KR20170064143A - Cultivating method of Cordyceps staphylinidicola for increasing its content of cordycepin - Google Patents

Abstract

The present invention relates to a method of culturing and growing a Chinese cabbage, especially a Chinese cabbage, and more particularly, to a method of cultivating and growing a Chinese cabbage. In the present invention, there is provided a method for culturing a Chinese cabbage, comprising: (a) inoculating a seed culture of a Chinese cabbage to a liquid culture medium and culturing for 5 to 10 days at a temperature of 18 to 25 ° C and a light irradiation dose of 800 to 1300 Lux; (b) culturing the strain cultivated in (a) in a sterilized liquid medium and cultivating it for 5 to 10 at a light irradiation dose of 800 to 1300 Lux while varying day and night air temperature in a range of 20 ± 2 ° C; And (c) inoculating the culture obtained in (b) into a sterilized solid medium and growing at 20 to 26 DEG C for 45 to 55 days. According to the present invention, not only is it possible to artificially cultivate Cordyceps with long-term viability and productivity, but also increase the content of cordycepin, thereby increasing the value of long-term viable Cordyceps.

Description

TECHNICAL FIELD The present invention relates to a method for cultivating and growing Cordyceps staphylinidicola,

The present invention relates to a method of culturing and growing a Chinese cabbage, especially a Chinese cabbage, and more particularly, to a method of cultivating and growing a Chinese cabbage.

Cordyceps is a kind of mushroom which is called insecticide in winter and grass like in summer. It is insecticide-producing medicinal mushroom which is a type of fungus that enters into the body of living insect. Cordyceps sinensis, Codyceps militaris, Paecilomyces japonica, etc. are well known as Cordyceps, Cordyceps staphylinidicola (Cordyceps staphylinidicola ) Are found in nature as strains containing higher codistin than the most commonly used pupae Cordyceps miliatris.

Cordyceps has been widely used for the treatment and health maintenance of various diseases caused by Donguibogam. In China, athletes are taking the Chinese caterpillar fungus and representing the miraculous records, thus recognizing the Chinese caterpillar fungus as the three major natural food health claims.

Cordyceps are among the highest protein content (more than 28% of dry matter) of crops and contain large amounts of substances that enhance immunity. Cordycepin (3'-deoxy-adenosine), which is an isomer of quinic acid, is contained in the immune active substance of Cordyceps, and the content of cordycepin is higher than that of other Cordyceps Is much higher. Codisepin is a nucleic acid substance that acts on genetic information of cells and activates degraded immune function to prevent normal cells from being converted into cancer cells. In clinical trials in which Cordyceps was injected, the amount of NK cells (natural killer cells) and cytokines secreted from immune cells increased by 18% ~ 25%, which is an immune cell that kills cancer cells. And has received much attention as a health functional food. Based on these results, Cordycepsin - containing Cordyceps was the only mushroom in Korea to be certified as a health food. In addition, the results of clinical studies have been published that the Chinese caterpillar fungus is very helpful in controlling blood pressure and diabetes, lung function and liver function.

Although caterpillar fungus is consumed much among mushrooms, the quantity collected from natural is very low and can not meet the demand, so many farmers around the world make artificial cultivation methods and cultivate them in large quantities. However, it is very difficult to cultivate because it is a plant that grows slowly and is sensitive to external environment and can not spray pesticides. Although it succeeded in large-scale cultivation, it has been known that the number of cultivated caterpillar fungus is low even in a farm where it has been cultivated for a long time. In other words, when 10 plants are cultured and harvested, about 1-2 cases are contaminated or abnormal strains grow and the probability of watering is lowered.

Currently, the most commonly used methods for the cultivation of Chinese caterpillar fungus include the method of cultivating caterpillar pupa caterpillar fungus which is inoculated directly with silkworm larvae, the method of cultivating brown rice + pupa singer caterpillar fungus by inoculating the fungus in a mixture of silkworm powder and brown rice, Method is to grow the germanium components, etc., and inoculated in the sterilized state, it is common to grow on a flat plate or in a bottle like other mushrooms.

1. Korean Registered Patent Application No. 10-1264441 2. Korean Patent Registration No. 10-1421742 3. Korean Patent Publication No. 10-1036054 4. Korean Patent Registration No. 10-0365041 5. Korean Patent Publication No. 10-2004-0032001

The present invention provides a method for culturing and growing Cordyceps staphylinidicola strain in nature, and in particular, it provides a method for stable cultivation and growth of a long-lived chrysanthemum, in which artificial cultivation and growth methods have not been established It is intended to provide a method for cultivation and growth which greatly increases the content of cordine sphin of Cordyceps sinensis.

