KR100383558B1 - Method and apparatus for seed culture of Cordeceps sp. strains using liquid medium for mass production of its fruit body - Google Patents

Abstract

The present invention relates to a liquid seed culture method and apparatus for mass production of Cordyceps fruit fruit, inoculated with Cordyceps fungi in an inoculator medium consisting of a carbon source, a nitrogen source and distilled water after expansion of Cordyceps fungi in a curry and 5 days in a 24 ° C. incubator. After culturing, the inoculation of Cordyceps sinensis inoculation source was aseptically inoculated so that the ratio between the liquid medium and the inoculation source was 100: 1 using the inoculation device of the present invention, and then, at 25 ° C. at a rate of 0.3vvm using the liquid seed culture apparatus of the present invention. After incubating for 5 days while injecting the sterilized air to prepare a liquid seed, inoculate the liquid seed evenly over a grain medium consisting of brown rice and silkworm chrysalis to fluoresce at 70-80% humidity and a temperature of 24 ° C. Incubate for 10 to 15 days in a culture room that can maintain the brightness of light by turning on humidity of 80 to 90%, temperature of 20 ℃, and brightness of light 500 In the growth chamber is maintained at a 1000Lux 15-18 days incubation by way of easily without contamination of the bacteria there is a great effect in producing fungus fruit bodies in large quantities.

Description

Liquid seed culture method and apparatus for mass production of Cordyceps fruit fruit {Method and apparatus for seed culture of Cordeceps sp. strains using liquid medium for mass production of its fruit body}

The present invention relates to a liquid seed culture method and apparatus for mass production of Cordyceps sinensis. More specifically, the present invention is to prepare a liquid medium consisting of dextrose, yeast extract, peptone and sterile water for mass production of Cordyceps fruit fruit body and the present inoculation device 10 and the liquid seed culture apparatus (20) To cultivate the liquid spawn easily and aseptically.

Common mushrooms mainly take vegetable organics as nutrients, while cordyceps is characterized by targeting insects. Cordyceps sinensis is a type of mushroom named because of its appearance in the body of insects in winter and grass-like in summer. Some species of Cordyceps, which invade insects and form fruiting bodies on the larvae, have been used since ancient times in China as an elixir of tuberculosis, as an antidote for tuberculosis, asthma, jaundice and antidote for opioid poisoning, post-mortem and tonic, and immune enhancing agents. It has been an expensive herbal 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). On the other hand, Cordyceps sinensis is rarely found in nature and is artificially cultured. In the method of artificial culture, spawn culture includes a solid spawn culture method and a liquid spawn culture method. In the case of Cordyceps sinensis, liquid spawn cultivation methods are common, but most of them have been grown in Erlenmeyer flask cultures, and industrial cultivation methods have not been developed. In Korean Patent Publication No. 97-68810, there is a known method for culturing and cultivating seedlings of Millyris cordyceps using silkworm pupa. However, the mass culture method of the spawn has not yet been systematically established, there is no commercial level culture method.

The present inventors have completed the present invention by producing Cordyceps sinensis liquid spawn aseptically and easily producing Cordyceps sinensis fruit body using the present invention inoculation device 10 and the liquid culture device 20 in view of the above points.

Accordingly, it is an object of the present invention to provide a method for culturing liquid spawn for mass production of Cordyceps sinensis.

Another object of the present invention is to provide an apparatus for culturing liquid seed for mass production of Cordyceps sinensis.

The object of the present invention is to inoculate the Cordyceps fungi and expanded culture in the curry and inoculated in the inoculation medium of the fungus Cordyceps fungi incubated at 24 ℃ for 5 days to prepare the inoculum dextrose, yeast extract, peptone, distilled water and After preparing a liquid medium consisting of an antifoaming agent and inoculated with the inoculation device 10 of the present invention so that the ratio of the liquid medium and the inoculation source is 100: 1, using the liquid seed culture apparatus 20 of the present invention, the rate of 0.3vvm at 25 ° C. The liquid spawn was prepared by incubating for 5 days while injecting the sterilized air, followed by inoculating the liquid spawn in a grain medium consisting of brown rice and silkworm pupa, followed by culturing to produce a large amount of cordyceps fruiting bodies.

