KR20170043795A - Culture method for improving spore formation of Beauveria bassiana - Google Patents
Culture method for improving spore formation of Beauveria bassiana Download PDFInfo
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Abstract
본 발명은 곤충병원성 곰팡이균(Beauveria bassiana)의 포자형성 향상을 위한 액체배지 조성물 및 배양조건에 관한 것으로, 구체적으로 상기 곰팡이균의 배양 시, 기존의 SDY(glucose, peptone, yeast extract)배지와 GYMK(glucose, yeast extract, MgSO4.7H2O, K2HPO4) 및 ERBM(미강을 물(1:9)로 121℃에서 15분간 멸균하여 추출한 추출액, (NH4)2SO4)에서의 배양을 비교한 결과, pH 5.0, 25℃ GYMK 배지에서 배양 시, SDY 배지보다 균의 생육 및 포자 형성도가 10배 이상 우수함을 확인하였고, 또한 배양 시 분산성을 향상시키기 위해서는 2% 글리세롤(glycerol)을 첨가하는 것이 균의 생장에 안정적이고, 배지 색상 변화에 영향이 없으며, 포자 형성에도 효과적임을 확인하여, 이를 통해 상기 곰팡이균의 최적 액체배지 조성 및 배양조건으로 유용하게 사용될 수 있다.The present invention relates to a liquid medium composition and a culture condition for improving spore formation of an insect pathogenic fungus ( Beauveria bassiana ). More specifically, the present invention relates to a liquid medium composition and a culture condition for culturing the fungus, wherein a conventional SDY (glucose, peptone, yeast extract) (NH 4 ) 2 SO 4 ) extracted by sterilization of glucose, yeast extract, MgSO 4 .7H 2 O, K 2 HPO 4 and ERBM in water (1: 9) at 121 ° C for 15 minutes As a result of comparing the cultures, it was confirmed that the growth and spore formation of the germs were more than 10 times better than the SDY medium when cultured in the GYMK medium at pH 5.0 and 25 ° C. In order to improve the dispersibility in the culture, 2% glycerol ) Is stable to the growth of bacteria, has no effect on the color change of medium, and is effective for spore formation. Thus, it can be usefully used as the optimum liquid medium composition and culture condition of the fungus.
Description
본 발명은 곰팡이균 배지 조성물 및 배양방법에 관한것으로서 구체적으로, 곤충병원성 곰팡이균인 백강균(Beauveria bassiana)의 포자형성 향상을 위한 액체배지 조성물 및 최적 배양조건을 이용한 포자형성 향상을 위한 배양방법에 관한 것이다.The present invention relates to a fungus culture medium composition and a culture method, and more particularly, to a fungus culture medium composition comprising a fungus belonging to the genus Beauveria bassiana ) and a culture method for improving spore formation using optimal culture conditions.
백강균인 보베리아 바시아나(Beauveria bassiana)는 농촌진흥청 국립농업과학원 농업미생물과에서 분양받은 균주로서 진딧물에 대한 살충효과 확인된 균주이다. Beauveria bassiana (Beauveria bassiana) is a strain cultivated in the Department of Agricultural Microbiology, National Institute of Agricultural Science and Technology (RDA), and has been shown to have insecticidal effect against aphids.
일반적으로 보베리아 바시아나(Beauveria bassiana)균은 불완전세대 분생자형 자실체로서 분생포자가 곤충 외벽의 표면에 달라붙어 발아한 후 침입도구인 부착기로 큐티클층을 통과하거나 분생포자에서 발아한 발아관이 직접 큐티클층을 통과하여 곤충내부로 침입하게 되는데, 이때 물리적인 작용과 lipase, protease, chitinase 등 효소작용이 함께 일어난다. 곤충내부에 침입한 곰팡이는 균사체로 성장하거나 확장된 발아관으로부터 균사체가 격막화되어 출아포자를 형성한 후 독성 대사물질(cyclodepsipeptide, beauvericin)을 분비하거나 혈체강의 영양원을 고갈시켜 숙주를 죽인다. 곤충이 죽고 난 후에 곤충내부는 균사체가 계속해서 성장하여 균사체로 꽉 찬 미이라와 같은 곤충사체는 건조하거나 추운 환경에서는 균사체의 저장과 보존 창고로서 작용하다가 성장하기 좋은 환경이 되면 곤충의 큐티클을 뚫고 나와서 성장하여 사체 위에 포자층을 형성한다. 이렇게 성장한 곰팡이가 생산하는 분생포자는 물이나 바람 등에 의해서 다시 부근 지역의 다른 곤충을 감염시킨다. 감염된 곤충은 죽기도 하지만 생식능력이 감소되거나 또는 감염된 유충이 태어나기도 하는 이차적인 살충효과도 있다(한국생물공학회지 14(3), 365~370, 1999; 한국잠사학회지, 38(1), 31~35, 1996; 대한민국 등록특허 10-2011-0094749).In general, Beauveria bassiana is an incomplete generation of conidial fruiting body, which is attached to the outer surface of the insect outer wall and germinated. It passes through the cuticle layer and enters the inside of the insect. At this time, the action of lipase, protease, chitinase and other enzymes occurs simultaneously. Fungi that enter the inside of the insects may grow as mycelium or diapause of the mycelium from the extended germination tube to form spores and secrete toxic metabolites (cyclodepsipeptide, beauvericin) or deplete the nutrients of the bloodstream to kill the host. After the insect has died, insects such as mummies, which continue to grow and mycelium filled with insects, grow and store as mycelium in dry or cold environments. When the environment grows, they break through the cuticle of insects It grows and forms a spore layer on the carcass. The fungus that grows in this way infects other insects in the vicinity by water or wind. There is also a secondary insecticidal effect, in which the infected insects die but the fertility is reduced or the infected larvae are born (Korean Biotechnology Journal, 14 (3), 365 ~ 370, 1999; ~ 35, 1996; Korean Patent No. 10-2011-0094749).
