KR20150055650A - Cultivation method of Phellinus linteus and Phellinus linteus cultivated thereby - Google Patents

Abstract

The present invention relates to a method for manually cultivating the black hoof mushroom (Phellinus linteus) fruiting bodies and the black hoof mushroom fruiting bodies thereof. The present invention allows farmers to industrially grow the black hoof mushroom fruiting bodies which can be only acquired in nature before. Accordingly, the black hoof mushroom fruiting bodies having the anti- inflammatory, anti-cancer, and antioxidant effects can be used as the ingredients for various food and medicine products and bring revenues for a farm.

Description

[0001] The present invention relates to a method for cultivating mushroom fruiting body and a mushroom fruiting body cultivated thereby,

The present invention relates to a method for artificial cultivation of woody muddy mushroom fruiting body which can be cultivated industrially in a farmhouse and which is capable of only natural harvesting, and to a woody muddy mushroom fruiting body cultivated thereby.

Phellinus linteus , a woody mud mushroom, is also called mulberry, a yellowish mushroom growing in mulberry trees. It is a white rotifer belonging to aphylloporales mushroom (phellinaceae) It is native to Korea, Japan, Australia and North America, and is considered to be a wild mushroom.

In 1968, Dr. Chihara of the National Cancer Research Center of Japan conducted a survey on Sarcoma 180 solid tumors. The result of the tumor suppression rate of 96.7% for the case of mushroom Pelinus Linheus was 7 out of 8 tumor infections in rats, Magazine was introduced. Since then, researches on anticancer and immunological activity using Linseus mushroom have been conducted all over the world, including Korea, and proved to be effective in anti-cancer, human immunity enhancement and antioxidant. Pelinus lentheus was selected as the world's top 10 anticancer food , 2002. 06).

Thus, most of the efficacy of pharmacologically active substances in domestic and foreign mushrooms was proved by Pelinus Linheus.

Rural Development Administration has cultivated Perinus Linheus fruit body produced by performing the artificial cultivation experiment of Ferinus Linheusus as a "consideration situation" and distributed it to the farmers. However, it takes 3 ~ 4 years for fruit body cultivation and production of fruiting body is extremely limited Economic mass production is difficult.

In Korea Perinus Linentus Phellinus baumii has been cultivated in a general farmhouse under the name "Longevity Situ mushroom" as a result of the successful introduction of artificial cultivation.

In 2003, the Korea Food & Drug Administration established the name of " Phellinus linteus , Phellinus mushroom" baumii "and was notified to use.

Therefore, it is required to produce pure mushroom fruiting body which is shortened in comparison with fruiting body mushroom fruiting body which is ferinerus linteus and cultivation period.

Korea Patent No. 1069686 Korean Patent No. 0361681 Korea Patent Publication No. 2005-0100718

It is an object of the present invention to provide a method for artificial cultivation of fruiting bodies of mushroom mushroom which can industrially grow woody mushroom fruiting bodies,

Another object of the present invention is to provide a woody mushroom fruiting body cultivated by the artificial cultivation method.

It is still another object of the present invention to provide an antioxidative composition comprising the mushroom fruit body extract as an active ingredient, a composition for promoting an anti-cancer activity, or a composition for prevention and treatment of anti-inflammatory diseases.

In order to achieve the above object, the present invention provides a method for artificial cultivation of mushroom fruiting bodies of woody mud, comprising the steps of: Phellinus linteus strain [Accession No .: KACC93057P] and then inoculated on hardwood logs.

Specifically, the method for artificial cultivation of mushroom fruiting bodies comprises: (A) culturing the cultured Phellinus linteus strain in a culture bottle containing hardwood sawdust; (B) sterilizing hardwood logs; (C) inoculating the sawdust cultivated in the step (A) into the sterilized hardwood logs; And (D) planting the inoculated hardwood logs by ground cultivation.

Phellinus cultured in step (A) linteus strain may be obtained by culturing the strain of Pelinus linteus in a potato glucose agar medium (PDA) and then pulverizing it by adding distilled water.

In the step (A), the culture bottle containing the Pelinus linteus strain and the hardwood sawdust may be cultured at a temperature of 18 to 28 ° C and a humidity of 50 to 80%.

