JP2001017158A - Culture medium composition for culturing phellinus linteus mycelium and culture of phellinus linteus mycelium using the same composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture medium composition for culturing Phellinus linteus mycelium in which the culture of Phellinus linteus mycelium is easily conducted and a special process for extracting mycelium is needless, and to provide a method for producing Phellinus linteus mycelium using the same culture medium. SOLUTION: This method comprises the following steps: preparing a culture medium by formulating 40-60 wt.% of at least one kind selected from the group consisting of brown rice, roasted brown rice, barley (or wheat), its powder, and uncracked and roasted grains of barley with 40-60 wt.% of underground water, by pouring the mixture into a bottle for culturing spawn, by heating them at a steam pressure of 15 lb/cm2 (120-121 deg.C) for 50-60 min to perform high pressure sterilization and by cooling them; separating tissue from carpophore of natural Phellinus linteus, subculturing the tissue in a medium extracted from mulberry trees and then performing seed culture in a similar medium; and inoculating the seed culture solution in the above formulated culture medium, followed by culturing it at 25±10 deg.C for 25-30 days.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a technique for culturing fungi.

[0002]

BACKGROUND OF THE INVENTION Phellinus linteu
s) is a rare pear tree that grows naturally on dead mulberry trees, and is reported to have the strongest antitumor properties among mushrooms.

[0003] Currently, cancer treatment includes chemotherapy, radiation therapy, or surgical treatment by surgery, but the treatment range is limited due to therapeutic limitations and side effects. Therefore, alternative or parallel treatment with existing chemotherapy that has direct cytotoxicity has been tried to restore the host's immune activity against cancer cells, and a more efficient treatment that is harmless to the human body has been tried. ing.

It is already known that bacterial preparations such as BCG and Zymosan, a cell wall polysaccharide of Saccaromyces cerevisiae, exhibit anticancer effects by stimulating host immunity. Also,
A lipopolysaccharide (lipopolysa) isolated from fungal, lichen and various botanical herbs used in Chinese medicine
ccaride), proteoglycan, and hemicellulose have been reported to have host-mediated antitumor effects. Recently, the lactolytic activity of polysaccharides or protein polysaccharides isolated from basidiomycetes, such as Lentinan from Shiitake mushrooms, PSK from Koshiba, copolang, and proteoglycan, has been revealed and used as an antitumor immunotherapy. Or has been developed.

[0005] Ikekawa et al. Have reported a comparative study on such anti-tumor polysaccharides of basidiomycetes,
We have reported that Mulberry has the strongest host-mediated antitumor effect. However, mulberry yellow is a mushroom that is parasitic on dead mulberry trees, and there are very few that grow naturally in the natural world, and as mulberry trees decrease, it becomes difficult to collect mulberry yellow fruit bodies,
It lacks resources and is extremely difficult to artificially culture, and has not been practically used until today.

[0006]

As a method of culturing and extracting the mycelium of Mulberry, the fruit body of Mulberry was collected, and the mycelium was separated and cultured on an agar medium.
0 g, peptone 3.0 g, KNO 35.0 g, CaCO
The mycelium was inoculated on a solid medium composed of 37.5 g and 1.8 to 2.0 liters of distilled water, cultured at 25 ° C. for 3 months in an incubator, and then dried in a hyphal mass 1
There is a method in which 00 g is reflux-extracted with 1,000 ml of hot water for 1 hour, the extract is concentrated under reduced pressure at a low temperature of 100 ° C. or less to 100 ml, and the concentrated liquid is freeze-dried to obtain a powder.

However, this conventional method of cultivating Mulberry mycelium is a method for using the mycelium for pharmaceuticals.
The method of culturing and extracting mycelium is complicated, and the extraction yield cannot be said to be high. In addition, since a non-edible medium component is used during the culture, another medium must be manufactured, and the medium component cannot be used, so that the cost of culturing and extracting mycelium is high. is there.

Accordingly, it is an object of the present invention to provide a medium composition for mulberry mycelium culture in which cultivation of mulberry mycelium is easy and a separate mycelium extraction step is not required. Further, the present invention provides a method for producing a Mulberry mycelium using the medium composition for culture of Mulberry mycelium.

[0009]

According to the present invention, brown rice,
It is composed of at least one of roasted brown rice, wheat, barley flour or roasted barley and 40 to 60% by weight of water and 40 to 60% by weight of water.
5 lb / cm ² , 120-121 ° C) for a predetermined time (50-
It is characterized in that a medium composition is obtained by heating, autoclaving, and cooling. Then, the medium is inoculated with a seed culture obtained by separating tissues from naturally occurring mulberry fruit bodies, subcultured on a mulberry tree extraction medium, and then seed-cultured on the same medium, for example, at 25 ° C. ± 1 ° C. 25-30 at temperature
Provided is a culturing method for culturing for a predetermined period such as days.