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

Figure 1 shows the inoculation device 10 for aseptically performing the Cordyceps sinensis liquid seed inoculation of the present invention.

Figure 2 shows a liquid seed culture apparatus 20 for facilitating the inoculation, culture and discharge of the liquid seed of the present invention.

Explanation of symbols on the main parts of the drawings

10: inoculation device 11: inoculation container

12: stopper portion 13: air injection pipe

14 air filter 15 discharge pipe

20: liquid seed culture apparatus 21: culture vessel

22: stopper 23: combined pipe

24: locking device 25: air filter

26: air discharge pipe 27: filter

The present invention comprises the step of inoculating the Cordyceps fungi, inoculated in a solid medium consisting of dextrose, yeast extract, peptone, agar and distilled water and culturing for 4 to 8 days in an incubator at 22 to 30 ° C .; Preparing an inoculum medium consisting of dextrose, yeast extract, peptone, and distilled water for inoculum culture of Cordyceps sinensis according to the present invention; Inoculating the inoculated fungus Cordyceps sinensis on the inoculum medium prepared in the step and culturing for 5 days at 24 ° C .; Preparing a liquid culture solution consisting of dextrose, yeast extract, peptone, distilled water and an antifoaming agent for liquid seed culture of the present invention; Discharging and inoculating 0.1 liter of the culture inoculation source into the liquid seed culture apparatus 20 of the present invention by using the inoculation device 10 of the present invention in 10 liter of the liquid culture solution of the above step; Obtaining a liquid seed by incubating the liquid culture solution inoculated with the inoculation source of the above step for 5 days while injecting 0.3vvm of sterile air at 25 ° C; And inoculate the liquid spawn prepared in the above step into a 1000 ml semi-transparent plastic container with 80 g of brown rice and 5 g of silkworm pupa pieces inoculated on a sterilized medium, and then turn on a fluorescent lamp at a humidity of 70 to 80% and a temperature of 24 ° C. to maintain light. After culturing for 10 to 15 days, and then incubated for 15 to 18 days while maintaining the temperature of the culture room at 20 ℃, humidity 80 to 90%, brightness of light 500 to 1000 Lux, consisting of mushrooms.

Inoculation of Cordyceps fungi for inoculating the inoculator in this step inoculates the mycelium fragment with a platinum ball so that other bacteria do not enter the culture medium in the culture vessel when the mycelium is grown to about 70% .In this case, the more inoculated mycelia are inoculated The shorter the incubation period and the higher the density of the inoculated mycelium, the smaller the diameter of the mycelium mass after culture. The inoculum culture was incubated at 22 to 30 ° C. for 5 to 8 days, preferably at 24 ° C. for 5 days. In the same Cordyceps sinensis, the incubation period tended to be shorter as the temperature of the culture solution approached the optimum temperature condition of the mycelium. When the inoculum was inoculated into the liquid culture solution of the above step, the liquid culture solution: the culture inoculation source is 20 to 200: 1 and preferably the liquid culture solution: the culture inoculation source is 100: 1. In addition, the culture of the liquid spawn in the above step was incubated while injecting the sterilized air at 0.1-1.0vvm for 4-10 days at 22-28 ° C. Preferably, the sterilized air of 0.3vvm at 25 ° C was injected. Incubated daily. The solid medium used in the expansion of the Cordyceps fungi in this step consists of 10 to 80 gm of dextrose, 2 to 20 gm of yeast extract, 2 to 20 gm of peptone, 10 to 20 gm of agar and 1000 ml of distilled water, preferably 40 gm of dextrose, yeast It consists of 10gm extract, 10gm peptone, 18gm agar and 1000ml distilled water. In addition, the culture medium for Cordyceps inoculum inoculum medium and liquid spawn culture medium consists of dextrose 10 ~ 80gm, yeast extract 2 ~ 20gm, peptone 2 ~ 20gm and distilled water 1000ml, preferably dextrose 40gm, yeast extract 10gm, peptone 10gm And 1000ml of distilled water