The step (c) may further include culturing the hardwood sprouted with the sawdust seedlings at 23 to 26 ° C for 110 to 120 days to allow the hyphae to be deposited on the entire upper surface of the hardwood sprout. The method may further include peeling off the mycelia that have been activated on the entire surface of the hardwood timber after cultivating the hardwood timber, and applying an impact to the hardwood timber.

The step (D) may further include maintaining the hardwood logs planted at 90% humidity and 25 to 32 ° C under 70% light-shielding conditions.

The ground to be cultivated in the step (D) may be sanded with sand having a thickness of 4 to 5 cm on the top surface of the gravel having an average diameter of 2 to 3 cm.

In step (D), 100 to 130 days after the plantation of the hardwood species, the mushroom fruit body of the woody mud can be formed. Further, the hardwood tree can be planted and the mushroom fruit body of the woody mud can be harvested one to two years later.

The above-mentioned broad-leaved tree may be one selected from the group consisting of oak, cherry tree, maple, mulberry, mulberry, beech, cirrus, and poplar.

According to another aspect of the present invention, there is provided a mushroom fruiting body produced by the artificial cultivation method of the mushroom of the present invention.

According to another aspect of the present invention, there is provided an antioxidant composition comprising the mushroom fruit body extract of the present invention as an active ingredient.

According to another aspect of the present invention, there is provided a composition for enhancing anticancer activity, which comprises an extract of fruiting body of mushroom mushroom of the present invention as an active ingredient.

According to another aspect of the present invention, there is provided a composition for preventing or treating an anti-inflammatory disease, which comprises the mushroom fruit body extract of woody mildew mushroom as an active ingredient.

Since the mushroom fruiting body of the present invention takes 1 to 2 years to be harvested, it can dramatically shorten the cultivation period compared to the conventional mushroom fruiting body (3 to 4 years old) and has a high fruiting body formation rate, To increase income and improve public health.

In addition, the fruiting body of the mushroom of the present invention has higher antioxidant, anti-cancer and anti-inflammatory activity than the mushroom mycelium of woody mud state (KACC93057P), mushroom fruiting body of municipal mushroom and mushroom fruiting body of longevity, .

FIG. 1 is a view showing a process (a, b) of inoculating and culturing sawdust cultured on sterilized hardwood logs in the course of cultivation of mushroom fruiting bodies of woody mud, according to an embodiment of the present invention, c).
FIG. 2 is a photograph showing a planting of broad-leaved timber in the cultivation area by ground cultivation in the course of growing the mushroom fruiting body of woody mud according to an embodiment of the present invention.
FIG. 3 is a photograph of a mushroom fruiting body of a mushroom grown in accordance with an embodiment of the present invention.
FIG. 4 is a graph showing the genetic relationship between the ITS rDNA sequence of the mushroom fruiting body grown in accordance with an embodiment of the present invention and the nucleotide sequence of the ITS region of various pelinus species registered in the NCBI GenBank.
FIG. 5 shows a detailed homology arrangement with ITS nucleotide sequences of Pelinus lavis, Pelinus baumi, and Pelinus linteus, which showed a fungicidal relationship with woody mushroom fruiting bodies grown according to an embodiment of the present invention.

The present invention relates to a method for artificial cultivation of woody muddy mushroom fruiting body which can be cultivated industrially in a farmhouse and which is capable of only natural harvesting, and to a woody muddy mushroom fruiting body cultivated thereby.

Hereinafter, the present invention will be described in detail.

The method of artificial cultivation of fruiting bodies of mushroom mushroom of the present invention is carried out by Phellinus linteus strain [Accession No .: KACC93057P] is cultivated and inoculated on hardwood logs. Specifically, (A) a step of culturing the cultured Phellinus linteus strain in a culture bottle containing hardwood sawdust; (B) sterilizing hardwood logs; (C) inoculating the sawdust cultivated in the step (A) into the sterilized hardwood logs; And (D) planting the inoculated broad-leaved timber by ground cultivation to artificially grow mushroom fruiting bodies of woody mud.

First, in step (A), Phellinus lenteus strain is cultivated in a potato glucose agar medium (PDA), and then Phellinus linteus strain pulverized by adding distilled water is dispensed into a cultivation bottle containing hardwood sawdust, and cultivated at a temperature of 8 to 28 캜 and 50 to 80 % ≪ / RTI >

The carbon source, nitrogen source and minerals for promoting the growth of the Pelinus linteus strain are preferably dissolved in the distilled water before use.