작물의 병해충 방제용 농약은 장기간 또는 과다사용 시 병해충의 저항성 발현과 환경오염 등의 부작용을 일으키는 것으로 알려져 있다. 이러한 문제 해결을 위해 미생물을 이용한 해충방제가 그 대안으로 제시되고 있고, 전 세계적으로 해충 방제용으로 등록된 곰팡이 살충제는 171품목으로 나방류, 딱정벌레목, 진딧물, 가루이 등의 해충방제에 많이 사용되고 있다. 국내에는 3종의 곰팡이 살충제가 등록되어 있다. 해충방제를 위해 곤충병원성 곰팡이를 작물에 살포할 때는 일반적으로 고농도의 포자를 살포한다. 따라서 살충성 곰팡이 포자의 대량생산은 곰팡이 살충제를 개발하는데 우선적으로 해결되어야만 하는 핵심 사항이다. 곤충병원성 곰팡이 포자의 대량 배양에는 일반적으로 3가지 방법[고체 배양, 액체 배양, 액체-고체 배양의 2단계 배양법(two-phase fermentation)]이 사용되고 있다. 고체 배양은 곤충병원성 곰팡이의 포자 생산에 가장 많이 사용되는 방법으로 기질로 보리, 쌀 등의 곡물을 사용하고 있다. 액체 배양은 출아포자(blastospore) 생산에 주로 사용되는 방법이고, 배지에 글루코스(glucose), 레시틴(lecithin) 등을 첨가하면 곤충병원성 곰팡이의 포자 생산량이 증가되는 것으로 보고되어 있다. 보베리아 브롱니아티(Beauveria brongniartii)는 효모 추출물(yeast extract)과 펩톤(peptone)을 액체 배지에 첨가하였을 때 포자 생산이 증가되었으며 카사미노산(casamino acid)을 첨가한 배지에서 메타리지움 아니소필래(Metarhizium anisopliae)는 포자 생산이 증가 될 뿐만 아니라 포자의 병원성도 증진되었다. 또한, 살충미생물의 대표적 균주인 백강균(Beauveria bassiana)과 같은 진균배양에 주로 이용되는 배지는 PD(potato dextrose broth)배지로 실험실 규모의 배양에서 주로 사용된다. 그렇지만 상용화를 위한 대량 배양조건에서는 PD배지를 이용하여 배양하는 것은 비경제적이기 때문에 경제성면에서 우수하고 쉽게 구할 수 있는 배지의 개발이 요구되고 있다.Pesticides for control of pests of crops are known to cause side effects such as the development of resistance to pests and environmental pollution during prolonged or excessive use. In order to solve these problems, pest control using microorganisms has been suggested as an alternative. Fungicide insecticides registered for pest control worldwide have been used for controlling insects such as moths, beetles, aphids, and powdery moths. Three types of fungicide are registered in Korea. When insect pathogenic fungi are applied to crops for pest control, spray with high concentrations of spores. Therefore, mass production of insecticidal fungus spores is a key issue that should be solved first in developing mold insecticides. In general, three methods (solid culture, liquid culture, two-phase fermentation of liquid culture and solid culture) are used for mass culture of insect pathogenic fungus spores. Solid culture is the most commonly used method to produce spores of insect pathogenic fungi. Grains such as barley and rice are used as substrates. Liquid culture is a method mainly used for production of blastospore. It has been reported that addition of glucose, lecithin to the medium increases the production of spores of insect pathogenic fungi. In the case of Beauveria brongniartii , when the yeast extract and peptone were added to the liquid medium, spore production was increased, and in the medium supplemented with casamino acid, ( Metarhizium anisopliae ) not only increased spore production but also increased the virulence of spores. In addition, a representative strain of insecticidal microorganism, Beauveria bassiana ) are mainly used in laboratory scale cultures with PD (potato dextrose broth) medium. However, in a large-scale culture condition for commercialization, it is not economical to cultivate using a PD medium. Therefore, it is required to develop a medium that is excellent in economy and can be easily obtained.
