KR100555299B1 - Culture medium of Phellinus gilvus containing sawdust and method for artificially culturing Phellinus gilvus using the same - Google Patents

Abstract

The present invention relates to a dry mud mushroom culture medium and a method for artificially cultivating dry mud mushrooms using the medium, and more specifically, to a dry mud mushroom mycelium and fruiting body culture medium and sawmedium containing sawdust as a main component Culturing the spawn of the dry mud mushrooms using to obtain a mycelium; Forming a fruiting body of the dried mud mushroom from the dried mud mushroom mycelium; And it relates to a method of artificial cultivation of dried mud mushrooms comprising the step of growing fruiting bodies from the fruiting body origin. Artificial cultivation of dried mud mushrooms using the medium of the present invention can be useful for producing large amounts of dried mud mushrooms having excellent pharmacological effects because the yield is increased and the number of days of cultivation is reduced compared to the conventional methods for cultivating solid wood have.

Description

Culture medium of Phellinus gilvus containing sawdust and method for artificially culturing Phellinus gilvus using the same}             

1 is a photograph of culturing dried mud mushroom mycelium using the sawdust medium of the present invention, the composition of the sawdust medium is different.

A: 100% oak sawdust, B: 95% oak sawdust + 5% rice bran,

C: oak sawdust 90% + rice bran 10%, D: oak sawdust 85% + rice bran 15%,

E: oak sawdust 80% + rice bran 20%, F: oak sawdust 75% + rice bran 25%,

G: oak sawdust 70% + rice bran 30%

Figure 2 is a photograph showing the grown form of dried mud mushroom fruiting body cultivated using the sawdust medium of the present invention, the composition of solid oak and sawdust medium.

A: solid oak, B: 100% oak sawdust,

C: oak sawdust 95% + rice bran 5%, D: oak sawdust 90% + rice bran 10%,

E: oak sawdust 85% + rice bran 15%, F: oak sawdust 80% + rice bran 20%,

G: oak sawdust 75% + rice bran 25%

Figure 3 is a graph comparing the anticancer activity of the extract from the fruiting body distilled water extracts of the dried mud mushrooms of the wood cultivation using the distilled water from the fruiting body of dried mud mushrooms cultured using the sawdust medium of the present invention.

Figure 4a is the anticancer activity of the ethanol extract of dried mud mushroom fruiting body ethanol extract of wood cultivation using 20%, 40% and 60% ethanol from the fruiting body of dried mud mushroom cultured using the sawdust medium of the present invention It is a graph comparing.

Figure 4b is a graph comparing the anticancer activity of the extract extracted with 80% ethanol from the fruiting body of dried mud mushrooms cultured using the sawdust medium of the present invention and the anticancer activity of the dried mud mushroom fruiting body ethanol extract of wood.

5 is a process chart showing a process of culturing dried mud mushrooms using the sawdust culture medium of the present invention.

The present invention relates to a medium for culturing dried mud mushrooms and a method of cultivating dried mud mushrooms using the same, and more specifically, to a method of cultivating dried mud mushrooms containing sawdust as a main component and artificially culturing dried mud mushrooms using the medium. It is about how to.

Phellinus gilvus, also known as situation mushroom, is a white fungus belonging to Aphylophorales, Hymnenochaetaceae and Phellinus , which occurs mainly on hardwoods such as oak and mulberry. However, shiitake mushroom cultivation occurs in oak alleys and causes disease (Tea et al., 1994, Latest Mushroom Cultivation Technology, Nongjinhoe, 325p). The fruiting bodies of dried mud mushrooms are known as perennial or perennial cork, and occur in a hemispherical shape attached to a tree, usually in a colony type, in which upper and lower sides are connected to each other, and the size is about 5 to 12 cm in diameter. Upper surface of fruiting body is dark yellowish brown with villi, edges are lighter in color than lampshade, and the surface of duct is dark brown with 6 to 8 per mm, spores are 4 to 5 × 3 to 3.5 ㎛ Surface is known to be smooth (Ryvardan, L., 1993, Europeon Polypores, Fungiflora, Norway).

