KR20230100890A - Method for producing spores of microbial agent for preventing fungal diseases of plant - Google Patents
Method for producing spores of microbial agent for preventing fungal diseases of plant Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
본 발명은 살균제 대체 미생물농약 및 생화학 농약에 관한 것으로서, 특히 Bacillus velezensis CE100 균주와 고체배양 기술을 이용하여 밀기울, 규조토, 제올라이트와 같은 담체를 사용한 포자 생산기술을 제공하는 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법에 관한 것으로서, 균주 Bacillus velezensis(이하, 균주)를 MS배지에서 액체배양하는 단계; 밀기울을 담체로 하는 고체배지를 멸균하는 단계; 및 상기 액체배양된 균주를 상기 고체배지에서 고체배양하는 단계;로 이루어지는 것을 특징으로 하는 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법을 제공한다. The present invention relates to microbial pesticides and biochemical pesticides as substitutes for fungicides, and in particular, microorganisms for fungal disease control in crops that provide spore production technology using carriers such as wheat bran, diatomaceous earth, and zeolite using Bacillus velezensis CE100 strain and solid culture technology It relates to a method for producing spores for agricultural chemicals, comprising: liquid-cultivating a strain Bacillus velezensis (hereinafter referred to as a strain) in an MS medium; Sterilizing a solid medium containing wheat bran as a carrier; and solid-cultivating the liquid-cultured strain in the solid medium.
Description
본 발명은 살균제 대체 미생물농약 및 생화학 농약에 관한 것으로서, 특히 Bacillus velezensis CE100 균주와 고체배양 기술을 이용하여 밀기울, 규조토, 제올라이트와 같은 담체를 사용한 포자 생산기술을 제공하는 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법에 관한 것이다.The present invention relates to microbial pesticides and biochemical pesticides as substitutes for fungicides, and in particular, microorganisms for fungal disease control in crops that provide spore production technology using carriers such as wheat bran, diatomaceous earth, and zeolite using Bacillus velezensis CE100 strain and solid culture technology It relates to a method for producing spores for pesticides.
본 발명은 농림축산식품부의 재원으로 농림식품기술기획평가원의 핵심농자재국산화기술개발사업(321055051HD030)의 지원으로 수행된 연구결과이다.The present invention is a research result performed with the support of the Core Agricultural Materials Localization Technology Development Project (321055051HD030) of the Agriculture, Forestry and Food Technology Planning and Evaluation Institute funded by the Ministry of Agriculture, Food and Rural Affairs.
현재 세계 작물보호제 시장은 화학농약 대비 생물농약이 전체 시장의 9.6%를 차지하고 있으나, 화학농약의 감축정책과 유기농 육성정책으로 연평균 6.6%의 성장률로 2023년에는 17.7%로 크게 성장할 것으로 예상된다.Currently, biological pesticides compared to chemical pesticides account for 9.6% of the global crop protection market, but it is expected to grow significantly to 17.7% in 2023 with an average annual growth rate of 6.6% due to the reduction policy of chemical pesticides and the organic cultivation policy.
특히 아시아 태평양 국가들은 인구증가로 인해 작물보호제 수요가 급증하고 있어 2023년에는 생물농약의 비중이 23.4%가 될 것으로 예측된다.In particular, in Asia Pacific countries, demand for crop protection agents is rapidly increasing due to population growth, and the share of biological pesticides is expected to be 23.4% by 2023.
따라서 작물보호제 시장의 다국적 기업과 중소기업에게는 생물농약이 새로운 블루오션으로 떠오르고 있는 실정이다. Therefore, biopesticides are emerging as a new blue ocean for multinational companies and small and medium-sized enterprises (SMEs) in the crop protection market.