곤충병원성 곰팡이 포자는 고체배양이나 액체배양 중 일지라도 정치배양을 하여야 표면에 균사가 성장하면서 포자가 형성되는 것이 일반적인 것으로 알려져 있다. 곤충병원성 곰팡이의 포자획득은 각 종마다 그리고 배지의 성분에 따라 포자농도의 정도가 다르며, 종래의 경우 곰팡이의 포자 생산방법으로 고체배양에 관하여 많이 연구가 되었으나(J. Life Science, 23(8), 1010~1018, 2013; 대한민국 등록특허 1998-0034989; 한국생물공학회지 14(3), 365~370, 1999; 강원대학교, 농학박사학위논문, 신상철, 2001년; Korean J. Microbiol. Biotechnol. 41(4), 398~406, 2013), 액체배양에서 포자형성이 잘되는 조건에 대한 선행기술은 거의 알려진 바가 없다.Insect pathogenic fungus spores are generally cultured in solid or liquid culture to form spores while growing hyphae on the surface. The spore concentration of the insect pathogenic fungi varies depending on the species of each species and the composition of the medium. Conventionally, a lot of studies have been conducted on the solid culture by the method of spore production of fungi (J. Life Science, 23 (8) Korea Institute of Bioscience and Biotechnology, Korea Institute of Bioscience and Biotechnology, 14 (3), 365 ~ 370, 1999, Kangwon National University, Doctoral thesis, Shin Sang Chul, 2001, Korean J. Microbiol. Biotechnol. 41 (4), 398-406, 2013), there is little known about the prior art for the conditions under which spore formation is good in liquid culture.
일반적으로 농업용 부산물인 미강과 같은 곡물에서 고체배양을 하면 포자 획득이 용이하나 농업용 해충방제제로 개발하기 위해서는 분무기 노즐의 막힘현상등의 문제점이 발생하므로 물에 완전히 희석되는 분말수화제형의 개발이 요구되고 있다.Generally, it is easy to obtain spores by cultivating solid from cereals such as rice bran, which is a byproduct of agricultural use. However, in order to develop as an agricultural pest control agent, problems such as clogging of the spray nozzle occur, so development of powder wettable powder have.
또한, 기존의 액체배지에서는 포자의 형성보다는 균사의 생장에 목적을 두고 있어 균사가 잘 배양된 액체배지를 미강과 같은 고체배지에 버무려서 포자를 배양하는 방법이 일반적이다. 그러나 수화제형으로 제제화를 하고자 위와 같은 방법으로 배양이 불가능하여 액체배지에서 높은 농도의 포자를 생성하고 단균사를 배양하는데 목적이 있어 이와 같은 조성의 조건으로 배양 하는 것이다.In addition, in the conventional liquid medium, it is generally aimed at the growth of mycelium rather than the formation of spores, so it is common to cultivate the spore by mixing the liquid medium with the mycelium in a solid medium such as rice bran. However, in order to formulate into a wettable powder, it is impossible to cultivate in the above manner, so that a high concentration of spores is produced in a liquid medium, and cultivation of a single mycelium is carried out.
곤충병원성 곰팡이균주를 대량 배양하여 제품화하기에는 기존 배지가 원료비용에서 경제적 부담이 발생되어 산업화에 지장을 초래하게 된다. 기존배지는 본 발명에서 개발된 배지와 비교하였을 때 배양 일수가 길어지기 때문에 PDB, SDB와 같은 기존 배지를 사용하는 것이 어려운 것이다. 여기서 배지의 성분차이에 따른 배양일수가 2일 정도 길어진다면 그에 따른 추가비용에 대한 문제도 무시할 수 없다. 따라서, 본 발명은 기존배지(조제배지)를 사용하지 못하고 저가의 높은 포자 농도를 유도할 수 있는 고효율의 성분들을 사용하여 포자형성이 최적화된 액체배지에서 살충균주를 배양하여 제품의 시가에 맞는 포자형성이 최적화된 대량 배양배지를 선정하는데 목적이 있다. In order to commercialize a large number of insect pathogenic fungus strains, the conventional culture medium will have an economic burden on raw material costs, which will hinder industrialization. It is difficult to use the existing medium such as PDB or SDB because the existing medium has a longer incubation period as compared with the medium developed in the present invention. In this case, if the culture days of the culture medium depend on the difference of the culture medium, the additional cost is not negligible. Accordingly, the present invention provides a spore culture suitable for culturing an insecticidal strain in a liquid medium in which spore formation is optimized by using high-efficiency components capable of inducing a low spore concentration without using an existing medium (preparation medium) The aim of this study was to select the optimal culture medium for growth.