In addition, the present invention relates to a liquid spawn incubator 20 used to easily perform aseptic, culture vessel 21 for receiving a liquid culture and a stopper portion 22 covering the culture vessel 21. ) And a stopper connected to the stopper, which combines air and inoculum injection and culture medium discharge, and the airtight at the ends of the locking device 24 and the combined pipe 23 for adjusting the sealing of the combined pipe 23. It is composed of an air filter 25 mounted for the purpose and an air discharge pipe 26 for discharging the air inside the culture vessel to the outside and a filter 27 for filtering various bacteria mounted in the air discharge pipe.

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 Isolation and Expansion of Cordyceps Probiotics

Prokaryote of Cordyceps sinensis of the present invention was used as received. The fungus was incubated with platelets, and then inoculated into a solid medium consisting of 40 gm of dextrose, 10 gm of yeast extract, 10 gm of peptone, 18 gm of agar, and 1000 ml of distilled water, and expanded to 4 to 8 days in a thermostat at 22 to 30 ° C.

Example 2 Culture of Cordyceps Sinensis Inoculator

Step 1: Preparation of Culture Solution for Cultivating Cordyceps Inoculum

To culture the Cordyceps inoculum, a culture solution was prepared as shown in Table 1 and sterilized at 121 ° C. for 20 minutes using an autoclave.

Composition ratio of liquid spawn culture medium of Cordyceps mushroom ingredient Liquid badge Liquid culture Dextrose 4 400 Yeast extract One 100 peptone One 100 Distilled water 0.1ℓ 10ℓ (Note) Unit: gm

2nd step: cultivation of Cordyceps inoculation source

The culture medium prepared in the first step of Example 2 was inoculated with the proliferative bacteria expanded in the sare. When the inoculation time was about 70% of the mycelium in the cherries were inoculated with mycelium pieces with platinum ball so that no other bacteria enter the culture medium in the culture vessel, and the inoculated culture was incubated at 24 ° C. for 5 days.

Example 3 Cultivation of Liquid Seedling of the Present Invention

First step: media production for liquid seed culture

The liquid culture solution was prepared as shown in Table 1 for liquid seed culture of the present invention. In addition, one or two types of household cooking oil, cottonseed oil, castor oil, olive oil, and palm oil were used together as an antifoaming agent to remove the foam generated during liquid culture. The amount of antifoam was added at 0.1 to 1.0% (w / v) relative to the liquid culture in order to spread throughout the upper portion of the culture.

Second step: inoculator inoculation for culturing the liquid seed of the present invention

Cordyceps inoculum inoculated in the second step of Example 2 was inoculated into the liquid culture solution prepared in the first step of Example 3. The inoculum was inoculated with 0.1 l of the inoculation source against 10 l of the liquid culture solution. At this time, the inoculation device 10 of the present invention was used as shown in FIG. 1 to prevent contamination of various bacteria. The inoculation mechanism 10 is composed of an inoculation vessel 11, a stopper portion 12, an air injection tube 13, an air filter 14, and a culture medium discharge tube 15. The stopper portion 12 of the inoculation container 11 is connected to the air inlet pipe 13 and the culture medium discharge pipe (15). The air injection pipe 13 is equipped with an air sterilizing air filter 14, one end of which is located at the bottom of the inoculation vessel 11, the other end is connected to the compressor. One end of the culture liquid discharge pipe 15 is located at the bottom of the inoculation vessel 11, and the other end thereof is connected to the combined pipe 23 installed in the liquid seed culture device 20. The culture medium discharge pipe 15 was connected to each other by varying the size of the tube, and the rubber pipes were connected and then covered with a foil to prevent contamination of various bacteria. Installed in 1cm. Therefore, when inoculating the inoculator into the liquid seed culture device 20, the air is injected into the air injection pipe 13 using a compressor, and the culture medium discharge pipe 15 is combined with the combined pipe 23 of the liquid seed culture device 20. The inoculum was discharged to the liquid seed culture apparatus 20 by the pressure of the air introduced into the air injection pipe 13 by connecting.