In order to achieve the above object, in the present invention,

(a) inoculating a seed culture of a Chinese cabbage to a liquid culture medium and culturing for 5 to 10 days at a temperature of 18 to 25 ° C and a light irradiation dose of 800 to 1300 Lux;

(b) culturing the strain cultivated in (a) in a sterilized liquid medium at a temperature of 20 ± 2 ° C for 5 to 10 days at a light irradiation dose of 800 to 1300 Lux at different day and night temperatures; And

(c) Inoculating the cultured medium obtained in (b) into a sterilized solid medium and growing at 20 to 26 DEG C for 45 to 55 days.

The culturing step (a) is preferably carried out for 7 +/- 1 days under the condition of 20 +/- 1 DEG C and a light irradiation amount of 1,000 +/- 100 Lux.

The solid medium preferably comprises 1-2 times water of brown rice and brown rice. In addition, the solid medium preferably contains 5 to 40% by weight of peptides having an average molecular weight of 400 to 10,000 in the whole medium. In a preferred embodiment, the solid medium may further comprise host insect powder.

The growing step (c) can be preferably grown under a light irradiation dose of 800 to 1300 Lux.

The growing step (c) is preferably carried out using a cultivation vessel with a lid on the top. The solid medium is placed in a culture vessel having a cover at the top, water is added, sterilized at a high temperature and a high pressure, and then the culture can be inoculated and allowed to grow for 45 to 55 days with only water of the medium itself. At this time, the cultivation vessel is preferably provided with a filter on the inside of the upper lid, and a vent hole is formed on the side, so that the air introduced through the vent hole is supplied into the cultivation vessel through the filter.

Further, in the present invention, there is provided a long-lived beetle caterpillar shucker with increased cordycepin content obtained by the above-mentioned culturing and growing method. The longevity of Cordyceps sinensis cultured and grown in accordance with the present invention is preferably not less than 1500 ppm, particularly preferably not less than 2000 ppm, preferably not less than 12 cm in height, when grown for 45 days or more.

The present invention provides a stable artificial culture and growth method for Cordyceps staphylinidicola, and particularly provides a culture and growth method capable of greatly increasing the content of cordisepin in Cordyceps sinensis. According to the present invention, the longevity of Cordyceps sinensis cultivated and grown according to the present invention is 2000 to 2500 ppm or more in terms of the content of cordycepin, and the height or thickness of fruiting body is also large, from 12 to 16 cm in height. According to the present invention, it is possible not only to artificially cultivate Cordyceps, but also to increase productivity of cordyceps, and to increase the content of cordycepin, thereby increasing the value of Cordyceps longensus.

1 is a preferred embodiment of a cultivating container according to the present invention.

The cultivation and growth method for increasing the content of cordine sphin of Cordyceps sinensis according to the present invention will be described step by step.

Seed culture step

Inoculation of the seedlings of Cordyceps mellifera to the liquid medium is carried out at 18 ~ 25 ° C for 5 ~ 10 days under the condition of 800 ~ 1300 lux of light irradiation.

The longevity of Cordyceps mellifera can be obtained by directly isolating the spores from the longevity celestial mongolian ferns collected from nature, or using the thus cultured seeds. In the examples of the present invention, spores were taken from the natural long-term coccolithic fruiting body of Fusarium oxysporum, and separated and cultured.

The liquid medium is not particularly limited, and a PDA medium is used in the embodiment of the present invention.

The culturing conditions are more preferably 20 ± 1 ° C and light irradiation 1,000 ± 100 Lux for 7 ± 1 days.

Mycelial culture step

The strains cultivated in the above step are transferred to a sterile liquid medium and incubated for 5 to 10 days at a light irradiation dose of 800 to 1300 Lux at a temperature of 20 ± 2 ° C with different day and night temperatures.

For example, in a constant temperature incubator, it is recommended to set the temperature to 18 ° C for 12 hours and 22 ° C for 12 hours to match the temperature difference between day and night.

The liquid medium is not particularly limited, and PDB medium is used in one embodiment of the present invention.

Fruit body growth stage

The culture (strain + medium) obtained in the above step is inoculated into a sterilized solid medium and grown at 20 to 26 DEG C for 45 to 55 days.

It is also possible to selectively irradiate light even at the growth stage of fruiting body. It is preferable to irradiate light even in the step of growing the fruiting body under the condition of a light irradiation dose of 800 to 1300 Lux.