In addition, rice bran and wheat bran can be used as a nutrient source in the culture bottle containing the hardwood sawdust, preferably 10 to 30 parts by weight of the rice bran and 10 to 30 parts by weight of the wheat bran per 100 parts by weight of the hardwood sawdust.

The hardwood used as sawdust may be one selected from the group consisting of oak, cherry, maple, mulberry, beech, beech, poplar.

Next, in step (B), the hardwood logs are cut, covered with vinyl foil, and sterilized at 100 to 121 ° C for 10 to 20 hours to prepare hardwood logs for mushroom cultivation.

Next, in step (C), the cultivated sawdust seedlings are inoculated into a sterilized hardwood tree (Fig. 1A). After that, the hardwood logs inoculated with the sawdust seeds are sealed in a plastic bag and cultured in a culture room at 23 to 26 DEG C for 110 to 120 days to allow the mycelium to be deposited on the entire upper surface (FIG.

The mycelium which is activated on the entire surface of the hardwood spruce is peeled off (Fig. 1 (c)), and the hardwood spruce is shocked 3-4 times for hyacinth.

The hardwoods used as the timber may be one selected from the group consisting of oak, cherry, maple, mulberry, beech, beech, poplar.

Next, in step (D), the impacted hardwood species is planted in a grower by ground cultivation (FIG. 2). In this case, when hardwood logs are directly planted on the ground, it is difficult to drain water and the logs are liable to be damaged by harmful insects and spoilage bacteria. Therefore, the ground on which the hardwood logs are planted is a surface on which the average diameter is 2 to 3 cm It is preferred that sand having a thickness of 4 to 5 cm is applied.

The cultivation is completed by putting one layer of vinyl and covering the upper surface with cacyimiron and vinyl sequentially.

When the planted hardwood timber is maintained at a temperature of 90% humidity and 25 to 32 ° C under a 70% light-shielding condition, a woody mud fruiting body is formed 100 to 130 days after planting the hardwood timber, and after 1 to 2 years Woody mud situation Mushroom Fruits can be harvested. This means that the cultivation period was shortened compared with the case where the fruiting body of the mushroom (Pelinus linteus line) was cultivated for 3 to 4 years.

The artificial cultivation method as described above can be used to grow mushroom fruiting bodies of woody mud.

In addition, an antioxidative composition, an anticancer composition, and an anti-inflammatory composition can be prepared using the extract obtained by extracting the mushroom fruit bodies of the above-mentioned woody mud as an active ingredient.

The antioxidant composition may be used as a medicine, a health supplement, a cosmetic, a food, a food additive. When used as a medicine, the antioxidant composition may further include an appropriate carrier, excipient and diluent commonly used in the production of a pharmaceutical composition. May be manufactured into a pharmaceutically acceptable formulation according to the method. In addition, when the antioxidant composition is used as a cosmetic, it can be formulated into wrinkle improving and whitening functional cosmetics, softening longevity, nutritional lotion, eye cream, nutritional cream, massage cream, cleansing cream, essence and the like.

The anticancer activity enhancing composition can prevent or treat diseases such as oral cancer, liver cancer, gastric cancer, or uterine cancer; The composition for the prophylaxis and treatment of anti-inflammatory diseases may be used for the treatment and prophylaxis of atherosclerosis, diabetes, arthritis, obesity, inflammatory bowel disease (IBD), Alzheimer's disease, multiple sclerosis, tuberculosis, sarcoidosis, , Inflammatory diseases such as hepatitis, cholecystitis, fungal infections, gastric ulcers, asthma, atopic dermatitis, tendinitis or nephritis can be prevented or treated.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

Example  One. Woody mud condition mushroom  Manufacture of fruit bodies

After culturing the Phellinus linteus strain (Accession No .: KACC93057P) for 14 days in a potato glucose agar medium (PDA), 500 ml of distilled water was added thereto, and 20 ml of the strain solution was cultured in the presence of 800 g of oak sawdust And the cells were cultured for 30 days at a temperature of 25 캜 and a humidity of 70%.