또한, 기존 배지는 대량배양하기에 부적합한 포자의 농도와 단균사가 배양되기 쉽지 않다는 문제점이 있어 포자 형성이 쉽지 않다.In addition, spore formation is not easy because the existing medium has a problem that the concentration of spores unsuitable for mass culture and single hyphae are not easy to cultivate.
이에, 본 발명자들은 백강균(Beauveria bassiana)의 포자 형성 향상을 위한 배양방식 개발을 위해 노력하던 중, 진탕 배양방식의 액체배양 중에도 곰팡이 포자를 형성하는 배지조성을 확립하기 위하여, 기존의 SDY(glucose, peptone, yeast extract)배지와 GYMK(glucose, yeast extract, MgSO4.7H2O, K2HPO4) 및 ERBM(미강을 물(1:9)로 121℃에서 15분간 멸균하여 추출한 추출액, (NH4)2SO4)에서의 배양을 비교한 결과, pH 5.0, 25℃ GYMK 배지에서 130rpm으로 5일간 배양 시 균의 생육과 포자 형성이 우수함을 확인하여, 상기 곰팡이균의 최적 배지의 조성 및 배양조건을 확립함으로써 본 발명을 완성하였다.Thus, the present inventors have baekganggyun (Beauveria In order to establish a culture medium for mold spores during liquid cultivation of shaking culture method, conventional SDY (glucose, peptone, yeast extract) medium and GYMK glucose, yeast extract, MgSO 4 .7H 2 O, K 2 HPO 4 ) and ERBM (1: 9) at 121 ° C for 15 minutes, (NH 4 ) 2 SO 4 ) As a result, it was confirmed that the growth of bacteria and spore formation were excellent when cultured at pH 5.0 and 25 ° C in GYMK medium at 130 rpm for 5 days, and the present inventors completed the present invention by establishing the optimal culture conditions and culture conditions for the fungus bacterium .
본 발명의 목적은 기존의 배지 및 배양조건에서 곤충병원성 곰팡이 포자의 대량생산에 비효율적이고 비경제적인 문제점을 극복하고자, 백강균(Beauveria bassiana)의 포자형성 향상을 위한 최적 액체배지 조성물 및 배양조건을 확립함으로써, 이를 이용한 백강균 포자의 대량 생산 방법에 관한 것이다.It is an object of the present invention to provide an optimal liquid medium composition and culture conditions for improving spore formation of Beauveria bassiana in order to overcome ineffective and uneconomical problems in the mass production of insect pathogenic fungus spores under existing culture medium and culture conditions , And a method for mass production of spore bacterium using the same.
상기 목적을 달성하기 위하여, 본 발명은 포자형성 향상을 위한 액체배지 조성물을 제공한다.In order to achieve the above object, the present invention provides a liquid medium composition for improving spore formation.
또 다른 목적은 본 발명에 따른 액체배지(GYMK(glucose, yeast extract, MgSO4.7H2O, K2HPO4) 배지)를 이용하여 pH 5.0, 25℃의 조건에서 배양하는 것을 포함하는 백강균의 포자형성 향상을 위한 배양방법을 제공한다.Yet another object of the present invention is to provide a method for producing a yeast extract which comprises culturing yeast extract in a liquid culture medium (GYMK (glucose, yeast extract, MgSO 4 .7H 2 O, K 2 HPO 4 ) medium according to the present invention at pH 5.0 and 25 ° C A culture method for improving spore formation is provided.