국내에서는 정부차원에서 친환경농업 육성방안에 따른 친환경농업 실천 확대와 화학비료 사용량 절감 및 토양 지력 증진을 위한 방안이 추진되고 있으며, 고독성 농약 등록 취소와 화학농약 사용 감축 및 미생물, 천연추출물 등 친환경 생물농약의 개발 및 사용이 확대되고 있고, 친환경 안전한 농산물에 대한 소비자의 요구 및 사회적 가치 상승에 따라 국내 친환경농산물의 수요 및 유기농산물의 시장규모가 증가하고 있으며, 유기농단지의 신규 조성과 함께 현재의 농약사용량을 매년 3%씩 감축하여 무농약 이상 농작물 재배면적을 전체 재배면적의 15%까지 육성하는 계획이 추진되고 있다. In Korea, measures to expand eco-friendly agriculture practices, reduce chemical fertilizer use, and improve soil fertility are being promoted at the government level according to the eco-friendly agriculture fostering plan, canceling registration of highly toxic pesticides, reducing the use of chemical pesticides, and eco-friendly biological pesticides such as microorganisms and natural extracts The development and use of agricultural products are expanding, and the domestic demand for eco-friendly agricultural products and the market size of organic products are increasing according to consumer demand for eco-friendly and safe agricultural products and the increase in social value. A plan is being implemented to reduce the amount of crops cultivated by pesticide-free crops up to 15% of the total cultivated area by reducing agricultural production by 3% every year.
또한, 소단위 미생물 제품의 희석 살포에 따른 비료효능 및 병해 방제능이 미미하고, 미생물 제품에 함유된 유효성분의 농도가 낮고 활성균체수가 경시적으로 불안정하며, 균체의 기질이나 기주 특이성이 취약하여 농가현장에 정착이 어려움이 있기 때문에 미생물 농약 증대를 위한 혁신적인 저가 고효율 제형화 기술과 국내 최초 생화학 살균제 농약 등록이 시급이 요망되고 있다.In addition, the fertilizer efficacy and disease control ability according to dilution and spraying of subunit microbial products are insignificant, the concentration of active ingredients contained in microbial products is low, the number of active cells is unstable over time, and the substrate or host specificity of the cells is weak. Since it is difficult to settle in the field, innovative low-cost, high-efficiency formulation technology for increasing microbial pesticides and the first biochemical disinfectant pesticide registration in Korea are urgently required.
현재, 미생물은 일반적으로 고가의 배지와 배양기로 액체 배양되며 동결건조, 분무건조, 통풍건조 등의 방법으로 건조된 후 부형제, 증점제 등을 혼합하고 분쇄되어 포장되고, 최소 1억원 이상의 배지와 10-20억 상당의 배양기, 건조기, 분쇄기를 사용하고 있기 때문에 제품은 높은 가격으로 생산 판매되고 있다. Currently, microorganisms are generally cultured in liquid with expensive media and incubators, dried by freeze-drying, spray drying, air drying, etc., then mixed with excipients and thickeners, crushed, and packaged, Because it uses incubators, dryers, and grinders worth 2 billion won, products are produced and sold at high prices.
따라서 국제적인 경쟁력을 가진 미생물농약 개발을 위해 가격이 저렴하고 병해충 방제능력이 화학농약 수준의 혁신적인 미생물농약 개발이 요구되고 있으며, 국내 등록된 살균제 농약들은 식물병원균의 세포막을 파괴하는 바 그룹이므로 다양한 살균작용 기작을 가진 다양한 생물농약 개발이 요구된다.Therefore, for the development of microbial pesticides with international competitiveness, it is required to develop innovative microbial pesticides that are inexpensive and have pest control capabilities comparable to those of chemical pesticides. Development of various biological pesticides with mechanisms is required.
현재의 미생물 농약제품에는 미생물의 숫자와 항균물질의 함량이 충분치 않아 효과가 미미한 문제점이 있으며, 식물유래 생화학 살균제농약 등록 제품은 전무한 실정이므로, 생물농약의 높은 가격과 불안정한 효과가 생물농약 시장 성장의 제약이 되고 있다.Current microbial pesticide products have a problem in that the number of microorganisms and the content of antibacterial substances are not sufficient, so the effect is insignificant. Since there is no registered product of plant-derived biochemical fungicide pesticides, the high price and unstable effect of biological pesticides are the driving force behind the growth of the biological pesticide market. becoming a constraint.
그러므로 가격이 저렴하고 방제능력이 화학농약과 동일한 혁신적인 생물농약 개발이 필요한 상황이다.Therefore, it is necessary to develop innovative biological pesticides that are inexpensive and have the same control ability as chemical pesticides.