Dry mud mushrooms have been used as a valuable herbal medicine since they have been shown to enhance immune function when combined with chemotherapy following resection of liver cancer including gastric cancer, esophageal cancer, duodenal cancer, colon cancer, and rectal cancer. In addition, it activates and detoxifies uterine bleeding and treatment, dysmenorrhea, intestinal bleeding, intestinal and gastrointestinal functions. (Ikegawa et al ., Gann , 59, 155, 1968) reported that dried mud mushrooms had higher tumor retardation rate than cloud mushrooms ( Coriolus versicolor ) and shiitake mushrooms ( lentinus edodes ), and protein nucleic acid. Enzyme , 21, 426, 1976) reported an anticancer activity of 96.7%. In addition, Chung et al. ( Journal of Pharmacy , 38, 158, 1994) reported that the protein polysaccharide of mycelium effectively inhibits Sarcoma 180 ascites and solid cancer, and Kim et al . ( Arc. Pharm. Res ., 17, 337, 1994). ) Analyzed the polysaccharides having antitumor activity and reported that glucose is the main ingredient, and that it consists of galactose, mannose, and arabinose. Then, the ball, etc. (Journal of Medicinal Plants, 22, 233, 1991) by a hot-water extract of the dry mud mushroom mycelium by acting on NK cell function, enhance non-specific immunity of the host cell exhibits anticancer activity, and the results of administration to the mouse LD ₅₀ This appeared to be 1,500 mg / kg or more and reported that there was stability.

As described above, dry mud mushrooms have been reported to be excellent in anticancer activity and immune enhancing effect. In Korea, dried mud mushrooms are commercially available at high prices for anticancer treatment. However, natural dry mud mushrooms are known to be difficult to obtain because they are rare and very expensive, and pharmacological effects are also known to vary depending on the region and spawn. Thus, a lot of research has been done to artificially cultivate dried mud mushrooms.

Conventional dry mud mushroom cultivation is usually grown in order to secure a strain, cultivate the mycelium from the strain in a mycelium culture medium, and then form a fruiting body from the mycelium in the fruiting body forming medium to grow the dry mud mushroom.

As a strain of dried mud mushrooms, there is a method of harvesting wild dried mud mushrooms to obtain a strain therefrom or using a strain obtained from a microbial deposit research institution. However, wild dry mud mushrooms are similarly distributed in the natural world, and because of the low number of occurrences, it is not easy to obtain the seed from them, and the situation is usually used to purchase the seed from a specific seed culture center in Korea.

The mycelial culture medium of dried mud mushrooms is prepared by adding mulberry or oak as sawdust to other trees, and adding rice bran to it. The culture medium serves to supply carbon source, nitrogen source and mineral source so that the dry mud mushrooms can grow sufficiently, and their proper mixing ratio greatly affects the quality and growth rate of the dried mud mushroom. Conventional culture medium for cultivating mycelium is based on the addition of 10% of rice bran to mulberry sawdust 30%, and mixes about 30% of cottonwood, oak or alder and supplies water to it. To create. In some cases, only pure edible grains based on rice are used. In addition, as a culture vessel for storing such a medium component, a bottle-shaped one made of glass, plastic, or polypropylene (PP) is frequently used.

When the mycelium of dried mud mushrooms is sufficiently cultured, it is possible to produce good quality mud mushrooms by forming the fruiting bodies by transferring them to the fruiting body-forming culture medium. To date, wood is mainly used as a culture medium for forming fruiting bodies. However, since the formation of fruiting bodies during the artificial cultivation is difficult, many of the biofilms in which the mycelium is agglomerated are traded in the private sector, and some artificially grown mushrooms are sold at high prices. However, up to now, the technique for cultivating dry mud mushrooms is not generally made, and there is a disadvantage in that the management of timber cultivation is difficult and pollution is easy.

Therefore, the present inventors tried to improve the culture medium used in the fruiting body formation process, which is the process of growing artificial mushrooms to artificially cultivate a large amount of dried mud mushrooms having excellent pharmacological effect and produce a large amount of wood instead of the conventional wood. As a result of artificial cultivation using a sawdust culture medium, the present invention was completed by confirming that the formation of fruiting bodies is superior to that of conventional wood cultivation.

An object of the present invention is to provide a medium for culturing dried mud mushroom mycelium and fruiting body containing sawdust as a main component.

Another object of the present invention is to provide a method for artificially cultivating dried mud mushrooms using the medium.

In order to achieve the above object, the present invention provides a dry mud mushroom mycelium and fruiting body culture medium containing sawdust as a main component.

In addition, the present invention

1) culturing the spawn of the dry mud mushroom using the medium to obtain a mycelium; And

2) provides a method of culturing dried mud mushrooms comprising culturing dried mud mushroom fruiting bodies from the mycelium of the dried mud mushrooms using the medium.

Hereinafter, the present invention will be described in detail.

The present invention provides a dry mud mushroom mycelium and fruiting medium culture medium containing sawdust as a main component.

In the medium of the present invention, the wood used as the material of the sawdust can be selected from the group consisting of oak, mulberry, elm and apple, it is preferable to use oak ( Quercus sp .). In the present invention, oak logs were harvested between November and February of the year when the movement of the sap was stopped, and the oak logs were harvested by drying the oak logs in the shade and using a sawdust maker when the moisture content is 35 to 45%. Prepared. The prepared sawdust was autoclaved at 121 ° C. for 90 minutes to maintain aseptic status.