On the other hand, oakwood was cut into 20 cm in diameter and 30 cm in length, covered with a sterilized vinyl foil, and sterilized at 110 ° C for 10 to 20 hours to prepare oakwood for mushroom cultivation. Then, And 50 g of the cultivated sawdust seeds are inoculated on top of the oak timber, sealed with vinyl, and cultured at 26 DEG C for 120 days. When the cultivation is completed, the mycelium that has been activated on the whole oak wood is peeled off with an iron brush, and the oak wood is shocked 3-4 times with a sieve for the mycelial foot. The above-mentioned impacted oak timber was planted in a grower by ground cultivation method and maintained at 90% humidity and 25 to 32 ° C under 70% light-shielding condition, artificially grown woody mud fruiting body after 400 days.

The ground on which the oak timber is planted is a sand having a thickness of 5 cm on the upper surface of the gravel having an average diameter of 2 to 3 cm and the oak timber is planted once a day for 15 minutes for 12 to 14 hours Lt; / RTI >

Test Example  1. Identification of species by morphological observation of fruiting body

Table 1 below shows the morphological observations of mushroom fruiting bodies (Example 1) and longevity mushroom fruiting bodies in the woody mud state according to the time after planting.

division Woody mud condition Mushroom fruiting body Longevity situation mushroom fruiting body 40 days after planting

125 days after planting

345 days after planting

As shown in Table 1 above, mycelium grows on the oak timber after 40 days of planting, and after 80 days, it becomes tender after lapse of 80 days. After 125 days, the oak woody mushroom fruiting body (Example 1) was attached to the oak wood, and after 345 days, the oak wood was solidly attached to the oak wood in a semicircular shape and grown in size.

On the other hand, after lapse of 345 days, mushroom fruiting body of longevity condition was formed as mycelial mass without any characteristic as fruiting bodies.

As shown in FIG. 3, the outer surface of the mushroom fruiting body of the woody mud grown as described above (Example 1) has a solid wood structure and has a dense brown concentric ring, . In addition, the rear part has a rusty yellow carpet shape, and the peripheries of the peripherals and the bottom part are initially light yellow, but soon become yellowish brown and form an unclear multi-layer structure.

Microscopic examination of the naked eye and microstructure for accurate classification showed that the fruiting bodies are perennial and adhered to the host without consideration, and the pores to be formed are circular or angular, and the tube tools form 5-7 per / ㎜. The mycelium type is 2 - branch type, spores are 4.5 ~ 6 × 4 ~ 5 ㎛, ovate or ovate, and have pale yellowish brown color. This was identified as a typical morphological feature of Pelinus Linthus described in the previous paper.

As a result, artificially grown mushroom fruiting bodies were identified as Ferinerus linteus strain based on the morphology of fruiting bodies.

The mushroom fruiting bodies of the present invention can be harvested within 1 to 2 years due to the rapid formation of fruiting body, and 4 to 8 fruiting bodies are formed per tree (more than 99% pore forming rate). This result is much better than that of mushroom fruiting body, which takes three to four years to harvest and shows a fruiting body formation rate of less than 10% per log.

Test Example  2. Woody mud condition mushroom  Genetic characterization of fruit bodies

100 μg of the powder was transferred to a 1.5-ml test tube, washed with 200 mM Tris-HCl (pH: 10), pH 400 ㎕ of Proteinase K (20 mg / ㎖) was added to the wells and incubated at 37 ℃ for 1 hour. To this mixture, 2 × CTAB buffer was added in the same volume, left at 65 ° C for 15 minutes, mixed with chloroform isoamylalcohol (24: 1), thoroughly mixed and centrifuged at 12,000 rpm. The supernatant was transferred to a new tube, added with 0.7 volume of isopropanol, allowed to stand at room temperature for 10 minutes, and centrifuged at 12,000 rpm for 5 minutes to precipitate the DNA. The DNA precipitate was washed with 70% ethanol, vacuum-dried and dissolved in 100 μl of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). 2 μl of 10 mg / ml RNase was added and the mixture was treated at 37 ° C for 30 minutes to remove the RNA contained in the solution and used as template DNA.