이에 본 발명자는 상술한 제반 사항을 종합적으로 고려하여 기존의 미생물 농약이 지닌 한계 및 문제점의 해결에 역점을 두어, 작물 난방제 병해인 고추 탄저병, 감자 더뎅이병, 토마토 잿빛곰팡이병 방제용 국산 소재를 이용한 저가 고효율 미생물 농약을 개발하고자 각고의 노력을 기울여 부단히 연구하던 중 그 결과로써 본 발명을 창안하게 되었다.Therefore, the present inventors comprehensively consider the above-mentioned matters and focus on solving the limitations and problems of existing microbial pesticides, and develop domestic materials for controlling crop heating disease such as pepper anthracnose, potato scab, and tomato gray mold. In order to develop a low-cost, high-efficiency microbial agricultural pesticide using the above, the present invention was created as a result of continuous research with great efforts.
따라서 본 발명이 해결하고자 하는 기술적 과제 및 목적은 국산 소재를 이용한 저가 및 고효율의 작물 곰팡이병 방제용 미생물 농약을 제조하기 위한 포자 생산기술을 제공하는 데 있는 것이다.Therefore, the technical problem and object to be solved by the present invention is to provide a spore production technology for producing a low-cost and high-efficiency microbial pesticide for fungal disease control in crops using domestic materials.
여기서 본 발명이 해결하고자 하는 기술적 과제 및 목적은 이상에서 언급한 기술적 과제 및 목적으로 국한하지 않으며, 언급하지 않은 또 다른 기술적 과제 및 목적들은 아래의 기재로부터 당업자가 명확하게 이해할 수 있을 것이다.Here, the technical problems and objects to be solved by the present invention are not limited to the technical problems and objects mentioned above, and other technical problems and objects not mentioned will be clearly understood by those skilled in the art from the following description.
상술한 목적을 달성하기 위한 본 발명에 따른 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법은, 균주 Bacillus velezensis(이하, 균주)를 MS배지에서 액체배양하는 단계; 밀기울을 담체로 하는 고체배지를 멸균하는 단계; 및 상기 액체배양된 균주를 상기 고체배지에서 고체배양하는 단계;로 이루어지는 것을 특징으로 한다.To achieve the above object, the method for producing spores for microbial pesticides for fungal disease control of crops according to the present invention comprises the steps of liquid-cultivating a strain Bacillus velezensis (hereinafter referred to as a strain) in an MS medium; Sterilizing a solid medium containing wheat bran as a carrier; and solid-cultivating the liquid-cultured strain in the solid medium.
또한, 본 발명에 따른 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법에 있어서, 상기 MS배지는 글루코스(Glucose) 20g/L, 효모추출물(Yeast extract) 15g/L, K2HPO4 2.5g/L, MgSO4 1g/L, MnSO4 0.1g/L 및 NaCl 1g/L를 포함하는 것을 특징으로 한다.In addition, in the method for producing spores for microbial agricultural chemicals for fungal disease control of crops according to the present invention, the MS medium contains 20 g/L of glucose, 15 g/L of yeast extract, and 2.5 g of K 2 HPO 4 /L, MgSO 4 1g/L, MnSO 4 0.1g/L and NaCl 1g/L.
또한, 본 발명에 따른 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법에 있어서, 상기 고체배지의 멸균은 고체발효기를 이용하여 1차 저온멸균(90℃, 4시간) 및 6시간 경과 후 2차 저온멸균을 실시하는 것을 특징으로 한다.In addition, in the method for producing spores for microbial pesticides for fungal disease control of crops according to the present invention, the solid medium is sterilized using a solid fermenter for primary pasteurization (90 ° C, 4 hours) and after 6 hours, 2 It is characterized by performing secondary pasteurization.
또한, 본 발명에 따른 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법에 있어서, 상기 균주의 접종량은 상기 담체 대비 액체배양액 10중량%이고, 상기 담체에 대한 수분 접종량은 담체 대비 수분 100중량%인 것을 특징으로 한다.In addition, in the method for producing spores for microbial pesticides for fungal disease control of crops according to the present invention, the inoculation amount of the strain is 10% by weight of the liquid culture medium compared to the carrier, and the water inoculation amount of the carrier is 100% by weight of water relative to the carrier It is characterized by being
전술한 바와 같이 본 발명은 국내 친환경 대단지에 주요 작물 병해충 방제의 매뉴얼로 활용되어 국내 친환경농업 활성화에 기여하는 효과가 있다.As described above, the present invention is used as a manual for controlling major crop diseases and pests in large domestic eco-friendly plants, and has an effect of contributing to the vitalization of domestic eco-friendly agriculture.