The medium of the present invention is characterized in that the sawdust is used as a main component, it is prepared by further mixing the nutrient source. The nutrient source is preferably at least one selected from the group consisting of rice bran, bran, corn cob, rice hull and peanut shell, and more preferably rice bran.

In the present invention, a culture medium was prepared by mixing rice bran with sawdust, and sawdust and rice bran are preferably mixed at a ratio of 8: 2 to 10: 0. In the present invention, oak sawdust and rice bran were mixed in a ratio of 10: 0, 9.5: 5, 9: 1, 8.5: 1.5, 8: 2, 7.5: 2.5, 7: 3, respectively, to prepare a sawdust medium. As a result of culturing dried mud mushrooms, it was confirmed that the spawn growth rate was excellent in the medium prepared by mixing at a ratio of 10: 0 to 8: 2, and the mixing ratio of oak sawdust and rice bran was 10: 0 to 8: 2. It was found that mixing is preferred.

In addition, the present invention

1) culturing the spawn of the dry mud mushroom with a medium containing the sawdust of the present invention to obtain a mycelium;

2) culturing the mycelium of step 1 in the medium containing the sawdust of the present invention to form fruiting body primitive; And

3) It provides an artificial cultivation method of dried mud mushrooms comprising culturing the fruiting body raw material formed in step 2 to the medium containing the sawdust of the present invention to grow fruiting bodies.

In the artificial cultivation method of the present invention, culturing the seed mite of the dry mud mushroom of step 1 is preferably incubated for 25 to 30 days at a temperature of 25 to 30 ℃ using the sawdust culture medium of the present invention.

In addition, it is preferable to incubate the fruiting body of step 2 for about 15 days at a humidity of 90% or more and a temperature of 22 to 27 ° C using the sawdust culture medium of the present invention.

In addition, the growth of the fruiting body of step 3 is preferably cultured at a humidity of 70 to 85%, temperature 25 to 32 ℃ using the sawdust culture medium of the present invention.

Detailed description of the dried mud mushroom artificial cultivation method of the present invention is as follows, the manufacturing process is briefly shown in FIG.

<First step> Preparation of spawn

The dry mud fungi were sterilely implanted in PDA, MEA medium, and the like, followed by completion of mycelial culture for 10 to 15 days in an incubator at a culture temperature of 25 to 30 ° C. After transplanting autoclaved oak sawdust + rice bran (8: 2) sawdust medium at 121 ℃ for 90 minutes, cultivate dried mud mushrooms. Sawdust spawn complete with germ transplantation is spawned for 25 to 30 days at a temperature of 25 to 30 ° C.

<Step 2> Preparation of medium material (sawdust, rice bran)

The wood used as the main media is oak (Quercus sp.), Which is used as sawdust and logs. Log felling is used between November and February of the next year when the movement of sap is stopped, and it is naturally dried in the shade after logging. Solid wood is cut to 12 to 15 cm in diameter and 20 cm in length (about 900 g) when the water content is 35 to 45%. Sawdust for sawdust cultivation is manufactured with a sawdust maker and used after a three-month dead stock. Use freshly dried rice bran.

<Step 3> Production and Sterilization of Medium

0%, 5%, 10%, 15%, 20%, 25%, 30 each of rice bran as a nutrient source in oak sawdust (900g before moisture control), the main ingredient in the production of dried mushroom medium Add by% and mix uniformly. After adjusting the water content of the medium to 65%, put about 2 kg in a heat-resistant polypropylene bag and close the stopper and autoclave it at 121 ℃ for 90 minutes. The autoclaved medium is cooled to 15 ° C. before the seed inoculation.

<Step 4> spawn inoculation and culture

The dry mud mushroom spawn grown in the first step in a clean room or aseptic phase is inoculated into a sawdust medium which has been sterilized and cooled in an inoculation room maintained at 15 ° C. Inoculation method is about 100g of spawn per 2kg sawdust medium. At this time, the opening and closing of the heat-resistant plastic bag should be carried out as soon as possible with the minimum exposure time to prevent contamination by various germs. Sawdust medium after the inoculation is incubated for 25 to 30 days in a culture chamber maintained at a temperature of 20 to 25 ℃. At the beginning of the cultivation, the temperature is maintained at about 20 to 22 ° C. for about 7 days, and then, after the dry mud mushrooms adhere to the sawdust medium, the culture is performed at about 25 ° C.

<Fifth Step> Induction of Mushroom Fruits

Soil for the generation of fruiting body selects sand that is favorable for drainage and water retention, and growers are installed based on the Ganoderma lucidum grower. After incubation of the mushroom spawn, the contaminated medium is selected from the sawdust medium and discarded. That is, after the mycelia culture is completed in the culture chamber, about 70% of the medium is removed from the upper layer, and then sufficiently watered to induce mushroom development. In order to induce the formation of mushroom fruit body, it is maintained for about 15 days at humidity above 90% and temperature of about 22 to 27 ℃. At this time, the fruiting body origin of dried mud mushroom is formed. Mushroom growth is carried out under the conditions of 25 to 32 ℃.

