KR100362902B1 - Process for mass production of Cordyceps bassiana - Google Patents

Abstract

The present invention relates to the mass production of yellow cordyceps bassiana fruiting body, by culturing the yellow bunched fungus strain in a culture medium containing a carbon source, a nitrogen source and water to prevent the incorporation of various bacteria and a liquid seed having excellent vitality in an easy and convenient way. There is an excellent effect to prepare the growth of the prepared liquid spawn in the appropriate medium is excellent effect of producing a large bunch of yellow bunching fungus fruiting body.

Description

Process for mass production of Cordyceps bassiana

The present invention relates to a mass production method of yellow cordyceps bassiana fruiting body. More specifically, the present invention relates to a method of artificially mass-producing fruiting bodies by culturing the liquid spawn of the Yellow Cordyceps sinensis strain and culturing in a medium to which grains and silkworm pupa are added.

Yellow bunched insects are a type of mushroom named because they are in the body of insects in winter and appear like grass in summer. Some species of yellow-capped fungus, which invade insects and form fruiting bodies on the larvae based on them, have been antiquated in ancient times as a elixir of tuberculosis, treatment of tuberculosis, asthma, jaundice and antidote for opioid poisoning, post-morbid and tonic, immune enhancing agents. It is an expensive herbal medicine that has been used as a medicine (Yiang, J., Mao, X., Ma. Q., Zong, Y. and Wen, H. 1989. Icons of medicinal fungi from China.Science Press.China.p. 575; Kobayasi, Y. 1940.The genus Cordyceps and its allies.Sci.Rept.Tokyo Bunrika Daikaku, Sect.B., 5: 53-260; Shimizu, D. 1994.Color iconography of vegetable wasps and plant worms.Seibundo Shinkosha. Japan.pp. 381). Representative yellow bunched cordyceps, which has been traditionally used for medicinal purposes, is Cordyceps bassiana , a yellow bunched cordyceps that forms fruiting bodies based on insect larvae. It has been shown that an antimicrobial substance, which is also oosparin (C14H10O8), is produced (Yiang, J., Mao, X., Ma. Q., Zong, Y. and Wen, H. 1989. Icons of medicinal fungi from China.Science Press.China.p. 575).

However, since the collection of fruiting bodies in the natural state is very difficult, the supply is limited, and as the natural harvesting of fruiting bodies becomes more difficult due to abnormal weather phenomena due to the destruction of ecosystems and pollutions, a stable artificial production technology of fruiting bodies is required (Basith). , M., and Madelin, MF 1968. Studies on the production of perithecial stromata by Cordyceps militaris in artificial culture.Canadian Journal of Botany. 46: 473-480).

As described above, as a method of mass-producing yellow bunched fungus weeds, it is difficult to collect them in the natural state, and there is a method of cultivation after spawn culture using chrysalis or a method of cultivating a large amount using artificial medium using brown rice. Mass production of Cordyceps sinensis is difficult.

The present inventors completed the present invention by culturing the mycelium cordyceps bassiana mycelium isolated from nature in liquid medium to prepare a liquid spawn and inoculating the spawn in a medium mixed with silkworm pupae and brown rice.

Accordingly, it is an object of the present invention to provide a mass production method through the liquid culture of yellow bunching fungus fruiting body.

The object of the present invention is to isolate the yellow bunched fungus mycelium from nature and inoculate the medium with carbon source, nitrogen source, inorganic salt and distilled water to prepare a liquid spawn and inoculate the liquid spawn in a medium containing silkworm pupa and brown rice. The temperature was maintained at 24 ° C. when the mycelium grew, and the temperature was maintained at 20 ° C. when the fruiting bodies were formed, thereby achieving a large amount of culture.

Hereinafter, the configuration of the present invention will be described.

Figure 1 is a process chart showing the present invention yellow bunching Chungchok fruit fruit body production method.

The present invention is to separate the cordyceps bassiana mycelium having excellent fruiting body formation from nature; For mass cultivation of the mycelium, inoculate in 500 mL inoculum medium containing 30 g / L per soybean meal and 3 g / L soybean meal and inoculate the inoculum into 10 L liquid medium containing 30 g / L per soybean meal and 3 g / L soybean meal. Inoculating to prepare a liquid seed; Preparing an optimal medium suitable for mycelial growth and fruiting by inoculating the liquid spawn on a medium containing silkworm pupae or silkworm pupae and brown rice; Inoculating the liquid spawn in the optimized medium consists of culturing in a culture room capable of maintaining light by turning on a fluorescent lamp while maintaining a humidity of 70 to 80%, temperature 20 to 24 ℃.