본 발명은 백강균(Beauveria bassiana)을 GYMK 배지(glucose, yeast extract, MgSO4.7H2O, K2HPO4)를 이용하여 pH 5.0, 25℃에서 배양한 결과, 기존의 SDY 배지(glucose, peptone, yeast extract)보다 균의 생육 및 포자 형성도가 10배 이상 우수함을 확인하였고, ERBM 배지(미강을 물(1:9)로 121℃에서 15분간 멸균하여 추출한 추출액, (NH4)2SO4)에서의 배양보다 포자형성이 우수하고 배지 오염도가 낮음을 확인하였으며, 또한 배양 시 분산성을 향상시키기 위해서 2% 글리세롤(glycerol)을 첨가함으로써 균의 생장에 안정적이고, 배지 색상 변화에 영향이 없으며, 포자 형성에도 효과적임을 확인함으로써, 이를 통해 본 발명은 상기 곰팡이균의 최적 액체배지의 조성 및 배양조건을 확립함으로써 살충성 곰팡이 포자의 대량 획득에 유용하게 사용될 수 있다.The invention baekganggyun (Beauveria bassiana ) were cultured at pH 5.0 and 25 ℃ using GYMK medium (glucose, yeast extract, MgSO 4 .7H 2 O, K 2 HPO 4 ), and compared to conventional SDY medium (glucose, peptone, yeast extract) (NH 4 ) 2 SO 4 ) than in the ERBM medium (water (1: 9) for 15 min at 121 ° C for 15 min.). And 2% glycerol was added to improve the dispersibility during culture, and it was stable to the growth of the microorganism, had no effect on the color change of the medium, and was also effective for spore formation. Thus, the present invention can be used to obtain a large amount of fungus spores by establishing the optimum liquid medium composition and culture conditions of the fungus.
도 1은 백양균(Beauveria bassiana)을 3개의 GYMK 배지에 배양하고 3일 및 4일차에 배지색상의 변화를 나타낸 도이다.Brief Description of the Drawings Fig. 1 is a graph showing the change of medium color on three days and four days after incubation of three kinds of GYMK medium on Beauveria bassiana .
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 글루코스(glucose) 1 내지 3%(v/v), 효모 추출물(yeast extract) 0.1 내지 0.3%(v/v), MgSO4.7H2O 0.004 내지 0.006%(v/v), K2HPO4 0.004 내지 0.006%(v/v)로 구성된 백강균(Beauveria bassiana)의 포자형성 향상용 액체 배지 조성물을 제공한다.The present invention relates to a pharmaceutical composition comprising 1 to 3% (v / v) glucose, 0.1 to 0.3% (v / v) yeast extract, 0.004 to 0.006% (v / v) MgSO 4 .7H 2 O, K 2 HPO 4 0.004 to 0.006% (v / v) ( Beauveria bassiana ). < / RTI >
상기 액체 배지 조성물은 글루코스 2%(v/v), 효모 추출물 0/2%(v/v), MgSO4.7H2O 0.005%(v/v), K2HPO4 0.05%(v/v)로 구성된 것이 더욱 바람직하다.The liquid medium composition glucose, 2% (v / v), yeast extract 0/2% (v / v ), MgSO 4 .7H 2 O 0.005% (v / v),
또한, 글리세롤 1.5 내지 2.5%(v/v)를 더 포함하는 것이 바람직하고, 2%로 첨가하는 것이 더욱 바람직하다.Further, it is preferable to further include 1.5 to 2.5% (v / v) of glycerol, more preferably 2%.
또한, 본 발명은 In addition,
1) 백강균을 글루코스 1 내지 3%(v/v), 효모 추출물 0.1 내지 0.3%(v/v), MgSO4.7H2O 0.004 내지 0.006%(v/v), K2HPO4 0.004 내지 0.006%(v/v)로 구성된 액체 배지에 접종하는 단계; 및1) a
2) 단계 1)의 액체 배지를 pH 4.7 내지 5.2, 22 내지 28℃에서 100 내지 150rpm으로 3 내지 7일간 배양하는 단계; 를 포함하는 백강균의 포자형성 향상을 위한 배양방법을 제공한다.2) culturing the liquid medium of step 1) at pH 4.7 to 5.2 and 22 to 28 ° C at 100 to 150 rpm for 3 to 7 days; The present invention provides a culture method for enhancing spore formation of a white germ strains.
상기 단계 1)의 액체 배지는 글루코스 2%(v/v), 효모 추출물 0/2%(v/v), MgSO4.7H2O 0.005%(v/v), K2HPO4 0.05%(v/v)로 구성된 것이 더욱 바람직하다.The liquid medium of step 1) was prepared by adding
본 발명의 구체적인 실시예에서, 백강균(Beauveria bassiana)의 대량 생산용 배지와 배양조건을 선정한 결과, 기존의 SDY 배지(glucose, peptone, yeast extract)보다는 GYMK 배지(glucose 2%, yeast extract 0.2%, MgSO4 ·7H2O 0.005%, K2HPO4 0.05%) 및 ERBM 배지(미강을 물(1:9)로 121℃에서 15분간 멸균하여 추출한 추출액, (NH4)2SO4)에서 균의 성장이 양호하였고, 배지의 pH 차이에서도 5.0으로 조절한 배지에서 균의 생육과 포자 형성이 우수한 것을 확인하였다(표 2 참조).In a particular embodiment of the invention, baekganggyun (Beauveria Mass production selected for the medium and culture conditions, the results for the bassiana), GYMK medium (
또한, GYMK 배지 및 ERBM 배지를 비교하였을 때, GYMK 배지를 사용한 배양 결과, 균수도 높게 측정되어, GYMK 배지가 백강균의 대량 배양배지로 적합함을 확인하였다.Also, when comparing GYMK medium and ERBM medium, it was confirmed that GYMK medium was suitable as a culture medium for massive culture of Bacillus thuringiensis as a result of culturing using GYMK medium.