또한, 본 발명은 기존의 유기농 자재와의 혼용방법 등 친환경과 관행 농가에서 활용 가능한 주요 작물 병해충 방제 매뉴얼을 제공하여 농가 소득 증대 및 친환경병 방제용 국산 자재의 선택폭을 넓히는데 기여하는 효과가 있다. In addition, the present invention provides a manual for controlling major crop pests that can be used in eco-friendly and conventional farms, such as methods of mixing with existing organic materials, thereby contributing to increasing farm household income and expanding the selection of domestic materials for eco-friendly disease control. .
또한, 본 발명은 해외 공인 기관(미국, 이스라엘 등)에서 포장 실증을 통해 제품화함으로써, 저가 고효율 미생물 농약 등록을 통하여 국내 미생물 살균제 시장의 절반 이상을 차지하고 있는 수입 미생물 살균제의 수입 대체 및 국산 친환경 농자재 수출을 통한 신성장 산업을 활성화하는데 기여하는 효과가 있다. In addition, the present invention is commercialized through packaging demonstration in overseas accredited institutions (USA, Israel, etc.), and through the registration of low-cost, high-efficiency microbial pesticides, import substitution of imported microbial disinfectants that account for more than half of the domestic microbial disinfectant market and export of domestic eco-friendly agricultural materials It has the effect of contributing to vitalizing new growth industries through
또한, 본 발명은 국내 살균제 농약 시장의 대부분을 차지하는 화학살균제 시장에서 저가 고효율 등록 제품을 통해 국내 미생물농약 및 생화학농약 시장을 크게 확대함으로써, 화학농약 대체용 제품과 매뉴얼을 활용하여 친환경농산물 생산 증대를 통한 농가 소득 증대 및 국민의 건강 증진에 기여하는 효과가 있다.In addition, the present invention greatly expands the domestic microbial pesticide and biochemical pesticide market through low-cost, high-efficiency registered products in the chemical disinfectant market, which accounts for most of the domestic disinfectant pesticide market, thereby increasing the production of eco-friendly agricultural products by utilizing alternative products and manuals for chemical pesticides. It has the effect of contributing to the increase of farm household income and the improvement of people's health.
도 1은 본 발명에 따른 미생물 농약의 균주 배양을 위한 액체배지 성분에 따른 생균수 조사 결과를 나타낸 그래프,
도 2는 본 발명에 따른 미생물 농약의 균주 배양을 위한 고체배지의 당 함량에 따른 생균수 조사 결과를 나타낸 그래프,
도 3은 본 발명에 따른 미생물 농약의 균주 배양을 위한 고체배지의 단백질 함량에 따른 생균수 조사 결과를 나타낸 그래프,
도 4는 본 발명에 따른 미생물 농약의 균주 배양을 위한 고체배양 담체에 따른 생균수 조사 결과를 나타낸 그래프,
도 5는 본 발명에 따른 미생물 농약의 균주 배양을 위한 배지 멸균 방법에 따른 균수 조사 결과를 나타낸 그래프,
도 6은 본 발명에 따른 미생물 농약의 균주 배양을 위한 초기균주 접종량에 따른 균수 조사 결과를 나타낸 그래프,
도 7은 본 발명에 따른 미생물 농약의 균주 배양을 위한 수분 접종량에 따른 균수 조사 결과를 나타낸 그래프,
도 8은 본 발명에 따른 미생물 농약의 균주 배양을 위한 고체배양에 따른 포자 및 수분변화 조사 결과를 나타낸 그래프. 1 is a graph showing the results of viable cell count investigation according to liquid medium components for culturing strains of microbial pesticides according to the present invention;
Figure 2 is a graph showing the results of viable cell counts according to the sugar content of the solid medium for culturing the strains of microbial pesticides according to the present invention;
Figure 3 is a graph showing the results of investigation of the number of viable cells according to the protein content of the solid medium for culturing strains of microbial pesticides according to the present invention;
Figure 4 is a graph showing the results of viable cell count investigation according to the solid culture carrier for culturing strains of microbial pesticides according to the present invention;
5 is a graph showing the results of investigation of the number of bacteria according to the medium sterilization method for culturing strains of microbial pesticides according to the present invention;
6 is a graph showing the results of investigating the number of bacteria according to the initial strain inoculation amount for culturing strains of microbial pesticides according to the present invention;
7 is a graph showing the results of investigating the number of bacteria according to the amount of water inoculation for culturing strains of microbial pesticides according to the present invention;
8 is a graph showing the results of investigation of spores and moisture changes according to solid culture for culturing strains of microbial pesticides according to the present invention.