ITS 1 (5'-TCCGTAGGTGAACCTCGGC-3 ') and ITS 4 (5'-TCCTCCGCTTATTGATATGC-3'), which were developed to amplify the fungal ITS rDNA region by PCR, were used to isolate the rDNA ITS region Respectively. For PCR amplification, the PCR reaction solution contained 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl ₂ , 0.01% gelatin, 100 ng prime, 50 ng template DNA, 200 쨉 m dNTP (dCTP, dTTP, dATP, dGTP), 20 pmol of 10 pmol of ITS 1 and 2 primers and 2.5 units of Taq polymerase (Promega) were added to make the total reaction solution 50 μl. The conditions for the PCR reaction were denaturation at 94 ° C. for 1 minute and denaturation at 94 ° C. for 4 minutes using a PCR instrument of PTC-200 (MJ Reasearch). Annealing was performed at 58 ° C. And DNA synthesis was performed at 72 ° C for 2 minutes in total, and final DNA synthesis was performed for 7 minutes. The nucleotide sequence of the PCR-amplified rDNA ITS region was determined as 5'-TCCGTAGGTGAACCTCGGC-3 'primer by Greengene Biotech.

As noted in the Sequence Listing, the rDNA ITS nucleotide sequence of the mushroom fruiting bodies of woody mud was composed of 730 bp and was registered with GenBank as the new nucleotide sequence (accession number KF589300). A homology search was performed in NCBI Blastn and the nucleotide sequence of the ITS region of the previously reported Phellinus species was input into the clustalW program of DNA star and aligned.

As shown in FIG. 4, the rDNA ITS nucleotide sequence of the mushroom fruiting body of woody mud and the ITS region of various ferrinus species registered in the NCBI GenBank were compared with each other. As a result, the mushroom fruiting body and the rDNA ITS nucleotide sequence were 100 There was no matching nucleotide sequence and 98% high homology to P. ribis , also known as brioche mushroom, but 95% of the mutants, Pelinus Baumi and Pelinus Linheus, And 96%, respectively. This indicates that the mushroom fruiting body of the woody mud is genetically closely related to Periners Leavis, Perrinus Linheus, and Perinus Baumi.

Fig. 5 shows the detailed homology alignment with the ITS nucleotide sequences of Pelinus lavis, Pelinus baumi and Pelinus linteus, which showed a fungicidal relationship with the woody mildew mushroom fruiting bodies. Mutations in seven nucleotide sequences were detected in 31-53 nucleotides, 41-42 nucleotides lacked only mushroom fruiting bodies and Pelinus linteus in woody mud, and in the highly mutated regions 177-181, high homology with pelinus linteus Respectively.

In conclusion, according to the genetic relationship of the rDNA ITS sequences, the woody mud fungus fruiting body constitutes the unique ITS nucleotide sequence and shows a genetic characteristic related to Perinus Leavis, Pelinus linteus and Pelinus baumi. In addition, the morphological characteristics of mushroom fruiting bodies of woody mud can be concluded to be typical Pelinus linteus, resulting in a new Pelinus linteus system.

Test Example  3. Woody mud condition mushroom  Antioxidant activity measurement of fruit body

10 g of each powder obtained by crushing the woody mushroom fruiting body, longevity mushroom fruiting body (Comparative Example 1), and mushroom fruiting body (Comparative Example 2) prepared according to Example 1 was mixed with 100 ml of water, and the mixture was homogenized After mixing at 2,500 rpm, 400 ml of ethanol was added and mixed. The ethanol extract was evaporated with a rotary evaporator and the remaining dried material was used in this experiment. The longevity condition mushroom fruiting body and the condition mushroom fruiting body were purchased from the Andong mushroom farm in Gyeongbuk.

Woody mud condition Phellinus (mycelium of mushroom fruiting bodies) Lactose Mycelium and mushroom mycelium of fruiting body were cut from the PDA-grown mycelium to obtain liquid type YGM (1% malt extract, 0.4% glucose, 0.4% yeast extract / water 1 L) were inoculated in plastic sterilized bottles (500 ml) at 120 ml each, and cultured for 25 days, and P. linteus (mycelium of mushroom fruiting bodies) was further cultured for 10 days. The same amount of mycelium was recovered from the culture solution, and 10 g of the same was pulverized and diluted with 10 mg / ml of ethanol extract according to the above fruiting body treatment method.