또한, 곰팡이 포자의 분산성을 향상시키기 위하여 글리세롤을 2% 처리한 결과, 배지의 색상 변화에 영향을 주지 않으면서 포자형성에도 효과적임을 확인하였다(표 4 참조).In addition, 2% treatment of glycerol to improve the dispersibility of fungal spores was found to be effective for spore formation without affecting the color change of the medium (see Table 4).
따라서, 본 발명을 통해 백강균의 최적 액체배지 조성물 및 배양방법으로서 GYMK 배지(glucose 2%, yeast extract 0.2%, MgSO4·7H2O 0.005%, K2HPO4 0.05%)조성물로 pH 5.0, 25℃에서 130rpm으로 5일간 배양하는 방법을 확립함으로써, 이는 살충성 곰팡이 포자의 대량 획득에 유용하게 사용될 수 있다.Thus, the optimum liquid medium composition and culturing method of the present invention was pH 5.0, 25 (g / 100 ml) with a composition of GYMK medium (
이하, 본 발명을 실시예에 의해서 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해서 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.
<< 실시예Example 1> 대량 1> Bulk 배양배지Culture medium 및 배양조건 확립 And establishment of culture conditions
백강균(Beauveria bassiana)의 대량 생산용 배지와 배양조건을 선정하고자 하기 실험을 수행하였다. 상기 백강균은 농촌진흥청 국립농업과학원 농업미생물과에서 분양받아 사용하였다. Beauveria bassiana ) for the mass production and culture conditions. The Bacillus thuringiensis was distributed from the Department of Agricultural Microbiology, National Institute of Agricultural Science, RDA.
구체적으로, SDY 배지를 대조군으로 사용하여 GYMK 배지 및 ERBM 배지에 곤충병원성 곰팡이인 백강균을 배양하여 포자 형성력을 비교하였다. 500ml 삼각플라스크에 각각의 배지를 하기 표 1과 같은 조성으로 넣고, 121℃에서 15분간 배지를 멸균하였다. 미리 백강균이 평판배양된 배지에서 포자를 백금이로 긁어 멸균증류수에 희석하여 포자현탁액을 만들고, 이를 멸균된 각각의 액체배지에 80㎕씩 접종하여 8일간 25℃, 130rpm에서 진탕 배양하였다. 배지의 pH 조건은 pH 5로 조절한 것과 순수 배지조성의 pH로 조건을 설정하여 배양하였다.Specifically, the spore forming ability of GYMK medium and ERBM medium was examined by cultivating an insect pathogenic fungus, Bacillus subtilis, using SDY medium as a control group. Each medium was placed in a 500 ml Erlenmeyer flask in the composition shown in Table 1 below, and the medium was sterilized at 121 ° C for 15 minutes. Spore suspension was prepared by scraping the spores with sterilized distilled water in advance in a medium in which platelets were cultured on a plate, and 80 쨉 l of each spore suspension was inoculated to each sterilized liquid medium. The suspension was cultured with shaking at 25 캜 and 130 rpm for 8 days. The pH of the culture medium was adjusted by adjusting the pH to 5 and the pH of the pure medium.
또한, 균의 생육정도는 혈구계수기로 포자수를 측정하는 방법과 단계희석평판도말법으로 생균수(cfu/ml) 측정하는 방법, 그리고 균체량(g)을 측정하여 최종 대량배양배지를 선발하였다.In addition, the final culture medium was selected by measuring the number of viable cells (cfu / ml) and measuring the amount of cells (g) by the method of measuring the number of spores using a hemocyte counter, the step dilution plate method.