Third step: cultivation of the liquid spawn of the present invention

Cultivation of the liquid seed of the present invention used the liquid culture apparatus 20 of the present invention as shown in FIG. The liquid seed culture device 20 is a culture vessel 21, the stopper 22, the combined pipe 23, the locking device 24, the air filter 25, the air discharge pipe 26 and the filter 27 Consists of. The culture vessel 21 accommodates a liquid culture solution, and the stopper portion 22 at the top of the culture vessel has a combined pipe 23 and an air discharge pipe 26 which serve to discharge the culture solution after the air, the inoculation source injection and the culture are completed, respectively. ) Is connected. The combined pipe 23 is equipped with a lock 24 to control the sealing of the tube and the air filter 25 for air disinfection, and the air discharge pipe 26 is discharged to the outside of the detoxifying harmful bacteria that may be generated in the incubator The filter 27 which prevents it is mounted. One end of the combined pipe 23 is located at the bottom of the culture vessel 21 and the other end is connected to the compressor to supply air into the culture vessel 21. One end of the air discharge pipe 26 is connected to the inside of the culture vessel 21, and the other end is open to the outside atmosphere. Accordingly, the present inventors incubated for 5 days while injecting 0.3 vvm of sterile air at 25 ° C. using the liquid seed culture device 20 as described above to obtain a liquid seed.

Example 4 Formation of Mushroom Using Liquid Cordyceps of the Present Invention

In order to form a mushroom using the Cordyceps sinensis seed of the present invention, a culture medium was prepared by putting 80 g of brown rice and 5 g of silkworm pupa pieces into a 1000 ml translucent plastic container. In this case, when using brown rice, brown rice: water content was added at a ratio of 1: 1.5, and when only silkworm pupa was used as a medium, water was not supplied. The mushroom culture medium was sterilized for 20-30 minutes at 121 ℃ using a high-pressure steam sterilizer. The sterilized mushroom culture medium was inoculated to cover the surface of the liquid spawn prepared in Example 3 evenly spread on the grain medium. At the time of inoculation, it was inoculated in a clean room to prevent the incorporation of various bacteria. The medium inoculated with the liquid spawn was transferred to a culture room capable of maintaining light by turning on a fluorescent lamp at a humidity of 70 to 80% and a temperature of 24 ° C. and incubated for 10 to 15 days. This was made to form. At 20 ℃, hyphae are initially milky, and gradually yellow areas develop throughout the hyphae. At this time, if the temperature of the culture room is maintained at 20 ℃, humidity 80-90%, and the brightness of the light is 500-1000 Lux, the color of hyphae growing on the surface of the medium is changed to fluffy state. After 15-18 days, the dark orange color on the surface of the pupa The formation of the locus began when the mycelium-shaped mycelial mass began to form. The development of the locus was initially elongated, and the upper part of the locus thickened in a club shape to form a pericardium. As the formed asthma matures, the growth of the mushroom stops and gradually degenerates, and the ascitic angle formed in the head of the mushroom is slightly attenuated than that found in nature, but the form of the ascetic capsule and follicle spores is the same as that found in nature. It became. Contrary to these general characteristics in strains with good mushroom formation, it was found that the growth of mushrooms was inhibited in strains with excessive growth of mycelia on the surface of the medium.