<Step 6> fruiting body growth and harvest

Dry mud mushroom fruit body freshness occurs, and when normal growth progresses, it grows about 3 to 10 cm in diameter when it is about 40 to 60 days, and the edge of growing fruit body is yellow, and the growing fruit body is almost yellow. It disappears and the fruiting body becomes dark brown. At this time, the mushrooms are harvested and the mushrooms are dried for 2 to 3 days in a 40 to 50 ° C. dryer.

Dry mud mushrooms cultivated using the sawdust culture medium of the present invention is not only about twice as fast as the mycelial growth rate compared to the dried mud mushrooms cultivated using conventional wood (see Table 3), the production efficiency of fruiting bodies Figure 2 is improved more than 2 times (see Table 4). In addition, the general and elemental components of the cultured mushroom fruiting body were also maintained (see Table 2 and Table 5).

In addition, the dry mud mushrooms have been found to have anti-cancer activity, the extract extracted with water or ethanol from the dried mud mushroom fruiting body cultured using the sawdust culture medium of the present invention is dried mud mushroom fruiting body cultured using the wood medium It was confirmed that the anticancer activity is much higher than the extract of (see Fig. 3, 4a and 4b).

Therefore, using the method of the present invention artificially cultivated using a sawdust culture medium compared to the conventional cultivation method using the wood can be seen to increase the yield and shorten the number of days required, cultivation using sawdust culture medium using wood Unlike cultivation, much of the working process can be mechanized, and two to three machines can be cultivated in the same place a year, so that dry mud mushrooms having various pharmacological effects can be produced in large quantities at low cost.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Cultivation of Dry Mud Mushroom Seedling

<1-1> Culture characteristics of dried mud mushrooms

Phellinus gilvus (KCTC 6653), 5 mm round × 5 mm thick, was transplanted into PDA (MERCK), MEA (MERCK), YEA (MERCK) or Czapek (MERCK) medium Then, mycelial incubation for 10 days in a thermostat of 10 to 35 ℃ culture temperature. After the mycelia were cultured in each culture medium, the mycelial growth was measured according to the culture medium and the culture temperature.

As a result, it was found that the dry mud mushroom mycelium grows well in PDA and MEA medium, and the fastest growing temperature is 25 to 30 ° C. (Table 1).

Culture Characteristics of Dried Fungi Temperature (℃) Mycelial growth (mm / 10 days) PDA MEA YEA Czapek 10 5 5 5 5 15 12 16 12 11 20 29 40 31 11 25 41 52 40 13 30 81 55 50 14 35 38 31 45 11

<1-2> spawn culture of dried mud mushrooms

As a seed culture medium, sawdust and rice bran of autoclaved oak (Quercus sp.) Were used at 90 ° C. for 90 minutes. Oak wood was used as a control. Logging of oak logs was used between November and February of the next year when the movement of sap was stopped, and was naturally dried in the shade after logging. When cutting the wood, the water content was 35 to 45%, and the diameter was 12 to 15 cm and the length was 20 cm (about 900 g). The sawdust for the sawdust was manufactured using a sawdust maker and used after 3 months of unloading. The added rice bran was freshly dried rice bran (chilgok pesticide, rice mill). First, physicochemical components of oak sawdust and rice bran were analyzed. Physicochemical analysis of the components was carried out according to the Soil Physicochemical Analysis (Han Ki-hak, 1988). CaO, MgO, and K ₂ O were weighed out in 10 g of the prepared sample in an Erlenmeyer flask, and then 50 ml of 0.1 N HCl solution was added.Then, the mixture was shaken with a rotary shaker at room temperature for 1 hour, filtered through filter paper, and the filtrate It was measured by an Atomic Absorption Spectrophotometer (Perkin Elmer 2380). Total carbohydrates were Tiurin method, total nitrogen was Geljal method, P ₂ O ₅ was colorimetric method, pH was immersed 5 g of dry sample in 25 ml of distilled water for 30 minutes and then pH meter (Fisher model) -50) (Han Ki Hak, 1988, Soil Physicochemical Analysis , Rural Development Administration, 26 ~ 214).

As a result, TC was similar in oak sawdust (32.2%) and rice bran (38.9%), and TN was higher in rice bran (2.2%) than oak sawdust (0.58%) and C / N ratio was 32.2% in oak sawdust. , the rice bran showed a _{17.7%, P 2 O 5,} MgO, K 2 O were rice bran contains more than oak sawdust (Table 2).