3-1. Electron donating ability measurement

Was measured by DPPH (1,1-diphenyl-2-picryl hydrazyl) free radical scavenging method according to the method of Blois (1958). 50 ml of water was added to the mycelium (5 g) and fruiting body (5 g) of mushroom (Example 1), mushroom and longevity condition of mushroom of woody mud, and 750 袖 l And 75 μl of distilled water was added to the control sample. After the reaction was carried out for 20 minutes at room temperature, the absorbance was measured at 517 nm. The degree of inhibition was calculated by the following equation (1).

division Example 1 The mycelium of Example 1 Comparative Example 1 The mycelium of Comparative Example 1 Comparative Example 2 The mycelium of Comparative Example 2 Inhibition rate (%) 46.4 24.2 42.1 20.5 45.1 9.8

* Final concentration is 200 ㎍ / ㎖.

As shown in Table 2 above, the inhibition rate of fruiting bodies of all three kinds of mushroom was superior to that of mycelia. In particular, it was confirmed that the degree of inhibition of mushroom fruit bodies grown according to Example 1 of the present invention was the best.

3-2. Xanthine oxidase ( Xanthine oxidase ) Low performance  Measure

Noro et al. (1983). (Example 1), mushroom mycelium, mushroom fruiting body and longevity mushroom fruity body extract and 1365 μl of phosphate buffer (pH 7.4) were mixed in a distilled water, 30 μl of xanthine solution (174 μg / ml in DW) was added, and 30 μl of a 250 mUnit / ml xanthine oxidase solution was added. For the control, distilled water was added instead of 75 μl of the sample. The difference in absorbance was measured at 295 nm for 3 minutes, and the enzyme inhibition was calculated by the following formula (2).

division Example 1 The mycelium of Example 1 Comparative Example 1 Comparative Example 2 Inhibition rate (%) × 1 68.4 40.1 49.7 78.1 × 2 99.9 80.4 90.0 97.4 × 4 78.3 60.2 76.8 80.4 × 8 13.2 4.9 22.4 47.6

As shown in the above Table 3, 100 μg / ml of fruit bodies and mycelium extracts were diluted 1, 2, 4 and 8 times, respectively. As a result, the woody mushroom fruiting bodies grown according to Example 1 of the present invention were 2 The antioxidant activity was 99.9% when diluted with water. The antioxidative activity of the mushroom was decreased as the concentration of mushroom fruiting body decreased. In addition, it was confirmed that the fruiting bodies of woody mushroom cultivated according to Example 1 were superior to those of woody mushroom mycelium, mushroom mushroom fruiting body and longevity mushroom fruiting body in the case of 2-fold dilution.

The xanthine enzyme is an enzyme that generates active oxygen, an enzyme that oxidizes hypoxanthine to xanthine and further oxidizes it to uric acid of xanthine (Noro et al ., 1983). Therefore, the enzyme inhibitory effect was measured by comparing the amount of uric acid oxidized by xanthine enzyme.

Test Example  4. Woody mud condition mushroom  Antiinflammatory Activity of Fruiting Body Nitrate Oxidation  assay

RAW264.7 cells were plated at 4 × 10 ⁴ per well (200 μl) in a 96-well plate and cultured at 37 ° C and 5% v / v CO ₂ for 24 hours. The cells were serially diluted twice with distilled water (Example 1), woody mud mushroom mycelium, mushroom fruiting body and mushroom fruiting body extract of long mushroom mushroom were added at a dose of 100 占 퐂 / ml. After inoculation, LPS (Lipopolysaccharide) was added at a concentration of 0.1 ㎍ / ㎖ for 30 minutes at 37 ℃ and 5% v / v CO ₂ for 18 hours to induce inflammation.

The cultured cells were taken out and 100 μl of the supernatant was transferred to a 96-well plate in each well. Greiss reagent A (0.1% NEDHC aqueous solution) and greiss reagent B (5% H ₃ PO ₄ , 1% Sulfanil amide mixed aqueous solution) : 1 < / RTI > After the reaction, the absorbance was measured at 540 nm using a microplate reader, and the standard value (control group) was obtained by diluting the value by 2 × with NaNO _{2. The} relative value (y = ax + b, x '= (y'-b) / a, x' is the NO generation value (uM) and y 'is the measured value of the comparative group.