이하에서는 본 발명에 따른 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법에 대한 실시 예를 도면을 참조하여 상세히 설명한다. 본 발명에 따른 작물 곰팡이병은 탄저병, 더뎅이병, 잿빛곰팡이병을 포함한다. Hereinafter, an embodiment of a method for producing spores for microbial pesticides for controlling fungal diseases of crops according to the present invention will be described in detail with reference to the drawings. Crop fungal diseases according to the present invention include anthracnose, scab, and gray mold.
이하에서 설명되는 실시 예는 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위하여 제공되는 것으로, 본 발명은 이하에서 개시되는 실시 예에 한정되지 않고 다양한 형태로 구현될 수 있다. The embodiments described below are provided to fully inform those skilled in the art of the scope of the invention, and the present invention may be implemented in various forms without being limited to the embodiments disclosed below.
도면들 중 동일한 구성들은 가능한 한 어느 곳에서든지 동일한 부호들을 나타낸다. 하기의 설명에서 구체적인 특정 사항들이 나타나고 있는데, 이는 본 발명의 보다 전반적인 이해를 돕기 위해 제공된 것일 뿐, 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변경, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 그리고 본 발명을 설명함에 있어, 관련된 공지 기능 혹은 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다. Like elements in the drawings indicate like reference numerals wherever possible. Specific details have been shown in the following description, which are only provided to help a more general understanding of the present invention, and are not intended to limit the present invention to specific embodiments, and all changes included in the spirit and technical scope of the present invention , it should be understood to include equivalents or substitutes. In the description of the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.
단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 출원에서, "포함하다" 또는 "가지다" 등의 용어는 명세서상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 본 발명에서의 기능을 고려하여 정의된 것으로서, 이는 본 발명의 기술적 사상에 부합되는 개념과 당해 기술분야에서 통용 또는 통상적으로 인식되는 의미로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries are defined in consideration of functions in the present invention, which should be interpreted as concepts consistent with the technical idea of the present invention and meanings commonly used or commonly recognized in the art. and, unless explicitly defined in this application, are not to be construed in an ideal or overly formal sense.
본 발명에 따른 작물의 곰팡이병 방제를 위한 미생물 농약에 사용되는 Bacillus velezensis는 많은 식물들에 대한 직간접적인 성장개선효과와 식물병원균에 대한 항균활성효과로 인해 많이 연구되고 사용되는 바실러스 종이다. Bacillus velezensis , which is used in microbial pesticides for fungal disease control of crops according to the present invention, is a Bacillus species that is widely studied and used due to its direct and indirect growth-improving effects on many plants and its antibacterial activity against plant pathogens.
이 중에서, Bacillus velezensis CE100은 전남대에서 분리한 균주로서 2020~21년 다양한 논문에서 선충 저해효과, 토마토 및 편백나무의 성장촉진, 2차대사산물인 Cyclic Tetrapeptide에 의한 딸기 탄저병균(Colletotrichum gloeosporioides)과 methyl hippurate에 의한 잿빛곰팡이균(Botrytis cinerea)에 대한 항균활성이 조사되었다.Among them, Bacillus velezensis CE100 is a strain isolated from Chonnam National University, and various papers in 2020-21 showed nematode inhibitory effect, growth promotion of tomatoes and cypress trees, strawberry anthracnose ( Colletotrichum gloeosporioides ) and methyl Antibacterial activity against gray mold fungus ( Botrytis cinerea ) by hippurate was investigated.
Bacillus 종은 식품, 의약, 농업 등의 분야에서 산업적으로 다양하게 이용되고 있다. Bacillus 종의 큰 특징 중의 하나인 포자형성은 영양소의 변동과 다양한 스트레스에 대응할 수 있으며 유전자를 보호하여 개체를 증가하는데 유리하게 사용되는 전략이다. 이러한 포자형성은 Bacillus 종의 생산과 보관 그리고 사용에 있어서 산업적으로 유용하게 사용되고 있다. Bacillus species are widely used industrially in the fields of food, medicine, and agriculture. Sporulation, one of the major characteristics of Bacillus species, can respond to nutrient fluctuations and various stresses, and is a strategy that is advantageously used to increase the population by protecting genes. This sporulation is used industrially in the production, storage and use of Bacillus species.