혈구계수기 측정법은 혈구계수기내에 표시되어 있는 구획에 존재하는 포자를 현미경으로 관찰하여 그 수를 측정하였으며 단계희석평판도말법은 배양액을 10배수로 희석한 후 적당한 희석농도를 PDA 배지에 도말, 배양 한 후 형성된 콜로니를 측정하였다. 균체량은 배양된 배양액을 필터페이퍼를 이용하여 균체를 걸러내고 이를 완전히 건조시켜 그 무게를 측정하였다.The hemocyte counting method was performed by observing the number of spores present in the compartment displayed in the hemocyte counter by a microscope and measuring the number of spores by diluting the culture with 10 times the amount of the culture solution and then culturing the appropriate dilution concentration on the PDA medium Colonies were measured. For the amount of cells, the cultured medium was filtered using a filter paper, and the cells were completely dried and weighed.
펩톤(peptone)
효모 추출물(yeast extract)Glucose
Peptone
Yeast extract
10
1040
10
10
효모 추출물
MgSO4.7H2O
K2HPO4 Glucose
Yeast extract
MgSO 4 .7H 2 O
K 2 HPO 4
2
0.05
0.520
2
0.05
0.5
(NH4)2SO4 Extract of rice bran by sterilization with water (1: 9) at 121 ℃ for 15 minutes
(NH 4) 2 SO 4
0.5%95.5%
0.5%
그 결과, 하기 표 2에 나타난 바와 같이, SDY 배지보다는 GYMK 배지 및 ERBM 배지에서 균의 성장이 양호한 것을 알 수 있었다. 또한, 배지의 pH 차이에서도 5.0으로 조절한 배지에서 균의 생육과 포자 형성이 우수한 것을 확인하였다(표 2).As a result, as shown in Table 2 below, it was found that the growth of the bacteria was good in the GYMK medium and the ERBM medium rather than the SDY medium. In addition, it was confirmed that the growth of bacteria and spore formation were excellent in the medium adjusted to pH 5.0 in the medium (Table 2).
또한, 육안적 차이는 SDY 배지와 GYMK 배지에서 배지의 색상이 엷은 황색(원래 배지의 색)에서 빨간색으로 변하였고, 포자 형성이 잘 된 배지는 빨간색이 유지되는 것을 확인하였으나 ERBM 배지에서는 그러한 현상은 일어나지 않음을 확인하였다.In addition, gross differences were observed in the color of pale yellow (original color of the medium) changed to red in SDY medium and GYMK medium, and in spore-forming medium, it was confirmed that red medium was maintained. In ERBM medium, .
또한, GYMK 배지 및 ERBM 배지를 비교하였을 때, GYMK 배지를 사용한 배양 결과, 균수도 높게 측정되었으며, 배지 색상의 변화로 오염여부와 포자형성의 최대치를 확인할 수 있는 장점을 가지고 있어 GYMK 배지를 대량 배양배지로 선발하였다.In addition, when the GYMK medium and ERBM medium were compared with each other, the number of bacteria was measured as a result of culturing using GYMK medium, and it was confirmed that contamination and maximum spore formation can be confirmed by changing the color of medium, And selected as a medium.
SDY
SDY
GYMK
GYMK
ERBM
ERBM
<< 실시예Example 2> 배지 색상 변화에 따른 포자형성량 및 배지 오염 여부의 관계 규명 2> Identification of the relationship between the amount of spore formation and the contamination of media with changes in medium color
GYMK 배지(glucose 2%, yeast extract 0.2%, MgSO4 ·7H2O 0.005%, K2HPO4 0.05%)의 색상은 6일간 배양하였을 때 변화가 나타났다. 따라서 상기 배지의 색상변화에 따른 균의 생장과 포자형성정도 그리고 오염도를 확인하였다.The color of GYMK medium (
구체적으로, 500ml 삼각플라스크에 GYMK 배지 300ml를 넣고, 121℃에서 15분간 배지를 멸균하고, 여기에 미리 만들어둔 포자현탁액 80㎕를 접종하여 25℃에서 130rpm으로 배양하였다. 그런 다음, 3번 반복하여 배양 3일째부터 배양 5일째까지 매일 균수, 포자수 그리고 오염여부를 확인하였다.Specifically, 300 ml of GYMK medium was placed in a 500 ml Erlenmeyer flask, and the medium was sterilized at 121 캜 for 15 minutes. 80 쨉 l of a previously prepared spore suspension was inoculated and cultured at 25 캜 at 130 rpm. Then, it was repeated 3 times, and the number of bacteria, the number of spores and the pollution were checked every day from the third culture day to the fifth culture day.
그 결과, 표 3 및 도 1에 나타난 바와 같이, 세균 오염이 있을 시에는 배양일자에 관계없이 배지의 색상 변화는 일어나지 않았다. 그러나 배지의 색상이 변하는 일자부터 그 다음 날까지 생균수에서는 큰 차이가 없었으나 혈구계수기의 포자수는 월등히 증가하는 것을 확인하였다. As a result, as shown in Table 3 and Fig. 1, when there was bacterial contamination, the color of the medium did not change regardless of the culture date. However, there was no significant difference in the viable cell count from the day when the color of the medium changed to the next day, but the number of cells of the hemocyte counter increased significantly.