Woody mud condition Mushroom fruiting body (Example 1), woody mud condition mushroom mycelium, mushroom fruiting body and longevity condition The mushroom fruiting body was used for the NO assay to examine inflammation inhibition mechanism. First, the NO assay was used to measure the production of nitric oxide, the major product in the immune system of mammalian cells, using RAW264.7 cell line, a macrophage. When LPS is added, it stimulates the macrophages via the tool-like receptor 4 and affects the expression of the inducible NO synthase (iNOs) to synthesize NO. LPS constitutes the cell wall of Gram staining (-) Bacteria and is an inflammation inducer (Yun et al ., 1996). At this time, the generated NO ₂ ^- , NEDHC, and sulfanilamide are reacted to form an azo coupling. These two ring structures have the maximum absorbance at a wavelength of 540 nM. Therefore, the production of NO can be indirectly measured by measuring this value (Kim et al ., 2010).

division Example 1 The mycelium of Example 1 Comparative Example 1 Comparative Example 2 NO induction
(uM) Control 0.0 0.0 0.0 0.0 LPS 22.7 19.8 20.8 18.4 × 1 7.4 9.1 7.8 8.8 × 2 11.4 13.4 8.8 10.2 × 4 13.2 14.6 10.1 13.1 × 8 14.5 15.4 13.4 14.1 × 16 15.5 16.5 15.1 14.5 × 32 17.8 18.4 17.7 16.2

As shown in Table 4 above, 100 쨉 g / ml of the fruit bodies and mycelium extracts were diluted 1, 2, 4, 8, 16 and 32 times, respectively. As a result, The mushroom fruiting bodies were found to be highly effective in inhibiting inflammation at a dilution of 1 to 7 μM (the undiluted solution itself), and the inflammation inhibitory effect was decreased as the concentration of mushroom fruiting bodies in the woody mud state decreased. In addition, it was confirmed that the fruit mushroom fruiting bodies grown according to Example 1 were superior to the mushroom mycelium of woody mud, mushroom fruiting bodies and long mushroom mushroom fruiting bodies by 1-fold dilution.

Test Example  5. Woody mud condition mushroom  Cell survival rate of fruit body MTT assay ) Measure

Cytotoxicity was measured by using 3-3 (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) of Green et al. RAW 264.7 cells were plated at 4 × 10 ⁴ per well (200 μl) on a 96-well plate and cultured at 37 ° C and 5% v / v CO ₂ for 24 hours. And 80 μl of each of them was inoculated to each well at a rate of 20 μl per well. The resultant was inoculated with 20 μl per well. The stock solution of mushroom fruiting body (Example 1), mushroom mycelium, mushroom fruiting body, / Ml. 50 μl of MTT (-20 ° C, 2 mg / ml) solution was added to the cultured cell culture medium, and the cells were again cultured for 4 hours under the same conditions as above to form formazan. After 4 hours, the supernatant of the medium is slowly removed and 150 μl of DMSO is added to each well. The formazan is sufficiently dissolved in a shaker for 30 minutes and the absorbance is measured at 540 nm in a microplate reader. Cell viability was calculated by the following formula (3).

The toxicity of mycelial fruiting bodies of RAW264.7 cells used for anti - inflammation (Example 1), mycelial mushroom mycelium, mushroom fruiting bodies and long - term mushroom fruiting body mycelium extracts were investigated by MTT assay. MTT assay is a method to measure the MTT assay by using MTT tetrazolium, a water-soluble substrate, through the metabolism of intracellular metabolites. MTT formazan, MTT formazan (3- (4,5-dimethylthiazol-2-yl) -tetrazolium bromide) to measure the number of living cells indirectly by measuring the concentration of promaza formed by living cells. It is a widely used method for measuring cell toxicity or viability (Green et al ., 1984).

division Example 1 The mycelium of Example 1 Comparative Example 1 Comparative Example 2 Cell viability (%)
Control 100.0 100.0 100.0 100.0 LPS 83.4 78.5 82.1 80.7 × 1 70.4 64.1 78.7 83.9 × 2 70.6 63.8 72.4 79.8 × 4 77.8 65.7 81.8 83.7 × 8 79.9 70.2 80.7 84.0 × 16 82.9 75.8 81.5 84.2 × 32 82.1 75.9 87.7 84.9

As shown in Table 5, when 200 ㎍ / ㎖ of fruit bodies and mycelium extracts were diluted with 1, 2, 4, 8, 16 and 32 times, respectively, the woody mud mushroom mycelium Both fruiting bodies (Example 1), mushroom fruiting body and long fruiting body mushroom fruiting bodies were found to have almost no cytotoxicity since they maintained a cell viability of 70% or more.