본 발명에 따른 작물 곰팡이병 방제용 미생물의 포자 생산을 위한 기계적 및 환경적 설정 조건을 하기와 같이 도출하였다. The mechanical and environmental conditions for producing spores of microorganisms for controlling fungal diseases in crops according to the present invention were derived as follows.
Bacillus velezensis 균주의 내생포자 생산을 위한 초기 배양액의 최적 조성을 확립하고 그 조성을 바탕으로 고체배양을 위한 담체를 선정하고 배양 조성 및 멸균, 배양 방법을 확립하였다.The optimal composition of the initial culture medium for the production of endospores of the Bacillus velezensis strain was established, the carrier for solid culture was selected based on the composition, and the culture composition, sterilization, and culture method were established.
1. 액체배지 조성1. Liquid medium composition
먼저, 포자생산을 위한 최적 배지 조건을 찾기 위해 하기의 표 1과 같은 배지 종류에 따라 균주 Bacillus velezensis(이하 '균주'라 함)를 0.2% 접종 후 30℃에서 120rpm으로 3일간 배양하고, 배양액 0.1ml를 처리하여 TSA(Tryptic soy agar)배지에서 101~109까지 희석 후 평판희석법을 이용해 미생물 개체수를 측정하였다. First, in order to find the optimal medium conditions for spore production, the strain Bacillus velezensis (hereinafter referred to as 'strain') was inoculated with 0.2% according to the type of medium shown in Table 1 below, and then cultured at 30 ° C. at 120 rpm for 3 days, and the culture medium 0.1 After treating ml and diluting to 10 1 to 10 9 in TSA (Tryptic soy agar) medium, the number of microorganisms was measured using a plate dilution method.
상기 표 1은 고체배지 접종을 위한 균주 배양에 적합한 2개의 배지조성(CN, MS)으로서, 이를 바탕으로 TSB 배지를 대조구로 당과 단백질 성분을 비롯하여 미량성분의 양을 수정한 CN1(Glucose 수정), MS(Glucose, Yeast extract 수정) 1~2의 배지조성에 대하여 30℃에서 120rpm으로 배양하여 균수를 측정하였다. 그 결과, TSB 처리구는 2.7 ×108 CFU/g의 균수를 보였으며 CN1과 MS 배지에서 1×109 CFU/g의 생균수가 확인되어 대조구에 비해 4.8배 높은 균수를 확인할 수 있었다. 이를 도 1에 나타내었다. 도 1은 액체배지 성분에 따른 생균수를 나타낸다. Table 1 above shows two medium compositions (CN, MS) suitable for culturing strains for solid medium inoculation. Based on this, TSB medium is used as a control, and the amount of sugar and protein components and minor components is corrected. CN1 (Glucose correction) , MS (Glucose, Yeast extract modified) 1 ~ 2 medium composition was cultured at 30 ℃ at 120 rpm and the number of bacteria was measured. As a result, the TSB-treated group showed a bacterial count of 2.7 × 10 8 CFU/g, and a viable cell count of 1 × 10 9 CFU/g was confirmed in the CN1 and MS medium, which was 4.8 times higher than that of the control group. This is shown in Figure 1. 1 shows the number of viable cells according to liquid medium components.
2. 고체배지 조성2. Solid medium composition
도 2 및 도 3은 고체배지의 당 및 단백질 함량에 따른 생균수를 각각 나타낸 것으로, 미생물 영양원이 미량인 질석을 담체로 하여 고체배양을 위해 당(1~4%)과 단백질(1~2%)의 성분량에 따른 생균수 변화를 3일간 34℃에서 배양하여 확인한 것이다. 도 2에 나타난 바와 같이 당함량은 Glucose(1%) 처리구에서 5.1×109 CFU/g의 생균수가 가장 높게 나왔으며, Glucose(2%) 처리구에서 배양 3일차에 3.6×109 CFU/g의 균수를 보였다. 도 3에 나타난 바와 같이 단백질 함량은 Yeast extract(1, 1.5%)에서 2.1×109 CFU/g의 생균수가 확인되었다. 이러한 시험 결과를 통해 균주배양을 위한 최적의 배지조성을 확립하였다.2 and 3 show the number of viable cells according to the sugar and protein content of the solid medium, respectively, sugar (1-4%) and protein (1-2%) for solid culture using vermiculite with a small amount of microbial nutrient as a carrier ) It was confirmed by culturing at 34 ℃ for 3 days according to the amount of components of viable cells. As shown in Figure 2, the sugar content was 5.1 × 10 9 CFU / g of the highest viable cell count in the Glucose (1%) treatment group, and 3.6 × 10 9 CFU / g on the third day of culture in the Glucose (2%) treatment group. showed the number of germs. As shown in FIG. 3, the protein content was 2.1×10 9 CFU/g of viable cells in yeast extract (1, 1.5%). Through these test results, the optimal medium composition for strain culture was established.