In the present invention, the spawn of Yellow Cordyceps bassiana may use both a solid spawn and a liquid spawn, but it is more preferable to use a liquid spawn rather than a solid spawn. This is because when the conventional solid spawn is used according to a conventional method, it takes a lot of time for the solid spawn to be inoculated on the surface of the culture medium to grow the mycelium and grow well. In addition, the liquid spawn of yellow bunched fungus can be used in the present invention, but the liquid spawn of yellow bunched fungus can be used by the method developed by the present inventors. There is an advantage to keep it.

In the step of incubating the liquid spawn in this step, inoculate the spawn in the inoculum medium to which the carbon source 2-7% (w / v) and the nitrogen source 0.1-1.0% (w / v) were added, and maintained at 22 to 30 ° C. while stirring at 80 to 200 rpm. And incubated for 6-8 days. Preferred conditions for incubation of the inoculum were inoculated with mycelium in a medium containing 3% of sulfur white per carbon as a carbon source and 0.3% of soybean powder as a nitrogen source, followed by shaking culture at 25 ± 1 ° C. with stirring at 80 to 150 rpm. The inoculum was inoculated in a liquid medium added with a carbon source of 2 to 7% (w / v) and a nitrogen source of 0.1 to 1.0% (w / v) at a ratio of 20 to 200: 1 in a volume ratio of the culture solution to the inoculation source. It was maintained at ˜28 ° C. and incubated for 5-12 days while the sterilized air was vented at a rate of 0.1-1.0vvm. When culturing the liquid spawn, inoculate the inoculum in a medium containing 3% per white sulfur as a carbon source and 0.3% soybean powder as a nitrogen source, maintain the temperature at 25 ± 1 ℃, and keep the sterilized air at 0.5vvm speed. Incubated for 7-10 days while ventilated with. In this step, as a carbon source for mycelial culture, malt, corn flour, corn starch, glucose, molasses, potato starch, wheat flour, and white sugar may be used, and the most mycelial growth was preferable when the white sugar was used. As a nitrogen source for mycelial culture, soybean meal, yeast extract, peptone, and corn leachate can be used, and soybean meal was most excellent in mycelial growth.

In the present invention, the cordyceps bassiana fruiting medium cultivation medium is added in a ratio of 60:40 grain to pupa, and grains are used alone or in brown rice or in barley, wheat, corn, soybeans, barley, sorghum. Either one can be added additionally. Therefore, unlike the conventional medium, since the grains that are easily obtained from the surroundings are used as the main material, there is an advantage in that it is easy to purchase the culture material and easily secure the culture material.

The grains used in the medium for cultivation of fruiting body of yellow bunched fungus vinegar of the present invention have the advantage of facilitating the formation of fruiting bodies by maintaining the hyphae in the medium and maintaining the moisture and aeration in the medium for the space existing between the grains and the grains in the incubator. have. In addition, the fruit growing medium contains moderate moisture enough to have a good influence on the growth of mycelia, but if the moisture in the medium is too much, the gap in the medium is reduced, which does not maintain proper aeration, so the surface hyphae promotes growth. Since the production of the fruiting body is inhibited, the water composition should be different depending on the type of grain.

When inoculating the liquid spawn of yellow bunched fungus of the present invention on the fruiting body cultivation medium, the surface of the cultivation medium may be inoculated so as to completely cover the fruiting body of the yellow bunched fungus vinegar, which can be mass-produced, and the fruiting body form is relatively similar to that found in the natural state. There is an advantage that can produce a healthy fruiting body.

Cordyceps bassiana fruiting body of the present invention produced by the present invention can be drunk directly by pouring water into a coffee pot or kettle after being collected in a culture vessel.

Yellow bunching fungus chok fruit fruit production method of the present invention is described in order in FIG.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1: Mass cultivation of the present invention Yellow Cordyceps Sinensis Mycelia

1st step: Separation of yellow bundle Cordyceps mycelium

Production of yellow bunching fungus herbaceous fruit according to the present invention was prepared by seed culture and proliferation of the isolated prokaryotic fungi were collected from nature and expanded culture. That is, the spawn preparation step of the yellow bunched fungus can be carried out in a conventional manner, and both the solid spawn cultured using a solid medium such as grains and the liquid spawn cultured by dissolving a predetermined nutrient in water can be used. In this step, liquid spawn was prepared.

