JP4054584B2 - Liquid inoculum culture device for mass production of Cordyceps fruit body - Google Patents

Description

【００１０】
第２段階：冬虫夏草接種原の培養
前記参考例２の第1段階で製造した培養液にシャーレで拡大培養した原菌を接種した。接種時期はシャーレで菌糸体が約70％程成長したとき、白金具で菌糸体片を培養容器内の培養液に他の菌が入らないように接種し、接種された培養液を24℃で5日間培養した。
【００１１】
実施例１：本発明の実施例１の液体種菌の培養
第1段階：液体種菌培養のための培地製造
本発明の実施例１の液体種菌培養のために、表１に示した通り、液体培養液を製造した。また、液体培養時に発生する泡除去のための消泡剤としては家庭用食用油、綿実油、蓖麻子油、オリーブ油および椰子油のうち1種または2種を共に用いた。消泡剤の添加量は培養液の上層部分に全体的に広がるようにするために液体培養液に対し0.1〜1.0％(ｗ/ｖ)添加した。
【００１２】
第２段階：本発明の実施例１の液体種菌培養のための接種原接種
前記参考例１の第１段階で製造した液体培養液に前記参考例２の第２段階で培養した冬虫夏草接種原を接種した。接種量は前記液体培養液10Lに対し前記培養接種原0.1Lを接種した。この際、雑菌の汚染を防止するために、図１に示した通り、接種器具(10)を用いた。接種器具(10)は接種容器(11)、栓部(12)、空気注入管(13)、エアフィルター(14)および培養液排出管(15)で構成されている。接種容器11の栓部(12)には空気注入管(13)と培養液排出管(15)が連結されている。空気注入管(13)には空気除菌用エアフィルター(14)が装着されており、その一端は接種容器(11)の底に位置し、その他端はコンプレッシャーに連結されている。培養液排出管(15)の一端は接種容器(11)の底に位置し、その他端は液体種菌培養装置(20)に設置された兼用管(23)に連結されている。培養液排出管(15)は管の大きさを異にして接続されるようにし、ゴム管を連結した後にホイルで塞ぎ雑菌の汚染を防止し、これら空気注入管(13)と培養液排出管(15)は全て栓部(12)から1cm内に設置した。従って、接種原を液体種菌培養装置(20)に接種するときには空気注入管(13)にコンプレッシャーを用いて空気を注入し、培養液排出管(15)を液体種菌培養装置(20)の兼用管(23)に連結して空気注入管に入って来た空気の圧力により接種原を液体種菌培養装置(20)に排出するようにした。
【００１３】
第３段階：本発明の実施例１の液体種菌の培養
本発明の実施例１の液体種菌の培養は、図２に示したような本発明の液体種菌培養装置(20)を用いた。前記液体種菌培養装置(20)は、培養容器(21)、栓部(22)、兼用管(23)、施錠装置(24)、エアフィルター(25)、空気排出管(26)および濾過器(27)で構成されている。前記培養容器(21)は液体培養液を収容しており、培養容器の上端の栓部(22)にはそれぞれ空気、接種原注入および培養が完了した後に培養液を排出する役割を兼ねる兼用管(23)と空気排出管(26)が連結されている。兼用管(23)には管の密閉を調節する施錠装置(24)と空気除菌用エアフィルター(25)が装着されており、空気排出管(26)には培養器内で発生する害毒性有害菌が外部へ放出されるのを防止する濾過器(27)が装着されている。兼用管(23)の一端は培養容器(21)の底に位置し、他端はコンプレッシャーに連結されて培養容器(21)内部に空気を供給するようになる。空気排出管(26)の一端は培養容器(21)の内部に連結されており、他端は外部の大気に開放されている。従って、本発明者は前記の如き液体種菌培養装置(20)を用いて25℃で0.3vvm程の速度で除菌された空気を注入しながら５日間培養して液体種菌を得た。
【００１４】
参考例３：冬虫夏草液体種菌を利用した茸の形成
本発明の参考例３の冬虫夏草液体種菌を利用して茸を形成するために1000mlの半透明プラスチック容器に玄米80gと蚕蛹片5gを入れて培養培地を製造した。この際、玄米を用いる場合には玄米：水分の含量を１：1.5の比率で添加し、蚕蛹のみを培地に用いる場合には水を供給しなかった。前記茸培養用培地を高圧蒸気滅菌器を用いて121℃で20〜30分間滅菌した。該滅菌された茸培養用培地に前記実施例１で製造した液体種菌を穀類培地上に均一に広げてその表面が覆われる程に接種した。接種するときには雑菌の混入を防止するために無菌室で接種した。前記液体種菌を接種した培地は湿度70〜80％、温度24℃内外の蛍光燈を燈して光を維持できる培養室に移し、10〜15日間培養して、菌糸が培地全体に広がれば20℃の培養室に移して茸が形成されるようにした。20℃では菌糸が初期に牛乳色を帯びているが、漸次に菌糸全般に亘って黄色を帯びる部分が多くなる。このときには培養室の温度を20℃、湿度80〜90％、光の明度を500〜1000Luxに維持してやると培地の表面に育つ菌糸の色が綿毛状態に変わり、15〜18日が過ぎると蛹の表面に濃い橙色を帯びる突起状の菌糸塊が形成され始まると子座の発生が始まった。子座の発達は初期に長さが伸張し、子座の上端部が棍棒状に厚くなりながらその部分で子嚢殻を形成した。形成された子嚢殻が成熟すると茸の成長は止まり、漸次に退化し始め、茸の頭部に形成された子嚢殻は自然状態で発見されるものより多少萎縮されているが、子嚢と子嚢胞子の形態は自然状態で発見されるものと同一のものと観察された。茸の形成が優れた菌株らにおいてこのような一般的な特徴とは逆に培地表面に菌糸の成長が旺盛過ぎる菌株らにおいては茸の発達が阻害されるものと明かされた。