The solid medium is preferably a cereal medium, more preferably a brown rice medium containing 1-2 times water of brown rice and brown rice. In the present invention, " brown rice medium " is defined as solid medium containing brown rice as a main component.

The solid medium preferably contains 5 to 40% by weight of the peptide of the average molecular weight of 400 to 10,000 in the whole medium. The inclusion of low molecular weight peptides (low molecular peptides, oligopeptides) promotes the growth of Cordyceps sinensis and greatly increases the content of cordisepin. The low-molecular peptide used in the present invention is not limited, but preferably one derived from collagen can be used. In addition, the low-molecular peptide may preferably be produced according to the method of Patent Application No. 10-2015-0095875, which is an elected cause of the present inventors.

In addition, the solid medium may further comprise powder of a long-lived beetle larva which is a host insect, if necessary. At this time, the larvae powder is preferably about 1/2 to 1/10 of the grain (brown rice) by weight.

In a preferred embodiment of the present invention, a cultivation vessel with a lid at the top is used in this fertilization step.

A preferred embodiment of the cultivation vessel is the same as in Fig. The cultivation vessel 100 includes a body 10 and an upper lid 20 and may be formed of a plastic material such as polypropylene (PP) or polyethylene (PE). The lid 20 is composed of an upper surface 21b formed in an upper and a lower two and a side surface 21a surrounding the upper surface. The filter 24 is provided between upper and lower portions of the upper surface of the upper surface, and a through hole 23 is formed in the lower portion So that air can pass through the filter and communicate with the inside and outside of the cultivation vessel. A vent hole 22 is formed at the lower end of the side surface 21a of the cover so that the air introduced through the vent hole 22 can be supplied into the cultivation vessel through the filter.

After the solid medium is contained in the cultivation vessel 10, water is added and sterilized at a high temperature and a high pressure, the culture can be inoculated only with water of the culture medium itself until the harvest time without additional water replenishment. When the cultivating container of the present invention is used, there is no fear of contamination by germs, and once put into a container, it is not necessary to supplement water separately until harvesting, and it is very simple and highly productive because it does not require human hands.

The growth period may vary depending on the environmental condition, but usually about 45 to 55 days is preferable in terms of appearance and codistin content.

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

< Example  1>

Fertilized Cordyceps sinensis collected from nature is fixed to Petri dish lid with sterile tape, and then closed in PDA medium for 12 hours and kept in clean bench. The spores dropped on the medium were separated, cultured in a thermostat at 20 ° C., and cultured for 17 days.

< Example  2>

Growth of mycelium by temperature

The strain obtained in Example 1 was inoculated into a brown rice medium, and the activation of the microorganism, the formation of mycelium and the content of cordine in accordance with the culture temperature were measured. After 27 days of inoculation, the content of cordycepin in the mycelium was measured, and the selected bacteria were used as the following test standards.

[Table 1]

Mycelial Growth Experiment with Temperature 27 days past  after)

The finished mycelium was yellow to red in color. Generally, in the initial stage where the mycelium is activated, the white microorganisms are activated between brown rice hairs on the apparent brown rice medium, and the density of the streak is increased. As time elapses, the color appears as pale red or yellow. As a result, hyphae were formed to such an extent that the shape of the brown rice medium was obscured.

&Lt; Example 3 >

Mycelial growth and codistin content according to light dose

A culture medium of 39 g / L of PDA and 20 g / L of glucose was used as the culture medium of the strain obtained in Example 1, and 24 g / L of PDB and 45 g / L of glucose liquid medium were used as mycelial culture media. The strain was inoculated on each solid medium and cultured for 20 days. The incubation time was changed on / off (using a timer) at intervals of 3 hours while the temperature of the incubator was changed to 12 hours at 18 ° C and 12 hours at 22 ° C (using a timer). The above temperature condition is to match the temperature difference between day and night as in nature.

Light irradiation was found to be closely related to the growth of Cordyceps, and the content of cordine. The results are shown in Table 2 below when all the conditions were the same and the irradiation time was different.

[Table 2]

When the light and irradiation intensity were strong, the mycelial growth was excellent. Therefore, it was found that around 1000 Lux was most suitable.

<Example 4>

Changes in the content of cordycepin depending on the type of medium and growth period

According to the results of Examples 2 and 3, the strain obtained in Example 1 was cultured under conditions of 20 ° C and 1000 Lux, which are the most suitable conditions for mycelial growth, and the cultured mycelium was transferred to a growth chamber, The content of codedine after completion was measured.