Analysis of Physicochemical Components in Media Discharge material pH content(%) T-C T-N C / N P ₂ O ₅ CaO MgO K ₂ O Oak sawdust 4.6 32.2 0.58 55.5 0.01 0.90 0.07 0.34 Rice bran 6.6 38.9 2.20 17.7 3.09 tr 1.83 2.86

In the above, tr indicates that the content is extremely small and cannot be measured.

After measuring the content of the medium composition, oak sawdust and rice bran were mixed to prepare a seed culture medium. Volumetric ratios of oak sawdust (900 g before moisture control) and rice bran as nutrients were 100: 0, 95: 5, 90:10, 85:15, 80:20, 75:25 and 70:30, respectively (v / v%). Mixed uniformly). The moisture content of the medium was adjusted to 65%, and then, about 2 kg were added to the heat-resistant polypropylene bag, and the stopper was closed. The solution was autoclaved at 121 ° C. for 90 minutes. The autoclaved medium was cooled to about 15 ° C. before spawn inoculation. 2 g of each sawdust medium thus prepared was inoculated with 100 g of prokaryotic mycelial culture at 25 to 30 ° C. for 10 days in PDA or MEA medium. Opening and closing the heat-resistant plastic bag during inoculation was carried out as soon as possible to minimize the exposure time to prevent contamination by various bacteria. After inoculation, the sawdust medium was cultured for 25 to 30 days in a culture chamber maintained at a temperature of 20 to 25 ℃. Initially, the temperature was maintained at about 20 to 22 ° C. for about 7 days, and the dried mud mushrooms were cultivated at sawdust medium and then cultured at about 25 ° C. In addition, as a control, the prokaryote was transplanted to oak logs and incubated for about 56 days. After incubation, the mycelial growth rate was confirmed by measuring the density of dried mud mushroom mycelia according to each culture medium.

As a result, the number of days of culture completion according to the sawdust medium prepared in the present invention is 25 to 30 days, the number of culture days required to reduce the number of days required to cultivation than 56 days of the control medium wood medium was reduced to about 1/2. The same culture days 25-30 days were higher than the mycelial density of wood medium. The growth rate of mycelia was the fastest with 25 days of sawdust medium containing 5% and 10% of rice bran. In addition, the density of hyphae was high in sawdust medium containing 20% and 25% of rice bran, and the higher the added density of rice bran, the higher the mycelial density. However, sawdust medium containing 30% of rice bran was contaminated with blue fungus ( Trichoderma sp.). This was judged to be easy to inoculate contaminating pathogens due to excessive nutrients in the medium (Table 3 and Figure 1).

Mycelial Growth Characteristics of Dry Mud Mushrooms by Different Media Materials (25 ℃) Culture medium Number of days of culture (day) Mycelial Density ^* Oak Sawdust (v / v,%) Rice bran (v / v,%) 100 0 28 +++ 95 5 25 +++ 90 10 25 +++ 85 15 28 +++ 80 20 28 ++++ 75 25 30 ++++ 70 30 － － Oak 56 +++

* Mycelial density: + + = low, + + + = medium, + + + + = high

Example 2 Cultivation of Dry Mud Mushroom Fruiting Body Using Sawdust Medium

After completion of the incubation of the dry mud mushroom spawn in Example 1, the contaminated medium in the sawdust medium was selected and discarded. The soil for cultivation of fruiting body was selected sand that is free for drainage and water retention, and the grower was installed based on the Ganoderma lucidum grower. After the mycelial culture was completed in the culture chamber, about 70% of the medium was removed from the upper layer of the heat-resistant polypropylene bag, and then sufficiently watered to induce mushroom development. To induce mushroom fruit body formation, the fruit body of the dried mud mushroom was incubated for about 15 days at a humidity of 90% or higher and a culture temperature of 22 to 27 ° C. for about 15 days. After confirming the primordial formation of the fruiting body, mushroom growth was carried out under the conditions of humidity of 70 to 85%, temperature 25 to 32 ℃. When the dried mud mushroom fruit body is formed and normal growth progresses, it grows about 3 to 10 cm in diameter when it is about 40 to 60 days, and the edge of the growing fruit body is yellow and the fruiting body that is almost finished growing disappears yellow. The fruiting body becomes dark brown, wherein the mushrooms were harvested and dried in the dryer at 40 to 50 ° C. for 2-3 days. After the mushroom was produced, the number of days required for the first shoot, fruiting body weight (g), fruiting body weight (g) was measured to confirm the production efficiency of the mushroom fruiting body according to the culture medium.