【００１５】
【発明の効果】
以上、前記参考例および実施例により説明した通り、本発明の液体培養装置を用いて液体種菌を培養することにより、雑菌の汚染を防止して雑菌の汚染による不良品の発生を著しく減少させ、容易に冬虫夏草茸を栽培し得る長点がある。
【図面の簡単な説明】
【００１６】
【図１】本発明を構成する冬虫夏草液体種菌の接種を無菌的に施行するための接種器具(10)を示したものである。
【図２】本発明を構成する液体主菌の接種・培養および排出を容易にするための液体種菌培養装置(20)を示したものである。
【符号の説明】
【００１７】
10：接種器具 11：接種容器
12：栓部 13：空気注入管
14：エアフィルター 15：排出管
20：液体種菌培養装置 21：培養容器
22：栓部 23：兼用管
24：施錠装置 25：エアフィルター
26：空気排出管 27：濾過器[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a liquid inoculum culture apparatus for mass production of Cordyceps fruit bodies, and more particularly, the present invention relates to a liquid composed of dextros, yeast extract, peptone and sterilized water for mass production of Cordyceps fruit bodies. to produce a medium, a device for culturing aseptically readily liquid seed culture using a liquid seed culture apparatus (20) of the present invention in the liquid medium.
[0002]
[Prior art]
Compared to the fact that common moths mainly consume plant organic matter as nutrients, Cordyceps is characterized by insects. Cordyceps is a kind of moth named after the parasite of insects in winter but coming out like grass in summer. Some species of Cordyceps insects that invade insects and form fruit bodies on the parasites from ancient times have been treated as anti-elderly mysterious drugs in China by treating tuberculosis, asthma and jaundice, antidote for opiate poisoning, post-disease It is an expensive Kampo medicine that has been used as a supplement, tonic, and immune function enhancer (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; Color iconography of vegetable wasps and plant worms Seibundo Shinkosha Japan pp. 381).
[0003]
On the other hand, Cordyceps sinensis is artificially cultivated because very few are found in nature. In the artificial culture method, the inoculum culture includes a solid inoculum culture method and a liquid inoculum culture method. In the case of Cordyceps sinensis, the liquid inoculum culture method is common, but most of the culture is confined to the Erlenmeyer flask culture, and no industrial culture method has been developed. Korean Patent Publication No. 97-68810 discloses a method for inoculating and cultivating Militaris cordyceps using a cocoon. However, the mass culture method of inoculum has not yet been systematically established, and there is no industrial level culture method.