Yellow bunched caterpillars were collected on the larvae on September 16, 1995 in Obongsan, Chuncheon, Gangwon-do. Collected Cordyceps sinensis was adhered to the lid of the curry with tape and then allowed to spore onto the medium after one day. When the spores germinated, they were removed from the medium (basic medium) in which potato agar medium and silkworm pupa powder were mixed. Yellow bundle Cordyceps was again selected strains excellent in fruiting body formation in the basic medium mixed with potato agar medium and silkworm pupae, wherein the selected Yellow Cordyceps fungus formed fruiting bodies as shown in Table 1. Test strains were inoculated on silkworm pupae and brown rice to induce fruiting body formation at 20 ℃ thermostat resulted in the fruiting body was formed. It takes one week for the mycelium to grow across the surface of the medium, and once the mycelium grows to white, the color becomes dark yellow. On the day of inoculation, the incidence began on the 25th day after the inoculation, and it took about 60 days after the inoculation until the mushroom, which was completely infiltrated.

Selection of excellent fruiting body strains in medium containing potato agar medium and silkworm pupa powder Strain Chair formation date Date of occurrence Fruiting body length (cm) Fruit body dry weight (g) / bottle after 60 days Yellow bunch cordyceps 18 25 6.5 80

Second Step: Preparation of Liquid Seedling Medium of the Yellow Multiple Cordyceps Mycelia

In order to select a medium for liquid seed culture of the strain of the present invention, various kinds of carbon sources, nitrogen sources, and inorganic salts were added and cultured. As a result, the mycelial growth was excellent in the carbon source test for mycelial growth as shown in Table 2 and the addition of sulfur white sugar, corn flour and wheat flour. Mycelial growth decreased in the order of yeast extract, peptone, and corn extract. In the test in which the inorganic salts were added, no clear effect was confirmed as shown in Table 4. Therefore, based on the results of Tables 2 to 4, as shown in Table 5, it was possible to prepare a medium for cultivating the liquid spawn of Cordyceps bassiana .

Effect of Carbon Sources on Mycelial Growth Type of carbon source Fucked Corn flour Corn starch glucose molasses Potato starch flour Yellow sugar Mycelial Growth ++ +++ ++ + ++ ++ +++ ++++ (Note) ++++: Very good, +++: Excellent, ++: Normal, +: Low

Effect of Nitrogen Sources on Mycelial Growth Nitrogen source type Corn Soak peptone Soy flour Yeast Juice Mycelial Growth ++ +++ ++++ +++ (Note) ++++: Very good, +++: Excellent, ++: Normal, +: Low

Effect of Inorganic Salts on Mycelial Growth Inorganic salt type Control Calcium chloride Calcium sulfate Calcium phosphate Magnesium sulfate Mycelial Growth +++ +++ +++ +++ +++ (Note) ++++: Very good, +++: Excellent, ++: Normal, +: Low

Composition Ratio for Cultivation of Liquid Seedlings of Yellow Cordyceps sinensis ingredient Inoculum (500 mL) Liquid Medium (10 L) Sulfur white sugar 15 300 Two minutes 1.5 30 Distilled water Remainder Remainder (Note) unit g

Third step: liquid spawn production of the present invention yellow bunched fungus

The mycelium obtained by incubating the progeny of the cordyceps bassiana selected from the first step and expanding the culture was inoculated into the inoculum medium having the composition ratio as shown in Table 5 prepared in the second step, and the temperature was 25 ± 1. The mixture was kept at 占 폚 and shaken with stirring at 80 to 150 rpm for 7 to 10 days. The pre-incubated inoculum was inoculated in the same culture medium as the inoculum medium and maintained at 25 ± 1 ° C, and cultured for 7 to 10 days while ventilating the filtered air at a rate of 0.5 vvm.

Fourth step: preparing a medium for cultivation of fruiting body

In this process, the culture medium for the production of Cordyceps bassiana fruiting body was not used as a silkworm pupa powder and a water supply medium as in the prior art, and a medium containing silkworm pupa powder was added to grains such as brown rice. As shown in Table 6, the addition of brown rice to silkworm pupa powder produced fast mycelial growth and good fruiting body formation, and the medium was prepared by paying attention to the good mycelial growth and fruiting body. That is, the culture medium was prepared by sterilizing 20 minutes at 121 ℃ using a grain 60g and silkworm pupa 40g for the mycelium culture of the present invention, each of the water content 1: 1.2 ratio.