As a result, the number of days required for the initial development of mushroom base after removing the heat-resistant polypropylene bag and inducing mushroom generation by irrigation was 11 to 13 days for each sawdust medium treatment. Similar. In addition, 231 g, 38.7 g of dry weight, and 25.6% of production efficiency in control wood logs, 577 g of sawdust medium with 10% of rice bran, 97 g of dry weight, and production efficiency. 64.1% showed higher production efficiency than using oak logs as it was, and rice bran contained in sawdust medium contained 0 to 20% by volume ratio (Table 4 and 2). Therefore, as shown in Figure 2, compared to the dry mud mushrooms cultivated using the oak wood medium medium, the fruit body weight of the dried mud mushrooms cultivated using the sawdust culture medium (sawdust itself or rice bran added medium) is much larger, the yield is excellent Able to know.

Fruiting Body Production Efficiency by Sawdust Culture Medium Culture medium Days to begin Fruiting body weight (g) Fruiting body building (g) Production efficiency ^a (%) Quantity index (in buildings) Oak Sawdust (v / v,%) Rice bran (v / v,%) 100 0 11 570 92 63.3 232 95 5 11 421 66 46.8 167 90 10 12 577 97 64.1 222 85 15 12 550 81 61.1 201 80 20 12 382 83 42.4 210 75 25 13 166 39 18.4 99 70 30 - - - - - Oak 12 231 38.7 25.6 100

A = (fruiting body weight / medium material building) × 100

Example 3 Investigation of Components of Dry Mud Mushroom Cultured in Sawdust Medium

Therefore, considering the situation of the mushroom (P. linteus) and long life situation mushroom (P. baumii) grown over at Kyungpook National Agricultural Technology 2-5 months dry mud mushroom (P. gilvus) grown in sawdust and three years The results are shown in Table 5 by comparing the composition ratio of the element content. Elemental contents of fruiting body for varieties of mud mushrooms were 45.6% to 46.8%, 40.1% to 42.1% oxygen, 5.3% to 5.5% hydrogen, 0.8% to 1.2% nitrogen, and no sulfur was analyzed. No difference was found, and the content of the ingredients was almost the same as that of the wood-grown mud mushrooms. In addition, the results of analyzing the general components are shown in Table 6. The moisture content of dried mud mushrooms was 10%, showing the lowest water content, and the total dietary fiber showed 29% to 33%, showing no difference among the varieties. In addition, there were no significant differences in all the content comparisons, including ash, protein, and vitamin C. Therefore, even when grown in sawdust medium, the general components and elemental components of the dried mud mushrooms were found to remain as it is.

Comparison of Elemental Components of Goryeo, Longevity and Dried Mud Mushrooms Bell carbon(%) Hydrogen(%) nitrogen(%) Oxygen(%) sulfur(%) Korean Situation Mushroom 46.8 5.5 0.8 42.0 ND Longevity Situation Mushroom 45.3 5.3 1.0 40.1 ND Dried Mud Mushrooms 45.6 5.5 1.2 40.3 ND

In the above, ND indicates that it was not detected.

Comparison of General Ingredients of Korea, Longevity and Dried Mud Mushrooms ingredient Korean Situation Mushroom Longevity Situation Mushroom Dried Mud Mushrooms Fat 0.23% 0.27% 0.22% Whole dietary fiber 33% 29% 31% Per full 0% 0% 0% moisture 135 15% 10% Ash 1.1% 1.1% One% Protein (% N × 6.25) 3.63% 4.32% 3.73% Vitamin c 1.5% 2.1% 1.5%

Example 4 Anticancer Activity of Dried Mud Mushroom Cultured in Sawdust Medium

Anticancer activity was measured by extracting the extract from dried mud mushroom fruiting body cultured in sawdust medium. First, the situation mushroom fruiting body was finely crushed and extracted by adding 30-fold volume of sterile distilled water. After extraction, the product was concentrated under reduced pressure 10 times and filtered through a 0.45 micron filter. Anthrone method was performed to determine the total sugar of the filtered situation mushroom concentrate (Pons A, et al ., J Biochem Biophys Methods , 1981, Mar, 4 (3-4): 227-31; Pollard MA, et al ., Caries Res , 1996, 30 (2): 132-7). To measure anticancer activity, we used sarcoma 180 cells (Korea Cell Line Bank, KCLB 40066) and p388 cells (Korea Cell Line Bank, KCLB 10046), which are mouse-derived cancer cells, and used SRB and MTT assays. SRB method can measure only the proliferation of cells, and MTT method is to know the proliferation of cells after metabolism. This is the difference between these two experimental methods.