3
생균수실험(cfu/ml)
Experiment of live cells (cfu / ml)
(세균오염으로 인해 백강균이 성장하지 않아 생균수 및 혈구계수기 시험이 불가능하였음)Not measurable
(Because bacteria did not grow due to bacterial contamination, viable counts and hemocyte count tests were not possible)
혈구계수기(spore/ml)
The hemocyte counter (spore / ml)
<< 실시예Example 3> 보조제 선발 3> Selection of supplements
곰팡이 포자의 표면은 소수성이 강하여 물에 분산이 어렵기 때문에 분산성을 향상시키기 위하여 계면활성제 등을 이용한다. 따라서 쉽게 사용이 가능한 계면활성제인 글리세롤(glycerol)과 트윈 20(Tween 20)가 균의 생장에 미치는 영향을 조사하였다.Since the surface of mold spores is highly hydrophobic and difficult to disperse in water, a surfactant or the like is used to improve the dispersibility. Therefore, the effect of glycerol and tween 20 (Tween 20), which are easy to use surfactants, on the growth of bacteria was investigated.
구체적으로, GYMK 배지를 대량 배양배지로 하여 보조제(글리세롤 및 트윈 20)를 농도별로 처리하고 21℃에서 15분간 배지를 멸균한 뒤, 포자현탁액 80㎕를 접종 후 25℃에서 130rpm으로 5일간 배양하였고, 그런 다음 단계희석평판도말법으로 생균수측정을 측정하고, 혈구계수기로 포자수를 측정하였다.Specifically, the adjuvant (glycerol and tween 20) was treated with GYMK medium as a large-scale culture medium, and the medium was sterilized at 21 ° C for 15 minutes. Then, 80 μl of spore suspension was inoculated and cultured at 25 ° C. for 15 days at 130 rpm , Then the viable cell count was measured by the stepwise dilution plate method, and the cell number was measured with a hemocyte counter.
그 결과, 표 4에 나타난 바와 같이, 글리세롤이 트윈 20보다 균의 생장에 더욱 안정적인 것을 확인하였고, 특히, 글리세롤을 2% 처리 시 배지의 색상 변화에 영향을 주지 않으면서 포자형성에도 효과적인 것을 확인하였다(표 4). As a result, as shown in Table 4, it was confirmed that glycerol was more stable to the growth of bacteria than tween 20, and in particular, it was confirmed that 2% treatment of glycerol was effective for spore formation without affecting the color change of medium (Table 4).
글리세롤(glycerol)
Glycerol
트윈 20 (Tween 20)
Tween 20 (Tween 20)
따라서, 상기 결과를 통해 곤충병원성 곰팡이균인 백강균(Beauveria bassiana)은 GYMK 배지(glucose 2%, yeast extract 0.2%, MgSO4·7H2O 0.005%, K2HPO4 0.05%)를 이용하여 pH 5.0, 25℃에서 130rpm으로 5일간 배양하였을 때, 곰팡이균의 포자형성 및 균의 생육 정도가 현저함을 확인함으로써, 상기 배양조건을 최적 배지 조성과 조건으로 확립하였다.Thus, the above-mentioned result shows that Beauveria bassiana , which is an insect pathogenic fungus, can be treated with GYMK medium (
Claims (3)
1 to 3% (v / v) glucose, 0.1 to 0.3% (v / v) yeast extract, 0.004 to 0.006% (v / v) MgSO 4 .7H 2 O, K 2 HPO 4 0.004 to 0.006% (v / v) baekganggyun liquid medium composition for the improvement of spore formation (Beauveria bassiana) consisting of.
The liquid medium composition for improving spore formation according to claim 1, further comprising 1.5 to 2.5% (v / v) of glycerol.
2) 단계 1)의 액체 배지를 pH 4.7 내지 5.2, 22 내지 28℃에서 100 내지 150rpm으로 3 내지 7일간 배양하는 단계; 를 포함하는 백강균의 포자형성 향상을 위한 배양방법.1) a glucose baekganggyun 1 to 3% (v / v), yeast extract from 0.1 to 0.3% (v / v), MgSO 4 .7H 2 O 0.004 to 0.006% (v / v), K 2 HPO 4 0.004 to 0.006% (v / v); And
2) culturing the liquid medium of step 1) at pH 4.7 to 5.2 and 22 to 28 ° C at 100 to 150 rpm for 3 to 7 days; Wherein the spore formation is carried out in a culture medium.
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