The inventor of the present invention pays attention to the above-mentioned points, and manufactures a liquid inoculum of Cordyceps aspen aseptically easily using the liquid culture apparatus (20) of the present invention to mass-produce Cordyceps fruit bodies. Was completed.
Accordingly, an object of the present invention is to provide a liquid inoculum culture apparatus for mass production of Cordyceps fruit bodies .
[0004]
[Problems to be solved by the invention]
The above-mentioned object of the present invention is to obtain a caterpillar of the caterpillar and expand it in a petri dish, inoculate the cell culture of the caterpillar of the caterpillar on the inoculum stock medium, and incubate at 24 ° C. for 5 days to produce an inoculum After that, a liquid medium composed of dextros, yeast extract, peptone, distilled water and antifoaming agent is manufactured, and the inoculum (10) is used so that the ratio of the liquid medium and the culture inoculum becomes 100: 1. After inoculation, the liquid inoculum is produced by culturing for 5 days while injecting air sterilized at a rate of 0.3 vvm at 25 ° C. using the liquid inoculum culture device (20) of the present invention. This was achieved by inoculating and cultivating a cereal medium consisting of cocoon and cocoon pieces to mass-produce Cordyceps fruit bodies.
[0005]
[Means for Solving the Problems]
The configuration of the present invention will be described below.
The present invention, inoculation instrument (10) liquid seed culture unit (20), the liquid seed culture apparatus for mass production of winter Mushinatsu drafts and implements entity consisting of,
The inoculation instrument (10),
An inoculation container (11) containing a liquid culture ;
A stopper ( 12 ) at the upper end of the inoculation container (11) ;
One end is connected to the stopper ( 12 ) and located at the bottom of the inoculation container (11) , and the other end is connected to a compressor to inject air into the inoculation container (11) (13 ) And
An air filter (14) for air sterilization mounted on the air injection pipe (13) ;
One end is located at the bottom of the inoculation vessel (11) , and the other end consists of a culture medium discharge pipe (15) opened to the outside atmosphere ,
The liquid seed culture device (20),
A culture vessel (21) containing a liquid culture ;
A stopper (22) at the upper end of the culture vessel (21) ;
One end is positioned at the bottom of the culture vessel (21) , and the other end is connected to a compressor through a removable air filter (25) to inject the inoculum obtained by cultivating the Cordyceps spp. A dual purpose tube (23) to be injected ,
A locking device (24) for adjusting the opening and closing of the dual-purpose pipe ( 23 ) ;
One end is connected to the inside of the culture vessel (21) , and the other end is an air discharge pipe (26) open to the outside atmosphere ,
A filter (27) attached to the air discharge pipe (26) ,
After injecting cordyceps inoculum from the culture solution discharge pipe (15) of the inoculation device (10) through the combined pipe (23 ) with the air filter (25) removed ,
The air filter (25) is attached to the dual purpose tube (23), and air sterilized from the outside is injected into the culture vessel (21) ,
Liquid inoculum culture for mass production of Cordyceps botanical bodies characterized by aeration culture while discharging the air in the culture vessel (21) to the outside through the filter (27) in the air discharge pipe (26) Device.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Reference Examples , but the scope of rights of the present invention is not limited to these Examples.
[0007]
Reference Example 1 : Isolation and expansion of cordyceps fungus. The caterpillar of Cordyceps sinensis in reference example 1 of the present invention was used after being sold. After plate culture of the caterpillar of the caterpillar, inoculate a solid medium composed of 40 g dextros, 10 g yeast extract, 10 g peptone, 18 g agar, and 1000 ml distilled water, and expand for 4-8 days at 22-30 ° C incubator. Cultured.
[0008]
Reference Example 2 : Culture of Cordyceps inoculum: Stage 1: Production of culture solution for Cordyceps inoculum Stock culture to produce Cordyceps inoculum was prepared as shown in Table 1, and autoclave And sterilized at 121 ° C. for 20 minutes.
[0009]
[Table 1]

[0010]
Second stage: Cultivation of Cordyceps inoculum: The culture solution produced in the first stage of Reference Example 2 was inoculated with the protozoa expanded in a petri dish. When the mycelium grows about 70% in a petri dish, inoculate the mycelium pieces with white metal fittings so that other bacteria do not enter the culture solution in the culture vessel, and inoculate the inoculated culture solution at 24 ° C. Cultured for 5 days.