<4-1> SRB Analysis

First, a sulforhodamine B (SRB) method was performed. Cancer cells were inoculated in a 96 well plate about 30,000 per well and then incubated in RPMI medium (GIBCO) for 24 hours. Cultured cancer cells were treated with extracts of distilled water from fruiting bodies of dried mud mushroom, Korean mushroom and longevity mushroom at concentrations of 7.5, 15 and 30 ㎍ / ml, respectively. In comparison, doxorubicin (Ildong Pharm., Korea), a type of anticancer agent, was treated at a concentration of 0.001, 0.01, 0.1, 1 or 10 μM. Each sample was treated with cells and then cultured with cancer cells for 48 hours, and then the cells were fixed at 4 ° C. for 2 hours by adding 50% TCA (SIGMA). After fixing, the plate was washed 5 times with distilled water and air dried. After air drying, 0.4% SRB dye (SIGMA) was added and stained for 30 minutes. After the dyeing was completed, washed 5 times with 1% acetic acid and air dried. After air drying, Tris salt was added to dissolve the dye. Thereafter, the absorbance was measured at 490 nm to determine the survival of cancer cells.

As a result, doxorubicin, an anticancer agent, showed a tendency to decrease the cell viability of cancer cells as the concentration of treatment increased, along with dried mud mushrooms cultured in sawdust medium, Korea mushroom and longevity mushroom Both extracts also decreased the cell viability of cancer cells as the treatment concentration increased (Table 7). Therefore, it was found that the dry mud mushrooms cultured in sawdust medium exhibited anticancer activity.

sample Cell survival rate (%) Mushroom extract Treatment concentration Sarcoma Solid Cancer 180 P388 Dried Mud Mushrooms (㎍ / ㎖) 7.5 89 88 15 87 83 30 68 76 Korean Mushrooms (㎍ / ㎖) 7.5 87 92 15 81 82 30 46 82 Longevity Situation Mushroom (㎍ / ㎖) 7.5 100 100 15 100 100 30 49 99 Doxorubicin (μM) 0.001 100 97 0.01 98 91 0.1 73 68 One 34 32 10 12 29

<4-2> MTT analysis

Next, MTT analysis was performed ( Cross P, et al., Radiat Oncol Investig , 1994, 1: 261-269). After inoculating about 5,000 cells per well into a 96 well plate, cancer extracts were extracted with water from fruiting bodies of dried mud mushrooms, Korean mushrooms, and longevity mushrooms grown in sawdust culture medium at 7.5, 15, and 30 ㎍ / ml, respectively. In the comparison group, doxorubicin, a type of anticancer agent, was treated at a concentration of 0.001, 0.01, 0.1, 1 or 10 μM. Incubated for 4 days after treatment. After the incubation, MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) was inoculated into each well and incubated for 4 hours. After the incubation was completed, the plate was centrifuged at 450 xg for 5 minutes, the medium in the plate was carefully removed, and 150 μl of DMSO was added. After adding DMSO, the cells were incubated for about 10 minutes to dissolve formazan crystal in MTT reagent. After that, the absorbance at 490 nm was measured to determine the survival of cancer cells.

As a result, doxorubicin, an anticancer agent, showed a tendency to decrease the cell viability of cancer cells as the concentration of treatment increased, along with dried mud mushrooms cultured in sawdust medium, Korea mushroom and longevity mushroom Both extracts also decreased the cell viability of cancer cells as the treatment concentration increased (Table 8). Therefore, it was found that the dry mud mushrooms cultured in sawdust medium exhibited anticancer activity.

sample Cell survival rate (%) extract Treatment concentration Sarcoma Solid Cancer 180 P388 Dried Mud Mushrooms (㎍ / ㎖) 7.5 84 92 15 86 70 30 55 35 Korean Mushrooms (㎍ / ㎖) 7.5 74 86 15 75 41 30 47 21 Longevity Situation Mushroom (㎍ / ㎖) 7.5 100 100 15 85 100 30 51 68 Doxorubicin (μM) 0.001 93 87 0.01 90 83 0.1 53 40 One 16 12 10 10 4

<4-3> Comparison of Anticancer Activity with Distilled Water Extract of Dried Mud Mushrooms Grown in Wood Culture Medium

In order to compare the anticancer activity of the dried mud mushroom fruiting bodies grown in sawdust culture medium with the anticancer activity of the dried mud mushroom fruiting bodies grown in oak logs, extracts were prepared from distilled water from the dried mud mushroom fruiting bodies. A dry mud mushroom distilled water extract was prepared by adding 30 times the amount of distilled water to the dried mud mushroom fruiting body produced in sawdust culture medium or wood culture medium for 8 hours. Each distilled water extract was treated with sarcoma solid cancer 180 cancer cells at the concentration of 18.75, 37.5, 75, 150 and 300 ㎍ / ㎖ and anticancer activity was measured by SRB analysis.

As a result, the distilled water extract of dried mud mushroom fruiting body grown in sawdust culture medium showed lower cell viability compared to the distilled water extract of dried mud mushroom fruiting body grown in wooden culture medium at the same treatment concentration. This decreases proportionally (Table 9 and FIG. 3). Therefore, it can be seen that the dry mud mushroom fruiting body distilled water extract grown in the sawdust culture medium has superior anticancer activity compared to the dry mud mushroom fruiting body distilled water extract grown in the wood culture medium.