[0010] Thereby, the mycelium of Mulberry can easily be prepared for a short time,
In addition to being able to be cultured at a high yield, it can be powdered by a simple drying method and can be used secondarily together with the medium components, so that it is economical and can obtain Mulberry mycelium containing various nutrients in a large amount. .

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The medium composition for mulberry mycelium culture of this example comprises 40 to 60% by weight of at least one of brown rice, roasted brown rice, barley, flour or roasted barley, and water (groundwater) 40. ~ 60w
%, and put in a container (bottle) for inoculum culture and sterilized by high-pressure sterilization and cooled. And the method of cultivating Mulberry mycelium of this example using the medium composition for Mulberry mycelium culture,
After separating the tissue from the fruiting body of naturally occurring mulberry and subculturing it in mulberry tree extraction medium, the seed culture solution cultivated in the same mulberry mycelium culture medium was added to the mulberry mycelium culture medium of this example. Inoculate,
25-30 ℃ at the temperature of 25 ℃ ± 1 ℃ in the incubator
They are cultured for days.

Brown rice is a rice milling process (threshing) in which only rice husks are peeled off, and white bran means rice left as it is, and it is preferable to use one with a grinding accuracy of less than 20%. This is possible by using brown rice which is generally commercially available. When a milling accuracy of 20% or more is used, there is a possibility that the mulberry mycelium cannot be sufficiently cultured.

[0013] The roasted brown rice is prepared by heating brown rice at a temperature of 100.degree.
Heat while stirring thoroughly for 10 minutes, or use a technique of gradually increasing the temperature in a heating chamber and maintaining 83-85 ° C. for 3-4 hours. Since roasted brown rice has a relatively lower moisture content than brown rice, it is preferable to increase the moisture content when forming the medium.

It is preferable to use wheat having a grinding accuracy of 30%. When the milling accuracy exceeds 30%, there is a possibility that the degree of cultivation of Mulberry yellow mycelium may be insufficient as in the case where the milling accuracy of brown rice is 20% or more.

[0015] The roasted wheat can also be treated in the same manner as roasted brown rice.

Barley has a lower milling accuracy than ordinary wheat, and is obtained by stripping only chaff.

The reason why the above brown rice, roasted brown rice, barley, roasted barley, and barley are suitable as a medium component for mulberry mycelium culture is that some of the nutrients in white bran or wheat core are mulberry mycelium. It is determined that the compound acts as an essential component for the growth of.
This can be guessed from the results that when white rice is used as a medium component, cultivation of mulberry mycelium is not possible or is insufficient.

The usage ratio of brown rice, roasted brown rice, wheat, roasted barley, barley is less than 40% by weight, or the usage ratio of groundwater is 60w
When the amount exceeds t%, the culture period of the Mulberry mycelium is prolonged, or the culture of the Mulberry mycelium becomes insufficient. On the other hand, if the usage ratio of brown rice, roasted brown rice, wheat, roasted barley, barley exceeds 60 wt%, or the usage ratio of groundwater is less than 40 wt%,
The state of the culture medium is dried, and the cultivation of Mulberry mycelium is not successful, or the density of mycelium is reduced, and the amount of mycelium obtained is reduced.

Brown rice, roasted brown rice, barley, roasted barley, barley and groundwater are put into a seed culture bottle and sterilized by high pressure to obtain a sterilizing effect. In addition to the sterilization effect, brown rice, roasted brown rice, barley, roasted barley, barley Is carried out, so that the mycelium of Mulberry can easily grow. Sterilization is performed by using a high-pressure sterilizer (autoclave) at a steam pressure of 15 lb / cm ² (about 120 ° C.) for 15 to 20 minutes to perform sterilization by heating for 50 to 60 minutes.
It is a technique to sterilize various microorganisms that can grow inside brown rice, roasted brown rice, barley, roasted barley, and barley by extending the length to minutes.

The strain to be used is obtained by isolating tissues from naturally occurring mulberry fruit bodies, subculturing them in a mulberry tree extraction medium, and placing 50 g of brown rice and water 5 in a 250 ml Erlenmeyer flask as a predetermined container.
0cc and autoclaved (121 ° C / 1.2kg / 50
The culture obtained by inoculating the above-mentioned subculture solution into the cooled medium after cooling is used as a culture inoculation source.

The naturally produced mulberry yellow used in the present example is obtained from those native to Mt. Seorak in Gangwon-do, Korea. Using this, tissue is separated from the fruiting body by a usual method, and subcultured by a stop culture method in a solid medium using dead mulberry trees as a material. Then, 50 g of brown rice and 50 cc of water are poured into a 250 ml Erlenmeyer flask, and sterilized by high pressure (121 ° C. /
(1.2 kg / 50 min) and cooled, and the subcultured mycelium was inoculated in a sterile state such as a sterile box or sterile room into a seed medium for seed culture, and used as an inoculum source.