<110> Hankyong Industry Academic Cooperation Center <120> Cultivation method of Phellinus linteus and Phellinus linteus          cultivated thereby <130> HPC4637 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 730 <212> DNA <213> phellinus linteus <400> 1 aaggatcatt atcgagtttt gaaagcgaga cttgctgctg gcgcgaaatt ttcgcgcatg 60 tgcacggcct tcgcgctcaa atccaactca aacccctgtg cacctttata tatcgcaagt 120 cgaagttagt agtctgaggt tcttgtaagt aatcggtagg aaggcgaaag cgagtgttac 180 tcgttaggta acctttcgaa aatgaaagcg agagcgtcgg gtgaagactt cggcttgttg 240 ttattacaca aacaccttat attgtcattg tgaatgtaat gctcctcgtg ggcgaaaagg 300 aatacaactt tcaacaacgg atctcttggc tctcgcatcg atgaagaacg cagcgaaatg 360 cgataagtaa tgtgaattgc agaattcagt gaatcatcga atctttgaac gcaccttgcg 420 ccccttggta ttccgagggg catgcctgtt tgagtgtcat gttaatctca aaccgctcgt 480 ctttcttaat cgaagggctt gcggtttgga cttggagggt tactgctggc gcctcttgag 540 gggtcggctc ctcttaaata tattagctgg gttttggctc gcgtttacgg tgtaatagtt 600 gattccattc accaacgagc gcttgcctga cgagcctgct tgaaaatcgt ccgcgtcgtc 660 ggacaaggag ttttacctcc ttcttgacac ctttgacctc aattcaggta ggattacccg 720 ctgaacttaa 730

Claims (15)

Phellinus linteus strain [accession no.: KACC93057P], and then cultivated by inoculating the hardwood logs. The method of artificial cultivation of mushroom fruiting bodies of woody mud. The method according to claim 1,
(A) The cultured Phellinus linteus ) in a culture bottle containing hardwood sawdust;
(B) sterilizing hardwood logs;
(C) inoculating the sawdust cultivated in the step (A) into the sterilized hardwood logs; And
(D) planting the inoculated broad-leaved timber by ground cultivation. The method according to claim 2, wherein the Phellinus cultured in step (A) wherein the strain is cultivated in a potato glucose agar medium (PDA) and then pulverized by adding distilled water. The method of artificial cultivation of mushroom fruiting body of woody mud. [3] The method according to claim 2, wherein in step (A), the culture bottle containing the Pelinus linteus strain and the hardwood sawdust is cultured at a temperature of 18 to 28 DEG C and a humidity of 50 to 80% Of artificial cultivation method. The method according to claim 2, further comprising the step of culturing the hardwood sprout seeded with the sawdust seedlings at 23 to 26 ° C for 110 to 120 days after the step (C) A method of artificial cultivation of mushroom fruiting body. 6. The method according to claim 5, further comprising the step of culturing the hardwood sprouts inoculated with the sawdust seeds, followed by peeling off the mycelial sprouted on the whole surface of the hardwood sprouts and applying an impact to the hardwood sprouts. Of artificial cultivation method. 3. The method according to claim 2, further comprising the step of maintaining the hardwood logs planted after step (D) at a temperature of 90% humidity and a temperature of 25 to 32 DEG C under 70% light-shielding conditions. Of artificial cultivation method. [3] The method according to claim 2, wherein the ground to be cultivated in step (D) is sanded with sand having a thickness of 4 to 5 cm on the upper surface of the gravel having an average diameter of 2 to 3 cm. Cultivation method. [3] The method according to claim 2, wherein in step (D), 100 to 130 days after the plantation of the hardwood species, the mushroom fruiting body of woody mud is formed. [3] The method according to claim 2, wherein the hardwood species is planted in step (D) and the mushroom fruit bodies are harvested one to two years later. [3] The method according to claim 1, wherein the broad-leaved tree is selected from the group consisting of oak, cherry, maple, mulberry, beech, beech, poplar. A woody mushroom fruiting body produced by a cultivation method according to any one of claims 1 to 11. An antioxidative composition comprising the fruit body extract of mushroom mushroom according to claim 12 as an active ingredient. A composition for enhancing anticancer activity comprising the fruit mushroom extract of the present invention as an active ingredient. A composition for preventing and treating an anti-inflammatory disease, which comprises the mushroom fruiting body extract of the present invention as an active ingredient.

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