Water extract Concentration (μg / ml) Cell survival rate (%) Sawdust Cultivation Extract 18.75 89.25 37.5 75.25 75 40 150 14.5 300 10.75 Wood Cultivation Extract 18.75 96.5 37.5 87.75 75 78.5 150 49.5 300 31.25

<4-4> Anticancer Activity of Ethanol Extract of Dried Mud Mushroom

In addition to the distilled water extracts of dried mud mushroom fruiting bodies cultured in sawdust culture medium, the extracts extracted with ethanol as a solvent showed anticancer activity and the difference between the anticancer activity of dried mud mushroom fruiting bodies grown from oak logs. 20%, 40%, 60%, and 80% of ethanol was added to the dried mud mushroom fruiting bodies produced in sawdust culture medium or wood culture medium, and extracted for 4 hours. The ethanol extracts were treated with sarcoma solid cancer 180 cancer cells at concentrations of 6.25 to 100 μg / ml or 0.625 to 10 μg / ml, respectively, to determine anticancer activity by SRB analysis.

As a result, the ethanol extract of dried mud mushroom fruiting body grown in sawdust culture medium showed lower cell viability compared to the ethanol extract of dried mud mushroom fruiting body grown in wood culture medium at the same treatment concentration. And as the concentration of the ethanol extractant increased, the survival rate of the cells was proportionally decreased (Table 10, Figure 4a and 4b). Therefore, the dry mud mushroom fruiting body ethanol extract grown in sawdust culture medium had anticancer activity with distilled water extract, and the anti-cancer activity was superior to the ethanol extract of dried mud mushroom fruiting body grown in wood culture medium.

Ethanol extract Concentration (μg / ml) Cell survival rate (%) Ethanol extract Concentration (μg / ml) Cell survival rate (%) Sawdust Cultivation Extract (20% Ethanol) 6.25 95.75 Log cultivation extract (20% ethanol) 6.25 93.5 12.5 85.25 12.5 89 25 58.5 25 88.5 50 29.25 50 87 100 16.5 100 49 Sawdust Cultivation Extract (40% Ethanol) 6.25 91.25 Log cultivation extract (40% ethanol) 6.25 93.75 12.5 51.25 12.5 84.75 25 25 25 76.5 50 18.5 50 68.75 100 11.25 100 40.25 Sawdust Cultivation Extract (60% Ethanol) 6.25 80.5 Solid Wood Cultivation Extract (60% Ethanol) 6.25 99.75 12.5 45.5 12.5 82.5 25 22.5 25 77.75 50 16.25 50 67.25 100 22.5 100 32.25 Sawdust Cultivation Extract (80% Ethanol) 0.625 89.25 Log cultivation extract (80% ethanol) 0.625 94.5 1.25 67.25 1.25 80 2.5 26.25 2.5 73.5 5 21.5 5 39 10 19.75 10 23.5

As described above, cultivating dried mud mushrooms using a medium containing sawdust as a main ingredient produces a large amount of dried mud mushrooms having various pharmacological effects due to shorter culture days and higher yields compared to conventional wood-grown mushroom fruiting bodies. It can be useful to

Claims (12)

delete delete delete delete delete delete 1) culturing the spawn of the dry mud mushroom in a culture medium containing sawdust as a main component; 2) inoculating the seedlings of step 1 into a sawdust medium for forming fruiting bodies, the main component of which is sawdust, followed by culturing the mycelia to adhere to the medium; 3) forming a fruiting body primitive by transplanting the sawdust medium for fruiting body formation in which the mycelium of step 2 is completely adhered to the grower; And 4) Artificial cultivation method of dried mud mushrooms comprising the step of growing the fruiting body original body formed in step 3 to fruiting body. 8. The method of claim 7, wherein the cultivation of the seedlings of the dry mud mushroom of step 1 is characterized in that the culturing for 25 to 30 days at 25 to 30 ℃. 8. The method of claim 7, wherein the priming of the fruiting body of step 3 is grown for 15 days at a humidity of at least 90% and a temperature of 22 to 27 ° C.      8. The method of claim 7, wherein the growing fruiting body of step 4 is carried out at a humidity of 70 to 85%, a temperature of 25 to 32 ° C. The method according to claim 7, wherein the seed of step 2 is inoculated in a sawdust medium for fruiting body formation and adhered to the medium, wherein the temperature is incubated at 20 to 25 ° C for 25 days to 30 days. The method according to claim 7, characterized in that the sawdust medium for fruiting in step 2 is mixed with oak sawdust as a main component and rice bran as auxiliary components in a volume ratio of 10: 0 to 8: 2.

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