As described above through the above examples, the method for producing mushrooms using the liquid species of Cordyceps sinensis according to the present invention has an effect of significantly reducing the occurrence of defective products due to the contamination of various bacteria compared to the conventional solid seed. In addition, since the mushroom is produced in a state that completely covers the entire surface of the culture medium, the production efficiency is increased, thereby increasing the mass production, and in particular, by culturing the liquid spawn using the inoculation device and the liquid culture device of the present invention, contamination of various bacteria is prevented. There is an advantage that can be easily cultivated Cordyceps Mushroom.

Claims (5)

(a) Cordyceps sinensis is inoculated into a solid medium consisting of 10 to 80 gm of dextrose, 2 to 20 gm of yeast extract, 2 to 20 gm of peptone, 10 to 20 gm of agar, and 1000 ml of distilled water, followed by expanded culture for 4 to 8 days in a thermostat at 22 to 30 ° C. Doing; (b) preparing an inoculum medium consisting of 10 to 80 gm of dextrose, 2 to 20 gm of yeast extract, 2 to 20 gm of peptone, and 1000 ml of distilled water; (c) inoculating the inoculum medium prepared in step (b) into the inoculation device 10 and inoculating the inoculated fungal cordyceps in step (a) to incubate the inoculum for 4 to 8 days in a thermostat at 22 to 30 ° C. step; (d) preparing a liquid culture solution by adding 10 to 80 gm of dextrose, 2 to 20 gm of yeast extract, 2 to 20 gm of peptone, 0.1 to 1.0% (w / v) antifoam, and 1000 ml of distilled water for liquid seed culture; (e) inoculating the culture inoculum of step (c) so that the ratio of the liquid culture solution: the inoculum is 20 to 200: 1; (f) In the liquid culture apparatus 20 of the present invention, the liquid culture solution inoculated with the inoculator of step (e) is incubated for 5 to 12 days while injecting the sterilized air at a rate of 0.1 to 1.0 vvm at 22 to 28 ° C. Culturing the seed; (g) inoculate the liquid spawn incubated in step (e) onto a medium prepared by sterilizing brown rice and silkworm pupa pieces in a 1000 ml translucent plastic container and turning on a fluorescent lamp at a humidity of 70 to 80% and a temperature of 24 ° C. After culturing for 10 to 15 days, and then incubated for 15 to 18 days while maintaining the culture room temperature at 20 ℃, humidity 80 to 90%, light brightness 500 to 1000 Lux, characterized in that to form a mushroom Liquid seed culture method for mass production of Cordyceps sinensis fruit. According to claim 1, wherein the antifoaming agent for defoam during the culture of Cordyceps sinensis in step (d), any one or two of household cooking oil, cottonseed oil, castor oil, olive oil and palm oil are used together. Liquid seed culture method for mass production of Cordyceps sinensis. According to claim 1, wherein the inoculation device (10) of the step (e) is the inoculation vessel 11 containing the liquid culture and the stopper 20 of the upper end of the inoculation vessel (11) and one end of the inoculation portion Is connected to the bottom of the inoculation vessel (11) and the other end is connected to the compressor air inlet tube 13 for injecting air into the inoculation vessel (11) and the air filter for air sterilization mounted on the air injection tube (13) 14) and liquid seed culture method for mass production of cordyceps fruiting body characterized in that the one end is located on the bottom of the inoculation vessel (11) and the other end is composed of a culture medium discharge pipe (15) connected to the aeration culture apparatus. According to claim 1, wherein the liquid seed culture device 20 of the step (f) is a lock for controlling the opening and closing of the culture vessel 21 and the stopper portion 22 and the combined pipe 23 and the combined pipe at the top of the culture vessel It consists of the device 24, the air filter 25 for air sterilization, the air discharge pipe 26 and the filtration device 27, injecting Cordyceps inoculum through the combined pipe 23 and then through the air filter 25 from the outside Mass production of Cordyceps sinensis characterized by injecting the sterile air into the culture vessel 21 and venting the air in the culture vessel 21 to the outside through the filtration device 27 with the air discharge pipe 26. Liquid seed culture method for. delete