A reflection film was attached to the whole growth chamber and a timer was attached to the fluorescent lamp to control temperature for 6 hours on / off, temperature for 12 hours at 18 ° C, and temperature for 12 hours at 22 ° C.

The effects of different media additives on the codisin content were analyzed under the same conditions. As a result, the content of cordisepin was observed as shown in Table 3 below, and different appearance was observed at the growth stage of hyphae and lettuce.

(1) Strain and growth state of the microorganism according to the condition of medium

The strain was inoculated into a 200 mml PDB liquid medium of a 500 mm Erlenmeyer flask and homogenized on the activated medium after 7 days of incubation at 20 ° C and on / off (using a timer) with light irradiation (1000 lux) every 3 hours, (121 ° C, 1.5 psi, 60 min), and then transferred to a soybean paste medium (water 160 L, defatted soybean meal 48 g, sugar 4.8 kg, ferrous phosphate 80 g, magnesium sulfate 80 g, edible oil 160 cc) Lt; / RTI &gt;

 The cultured inoculum was inoculated with 15 cc of the experimental medium in a liquid inoculation machine to a sterilized brown rice medium (65 g of water, 50 g of brown rice, 2.5 g of each of the medium additives each) at sterilization (101 ° C for 4 hours).

As a polymeric peptide in the medium additives, a 10% sterilized purified water solution of a polypeptide having an average molecular weight of 4,000 to 10,000 was used, and a 10% sterilized purified water solution of an oligopeptide having an average molecular weight of 400 to 800 was used as a low molecular peptide. The oligopeptide prepared in Example 1 was used as the oligopeptide used in Experimental Group 2. A polypeptide having an average molecular weight of 4,000 to 10,000 was obtained by adding 0.1% by weight of an alkaline colloid to Donkey collagen and decomposing at 55 占 폚 for 3 to 6 hours.

An oligopeptide having an average molecular weight of 400 to 800 was used which was prepared according to the method of Patent Application No. 10-2015-0095875.

Growth level and cordycepin content were measured after a certain period of growth, and the results are shown in Table 3 below.

[Table 3]

It was confirmed that the Cordyceps sinensis grows well on the brown rice medium, and it was confirmed that the content of cordycepin was greatly changed by adding the amino acid to the peptide by adding the peptide under the same condition as the growth of Cordyceps.

(2) Growing date suitable for growth period (harvesting period)

Conditions of the medium were grown on a brown rice medium under the conditions of addition of low molecular weight peptide among the medium additives of Table 3. Table 4 shows the appearance, the height of the uterus and the content of cordine sebine according to the growth period.

After a certain period of growth, the water was formed under the mycelia and the flesh of the mycelium, and the overall bright red color was also faded and darkened. Therefore, the appropriate growth period will depend on environmental conditions, but generally 45 to 55 days are appropriate.

[Table 4]

Claims (8)

(a) inoculating a seed culture of a Chinese cabbage to a liquid culture medium and culturing for 5 to 10 days at a temperature of 18 to 25 ° C and a light irradiation dose of 800 to 1300 Lux;
(b) culturing the strain cultivated in (a) in a sterilized liquid medium at a temperature of 20 ± 2 ° C for 5 to 10 days at a light irradiation dose of 800 to 1300 Lux at different day and night temperatures; And
(c) inoculating the culture obtained in (b) into a sterilized solid medium and growing at 20 to 26 DEG C for 45 to 55 days. The method according to claim 1,
Wherein the growing step (c) is carried out at a light irradiation dose of 800 to 1300 Lux. 3. The method of claim 2,
The growing step (c) comprises growing the solid medium in a cultivation vessel having a lid at the top, adding water, sterilizing the medium at high temperature and high pressure, and inoculating the culture medium for 45 to 55 days in water alone A method of cultivating and growing a caterpillar fungus. 4. The method according to any one of claims 1 to 3,
Wherein the culturing step (a) is carried out for 7 +/- 1 days under the condition of 20 +/- 1 DEG C and a light irradiation amount of 1,000 +/- 100 Lux. 4. The method according to any one of claims 1 to 3,
Wherein the solid medium comprises 5 to 40% by weight of peptides having an average molecular weight of 400 to 10,000 in the whole medium. 4. The method according to any one of claims 1 to 3,
Wherein the solid medium further comprises host insect powder. The method of claim 3,
Wherein the lid is provided with a filter on the inside thereof and a vent hole formed on the side thereof so that the air introduced through the vent hole is supplied to the interior of the cultivation vessel through the filter. A long-term broad-leaved caterpillar fungus with increased codistin content obtained by the method for cultivation and growth according to any one of claims 1 to 3.

Images