich in effective pharmacological components can be obtained. Can be.

Claims (7)

After culturing the fruiting body tissue or spores of Felinus linteus strain in agar medium, mulberry extract is added to a mixture of bran and bran and peptone selected from lumber, lumber, and nutrients. Method for solid culture of Felinus linteus, characterized by inoculating and incubating in a solid medium prepared by. The method of claim 1, wherein the strain is Felinus linteus KCTC 0173BP Solid culture method of Felinus linteus, characterized in that. The solid culture method of Felinus linteus according to claim 1, wherein the weight ratio of one species: bran: peptone selected from lumber, lumber sawdust, and immersion of lumber is 10: 0.5-1.8: 0.2-0.9. The method of claim 1, wherein the culture temperature in agar medium and solid medium is a solid culture method of Felinus linteus, characterized in that 13 ~ 36 ℃. The method of claim 1, the mulberry extract is a solid culture method of Felinus linteus, characterized in that the addition of 30 to 50% by weight. A method for extracting the immunoactive component from the Felinus linteus mycelium cultured by the method of claim 1 using an aqueous solution containing water, acid, alkali, or the like under heating or pressurization. An immunoactive component substance showing B cell specific immunopotentiating activity prepared from the solid culture of Felinus linteus by the method of claim 6.