[0011]
Example 1 : Cultivation of liquid inoculum of Example 1 of the present invention First stage: Production of medium for liquid inoculum culture For liquid inoculum culture of Example 1 of the present invention, as shown in Table 1, liquid culture A liquid was produced. In addition, as an antifoaming agent for removing bubbles generated during liquid culture, one or two of edible oil for household use, cottonseed oil, coconut oil, olive oil and coconut oil were used. The antifoaming agent was added in an amount of 0.1 to 1.0% (w / v) with respect to the liquid culture solution so as to spread over the entire upper layer of the culture solution.
[0012]
Second stage: Inoculum inoculation for liquid inoculum culture of Example 1 of the present invention The Cordyceps inoculum inoculated in the second stage of Reference Example 2 is added to the liquid culture solution produced in the first stage of Reference Example 1 . Vaccinated. The inoculum was inoculated with 0.1 L of the culture inoculum for 10 L of the liquid culture solution. At this time, in order to prevent contamination with germs, an inoculation device (10) was used as shown in FIG. The inoculation device (10) includes an inoculation container (11), a stopper (12), an air injection tube (13), an air filter (14), and a culture solution discharge tube (15). An air injection pipe (13) and a culture solution discharge pipe (15) are connected to the stopper (12) of the inoculation container 11. An air filter (14) for air sterilization is attached to the air injection pipe (13), and one end thereof is located at the bottom of the inoculation container (11) and the other end is connected to a compressor. One end of the culture solution discharge pipe (15) is located at the bottom of the inoculation container (11), and the other end is connected to a dual-purpose pipe (23) installed in the liquid inoculum culture device (20). The culture medium discharge pipe (15) is connected with different pipe sizes, and after the rubber pipe is connected, it is closed with foil to prevent contamination of germs. These air injection pipe (13) and the culture liquid discharge pipe All (15) were installed within 1 cm from the plug (12). Therefore, when inoculating the inoculum into the liquid inoculum culture device (20), air is injected into the air injection tube (13) using a compressor, and the culture solution discharge tube (15) is also used as the liquid inoculum culture device (20). The inoculum was discharged into the liquid inoculum culture device (20) by the pressure of the air that was connected to the tube (23) and entered the air injection tube.
[0013]
Third stage: Cultivation of liquid inoculum of Example 1 of the present invention The liquid inoculum of Example 1 of the present invention was cultured using the liquid inoculum culture apparatus (20) of the present invention as shown in FIG. The liquid inoculum culture device (20) includes a culture vessel (21), a stopper (22), a dual-purpose tube (23), a locking device (24), an air filter (25), an air discharge tube (26), and a filter ( 27). The culture vessel (21) contains a liquid culture solution, and the cap (22) at the upper end of the culture vessel is a dual-purpose tube that also serves to discharge the culture solution after air, inoculum injection, and culture are completed. (23) and the air discharge pipe (26) are connected. The dual-purpose pipe (23) is equipped with a locking device (24) that adjusts the sealing of the pipe and an air filter (25) for air sterilization, and the air discharge pipe (26) is harmful and harmful in the incubator A filter (27) is installed to prevent harmful bacteria from being released to the outside. One end of the dual-purpose tube (23) is located at the bottom of the culture vessel (21), and the other end is connected to a 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 opened to the outside atmosphere. Therefore, the present inventor obtained a liquid inoculum by culturing for 5 days while injecting air sterilized at 25 ° C. at a rate of about 0.3 vvm using the liquid inoculum culture apparatus (20) as described above.