The inoculum amount of the seed culture solution is preferably 1 to 10% based on the weight of brown rice, roasted brown rice, barley, roasted barley, and barley in the medium. The culture conditions for Mulberry mycelium were 2
The temperature is set to 5 ± 1 ° C., and the cells are cultured with naked eyes until the mulberry mycelium is completely filled in the inoculum culture bottle.

Next, the present invention will be described with reference to further specific examples, which do not limit the scope of the present invention.

First example:

300 g of brown rice (produced by the agricultural cooperative) and 250 cc of groundwater
Was placed in a 1,000 ml inoculum culture bottle, the lid was closed, and a high-pressure sterilization method using a high-pressure sterilizer (vapor pressure of 15 lb / cm ²⁾ was performed.
(20-121 ° C. for 60 minutes), followed by cooling to obtain a culture medium. On the other hand, natural mulberry that grows naturally at Mt.Seorak in Gangwon-do, Korea, is collected, tissues are separated from fruiting bodies by the usual method, and then subcultured by solid culture using dead wood of mulberry as a stop culture method. , This subcultured mycelium,
50 g of brown rice and 50 cc of water are poured into a 250 ml Erlenmeyer flask, subjected to high-pressure sterilization (121 ° C./1.2 kg / 50 minutes), and the cooled medium is inoculated in a sterile room. 15 g of the seed culture obtained by the seed culture is inoculated into the above-mentioned medium, and the temperature is 25 ± 1 °
At the following temperature.

The cultivation rate of the mycelium was measured thereafter, and the density of the mycelium was visually evaluated. The hyphal culture rate was 100% after 30 days, and the hyphal density was high.

Examples 2 to 18 and Comparative Examples 1 to 5:

The same method as in Example 1 was used except that the types and amounts of the medium components were changed as shown in Table 1. The culture rate and hyphal density of the mycelium were measured and evaluated while culturing the mulberry mycelium, and the results are shown in Table 1.

[Table 1]

As can be seen from Table 1, brown rice, roasted brown rice,
A medium in which at least one of wheat, roasted barley, and barley and groundwater are autoclaved has no significant difference in hyphal growth and hyphal density due to the medium components. Furthermore, roasted barley has a high porosity and a high mycelium density. On the other hand, roasted round corn comes into contact with the inner surface of the lid because the water inside the inoculum bottle boils up during sterilization,
It can be seen that the cultured species is contaminated.

Further, as a result of cultivating Mulberry mycelium using the white rice in the same manner as in the first example, the culture of Mulberry mycelium did not proceed as expected due to the depletion of nutrient components in the medium. Furthermore, when sterilization was not performed, contamination was caused by the growth of various bacteria, and when distilled water was used, the mycelium could not be cultured successfully due to a lack of inorganic substances.

Seventeenth to nineteenth examples:

The mycelium of Mulberry was cultured in the same manner as in Example 1 except that the inoculum amount of the seed culture was changed as shown in Table 2. As a result of measuring the hyphal density with a spectrophotometer, the culture was completed on the 25th day in the case of the first example, and the results shown in Table 2 were obtained in the case of the 17th to 19th examples.

[Table 2]

The inoculation amount of the tumor culture solution is preferably 1 to 10% by weight based on the weight of the solid content of the medium. The most effective medium for a 1,000 cc culture bottle is 300 g of brown rice and 250 g of groundwater.
cc.

[0034]

Industrial Applicability The medium composition for mulberry mycelium culture of the present invention and the method for culturing the mulberry mycelium using the same use cereals that can be easily obtained as a medium component, and thus the mulberry mycelium can be produced at a high level. Not only can it be easily obtained in density, but also after completion of cultivation, it becomes possible to use Mulberry mycelium together with the medium components, and there is no need for an extractant for Mulberry mycelium, which is economical and reduces the mycelium. The culturing process is simple.

Continued on the front page F term (reference) 2B011 AA07 BA08 BA13 GA04 GA12 KA04 4B065 AA71X BB26 BC36 BD08 CA44

Claims (2)

[Claims] 1. 40 to 60% by weight of at least one of brown rice, roasted brown rice, barley, flour or roasted barley and 40 to 60% by weight of water.
And a medium composition for cultivation of Pseudomonas aeruginosa mycelium obtained by putting into a culture vessel, sterilizing under high pressure, and cooling. 2. A mycelium obtained by separating tissue from the fruit body of Mulberry yellow and subcultured by a stop culture method using a solid medium containing dead wood of Mulberry tree, and adding 50 g of brown rice and 50 g of water to a 250 ml container.
Add 0cc and pasteurize (121 ℃ / 1.2kg / 50min)
The culture medium obtained by cooling and cooling is aseptically inoculated and seed-cultured, and the seed culture thus obtained is inoculated and cultured on the medium comprising the medium composition for mulberry mycelium culture of claim 1. A method for culturing the mycelium of Mulberry.