고체배양은 흐르는 물 없이 젖은 상태의 담체에서 미생물이 성장하게 되는데 고체 매트릭스 내에 존재하는 수분을 이용하고 충분한 산소전달이 용이하기 때문에 탱크배양과는 달리 미생물이 공기 중의 산소와 충분히 접촉하게 되며 미생물의 성장, 효소 및 생산을 증대할 수 있다. 이러한 고체배양은 비교적 저렴한 장비를 이용하여 낮은 에너지를 투입하여 생산할 수 있으며 농업 부산물과 같은 값싼 재료의 가치를 증대할 수 있는 장점을 가지고 있다.In solid culture, microorganisms grow on a carrier in a wet state without running water. Because it uses the moisture present in the solid matrix and allows sufficient oxygen transfer, unlike tank culture, microorganisms come into sufficient contact with oxygen in the air and grow microorganisms. , enzymes and production can be increased. This solid culture can be produced with low energy input using relatively inexpensive equipment and has the advantage of increasing the value of cheap materials such as agricultural by-products.
3. 고체배양 담체3. Solid culture carrier
고체배양에 적합한 담체를 찾기 위해 확립된 배지조건을 기반으로 제올라이트, 질석, 규조토, 밀기울 등을 담체로 하여 균주를 34℃에서 3일간 고체 배양한 결과 유기물 기반의 밀기울 배지에서 9.3×109 CFU/g으로 가장 높은 균수를 보였다. 도 4에 제올라이트, 질석, 규조토, 밀기울 등의 고체배양 담체에 따른 생균수를 나타내었다. 밀기울 배지가 기본적으로 가지고 있는 일정량의 탄소 영양원과 큰 입자량 때문에 확보된 공극이 균주가 성장할 수 있는 보다 좋은 조건으로 작용되어 가장 많은 균수를 보인 것으로 판단된다.Based on the established medium conditions to find a carrier suitable for solid culture, the strain was cultured at 34 ° C for 3 days using zeolite, vermiculite, diatomaceous earth, wheat bran, etc. as a carrier, resulting in 9.3 × 10 9 CFU / g showed the highest bacterial count. 4 shows the number of viable cells according to solid culture carriers such as zeolite, vermiculite, diatomaceous earth, and wheat bran. It is judged that the voids secured due to the amount of carbon nutrients and large particles that the bran medium basically has acted as a better condition for the growth of the strain, showing the highest number of bacteria.
4. 배지 멸균4. Media Sterilization
고체배양 시 배지 및 담체의 멸균정도는 중요한 배양요소이다. 효율적인 멸균 방법을 확립하기 위하여 기존의 멸균(121℃, 15분)을 대조구로 하여 간헐적 멸균(121℃, 15분, 6시간 후 재멸균)과 저온멸균(90℃, 4시간), 간헐적 저온멸균(90℃, 4시간, 6시간 후 다시 4시간멸균)등의 방법을 사용하여 고체 배양 담체인 밀기울의 멸균 정도를 확인하였다. 그 결과, 비멸균 시 2.3×105 CFU/g의 균수가 확인되었으며 저온멸균 시 약 2.3×105의 균이 남아있는 것으로 조사되었다. 간헐적 멸균(121℃, 15분 멸균 후 6시간 후 다시 재멸균)은 약 6.7×102 CFU/g으로 대부분의 조사에서 균이 관찰되지 않았으며 고체발효기를 이용하여 저온멸균(90℃, 4시간) 후 6시간이 지나고 다시 저온멸균을 실시하는 방법은 약 2.1×103 CFU/g의 균이 확인되었다. 도 5에 배지 멸균 방법에 따른 균수를 나타내었다.During solid culture, the degree of sterilization of the medium and carrier is an important culture factor. In order to establish an efficient sterilization method, the existing sterilization (121 ℃, 15 minutes) as a control, intermittent sterilization (121 ℃, 15 minutes, 6 hours re-sterilization), low temperature sterilization (90 ℃, 4 hours), intermittent pasteurization (90 ° C., 4 hours, 6 hours, then sterilization for 4 hours again) was used to confirm the degree of sterilization of wheat bran, which is a solid culture carrier. As a result, it was confirmed that the number of bacteria was 2.3×10 5 CFU/g when non-sterilized, and about 2.3×10 5 bacteria remained when pasteurized. Intermittent sterilization (121 ℃, 15 minutes sterilization, then resterilization after 6 hours) is about 6.7 × 10 2 CFU / g, and no bacteria were observed in most investigations, and low-temperature sterilization (90 ℃, 4 hours) using a solid fermenter ) After 6 hours of pasteurization, about 2.1×10 3 CFU/g of bacteria was confirmed. Figure 5 shows the number of bacteria according to the medium sterilization method.