Mycelial Growth and Fruiting Body Formation Test of Yellow Multiple Cordyceps Sinensis in Silkworm Pupa badge Mycelial Growth Fruiting body formation Silkworm pupa 40g ++ ++ Silkworm pupa 40g + brown rice 60g +++ +++ Silkworm pupa 20g ++ ++ (Note) +++: Excellent, ++: Normal, +: Low

5th step: liquid seed inoculation of the present invention yellow bunching fungus

In the culture medium prepared for the cultivation of Cordyceps bassiana fruiting body prepared in the fourth step was inoculated with the liquid seed of the yellow bunching fungus prepared in the third step. At this time, the liquid spawn was spread evenly on the grain medium to cover the surface and inoculated so as to inoculate the inoculation was carried out in a sterile chamber to prevent the mixing of bacteria.

6th step: cultivation and production of fruiting bodies

In the fifth step, the medium inoculated into the culture medium of the yellow bunched fungus sect. In the culture medium was transferred to a normal culture room, and when the culture was completed, the fruiting body was produced. At this time, as the culture conditions, the humidity was 70 ~ 80%, the temperature was initially maintained at 24 ℃ when the mycelium grows and incubated for 10 to 15 days while maintaining the light by turning on a fluorescent lamp. After incubation for 10 to 15 days, the culture room temperature is 20 ° C, humidity is 80 to 90%, and the brightness of light is maintained at 500 to 1000 Lux. As a result of incubation by the above method, the hyphae grown after 10 to 15 days are initially pale gray, and gradually yellow areas develop throughout the mycelia, and the color of the hyphae growing on the surface of the medium changes to a fluffy state. After 15 to 18 days, a dark yellow lump-shaped mycelium on the surface of the pupa begins to form. The development of the locus is initially elongated, and the conidia form at this point as the upper part of the locus thickens to form a club. Yellow bunched fungus produced in this way, peel off the container and take only the fruiting body part and dry.

As described above, the present invention has an excellent effect of producing a liquid seed having excellent vitality without contamination of various bacteria by incubating the Cordyceps bassiana bacteria in a medium containing a carbon source, a nitrogen source and water, The liquid spawn grown above is grown in a medium containing grains and silkworm pupa powder so that the fruiting body is completely covered on the surface of the medium, thereby producing a large quantity of yellow bunching fungus, and thus it is a very useful invention in yellow bunching fruiting plant fruiting industry. .

Claims (5)

(A) a process for separating and plate culture mycelium of Cordyceps bassiana ; (b) 2 to 7% (w / v) of carbon source, 0.1 to 1.0% (w / v) of nitrogen source, 0.1 to 0.15% (w / v) of KH ₂ PO ₄ , Preculturing for 6-8 days in a shaker at 80-200 rpm, 22-30 ° C. in a culture medium containing MgSO ₄ 7H ₂ O 0.1-0.15% (w / v) and the remaining distilled water; (c) inoculating the inoculum pre-cultured in the step (b) to the same culture medium used in the step (b) at a ratio of 20 to 200: 1 and maintaining the temperature at 22 to 28 ° C. Aeration for 5 to 12 days in an amount of 0.1 to 1.0 vmv of air to produce a liquid seed; (d) inoculating the culture medium containing grains and silkworm pupae in the aseptic chamber so that the liquid spawn prepared in (c) is evenly spread on the medium to cover the surface thereof; (e) In the method of producing Cordyceps sinensis, the medium inoculated with the liquid seed in step (d) is grown at 24 ° C. to grow mycelia, and then grows at 20 ° C. to form fruiting bodies. Cordyceps mycelium in the step (a) using the yellow bunch Cordyceps fruit body, the separation of cordyceps mycelium is to measure the date of occurrence of the locus, the date of formation of the locus, the length of the fruiting body, the fruiting body dry weight in a medium mixed with potato agar medium and silkworm pupa powder. Pure water separation (b) In the process (b) the carbon source is used alone or mixed with sulfur white sugar, wheat flour, corn flour, corn starch, malt, potato starch, molasses, glucose, nitrogen source alone or mixed with yeast juice, peptone, corn steep liquor (D) The content ratio of grain and silkworm pupae in the process (d) is in the range of 3: 2, and the yellow mulberry cordyceps fruiting body, characterized in that the grain is one of brown rice, barley, wheat, corn, soybean, yulmu, and sorghum. Mass production method. delete delete delete delete