[0014]
Reference Example 3 : Formation of cocoon using Cordyceps liquid seed fungus In order to form cocoon using Cordyceps liquid seed fungus of Reference Example 3 of the present invention, culturing 80 g of brown rice and 5 g of potato pieces in a 1000 ml translucent plastic container. A medium was prepared. At this time, when brown rice was used, the brown rice: water content was added at a ratio of 1: 1.5, and when only rice bran was used as the medium, no water was supplied. The anther culture medium was sterilized at 121 ° C. for 20-30 minutes using a high-pressure steam sterilizer. The sterilized koji culture medium was inoculated so that the liquid inoculum produced in Example 1 was spread evenly on the cereal medium and the surface thereof was covered. At the time of inoculation, inoculation was carried out in a sterile room in order to prevent contamination with germs. The medium inoculated with the liquid inoculum is transferred to a culture chamber in which 70 to 80% humidity and a temperature of 24 ° C. inside and outside the fluorescent lamp can be maintained to maintain light, and cultured for 10 to 15 days. It moved to the culture | cultivation room | chamber of 0 degreeC so that the wrinkle might be formed. At 20 ° C, the hyphae are initially milky in color, but gradually become yellowish throughout the hyphae. At this time, if the temperature of the culture chamber is maintained at 20 ° C., humidity 80-90%, and light intensity at 500-1000 Lux, the color of the mycelium that grows on the surface of the medium changes to a fluffy state, and after 15-18 days, Occurrence of the locus was started when a prominent mycelium of dark orange color started to form on the surface. As for the development of the child seat, the length of the child seat expanded in the early stage, and the upper end of the child seat was thickened like a stick, forming a cyst of that part. As the formed ascopae matures, the growth of the cocoon stops and gradually begins to degenerate, and the ascopae formed on the head of the cocoon is somewhat more atrophic than that found in the natural state, And the ascospore morphology was observed to be identical to that found in nature. Contrary to these general characteristics, strains with excellent cocoon formation have been shown to inhibit the development of cocoons in strains where hyphal growth is excessive on the medium surface.
[0015]
【The invention's effect】
As described above with reference examples and examples, by culturing liquid inoculum using the liquid culture apparatus of the present invention, contamination of various bacteria is prevented and the occurrence of defective products due to contamination with various bacteria is significantly reduced. There is a long point that can easily cultivate cordyceps.
[Brief description of the drawings]
[0016]
FIG. 1 shows an inoculation device (10) for aseptically inoculating a Cordyceps liquid inoculum constituting the present invention .
FIG. 2 shows a liquid inoculum culture device (20) for facilitating inoculation, culture and discharge of liquid main bacteria constituting the present invention.
[Explanation of symbols]
[0017]
10: Inoculation device 11: Inoculation container
12: Plug part 13: Air injection pipe
14: Air filter 15: Discharge pipe
20: Liquid inoculum culture device 21: Culture vessel
22: Plug part 23: Combined pipe
24: Locking device 25: Air filter
26: Air exhaust pipe 27: Filter

Claims (1)

Inoculum (Ten) And liquid inoculum culture equipment (20) In a liquid inoculum culture device for mass production of Cordyceps fruit body consisting of
The inoculum (Ten) Is
Inoculation container containing liquid culture (11) When,
The inoculation container (11) The top plug ( 12 )When,
One end is the stopper ( 12 Connected to the inoculation container) (11) Located at the bottom of the inoculation container with the other end connected to the compressor (11) Air injection pipe to inject air into (13) When,
Said air injection pipe (13) Air filter for air sterilization mounted on (14) When,
One end is the inoculation container (11) Is located at the bottom of the culture medium, and the other end is open to the outside atmosphere. (15) And consists of
Liquid inoculum culture device (20) Is
A culture vessel containing a liquid culture (twenty one) When,
The culture vessel (twenty one) Top plug part of (twenty two) When,
One end is the culture vessel (twenty one) The other end is a removable air filter (twenty five) Combined with a compressor through which the inoculum obtained by cultivating the caterpillar of Cordyceps sinensis is infused, or combined with air (twenty three) When,
The combined pipe ( twenty three ) Locking device to adjust opening and closing (twenty four) When,
One end is the culture vessel (twenty one) The air discharge pipe is connected to the inside and the other end is open to the outside atmosphere (26) When,
Air discharge pipe (26) Filter attached to (27) And consists of
Air filter (twenty five) The above-mentioned combined use tube in a state where it is removed (twenty three) Through the inoculum (Ten) Of the culture medium discharge tube (15) After injecting Cordyceps inoculum,
Air filter (twenty five) The combined use tube (twenty three) Attached to the culture container, the air sterilized from the outside (twenty one) Injected into the inside of
Air discharge pipe (26) In the culture vessel (twenty one) The air inside the filter (27) A liquid inoculum culture device for mass production of Cordyceps fruit bodies characterized by aeration culture while discharging through the outside.

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