5. 고체 배양 조건 및 포자변화5. Solid culture conditions and sporulation
따라서 전술한 바와 같이, 포자생산과 최적의 담체 및 배양 조건을 확립하기 위해 초기 미생물 배양액의 배지 조성을 확립하였다. 이를 바탕으로 최적의 접종액 양과 수분 함유량 등을 조사하였고, 최적의 담체를 선택하기 위하여 다양한 담체에 균주를 배양하여 포자생성 정도를 확인하였다.Therefore, as described above, the medium composition of the initial microbial culture was established in order to establish optimal carrier and culture conditions for spore production. Based on this, the optimal inoculum amount and moisture content were investigated, and the degree of sporulation was confirmed by culturing the strain on various carriers in order to select the optimal carrier.
고체배양 시 최적 균주 접종량을 설정하기 위해서는 접종 배양액과 전체 담체에 대한 수분량의 총량을 고려하여 접종해야 한다. 초기 접종량 시험은 액체배양액을 담체 중량 10% 접종 시 11.1×109 CFU/g으로 가장 높은 균수를 보였으며 담체 대비 수분 접종량은 100%에서 7.8×109 CFU/g으로 가장 많은 균수가 확인되었다. 도 6 및 도 7에 초기균주 접종량에 따른 균수 및 수분 접종량에 따른 균수를 각각 나타내었다.In order to set the optimal strain inoculation amount during solid culture, inoculation should be performed in consideration of the total amount of moisture for the inoculation culture medium and the entire carrier. In the initial inoculation test, the highest number of bacteria was 11.1 × 10 9 CFU / g when the liquid culture medium was inoculated with 10% of the carrier weight, and the highest number of bacteria was confirmed at 7.8 × 10 9 CFU / g at 100% water inoculation compared to the carrier. 6 and 7 show the number of bacteria according to the amount of inoculation of the initial strain and the number of bacteria according to the amount of water inoculation, respectively.
도 8은 본 발명에 따른 미생물 농약의 균주 배양을 위한 고체배양에 따른 포자 및 수분변화 조사 결과를 나타낸 그래프로서, 고체배양 시 배양온도 34℃, 초기접종량 10%, 수분접종량 100%일 때 접종일부터 건조까지의 균수와 포자율 그리고 수분함량을 측정하였다. 균수는 접종일에 6.3×107 CFU/g에서 접종 3일째에 4.8×109 CFU/g으로 확인되었다. 수분함량은 접종일에 57.8%에서 배양 3일째에 7.3%였다. 포자 형성율은 배양 1일에 42.6%였으며 배양 3일에는 90%의 포자 형성율을 보였다.Figure 8 is a graph showing the results of investigation of spores and moisture changes according to solid culture for culturing strains of microbial pesticides according to the present invention, inoculation date when the culture temperature is 34 ° C, the initial inoculation amount is 10%, and the water inoculation amount is 100% during solid culture From drying to drying, the number of bacteria, sporulation rate, and moisture content were measured. The number of bacteria was confirmed from 6.3×10 7 CFU/g on the inoculation day to 4.8×10 9 CFU/g on the 3rd day of inoculation. Moisture content was 57.8% on the day of inoculation and 7.3% on the third day of culture. The sporulation rate was 42.6% on the 1st day of culture and 90% on the 3rd day of culture.
배지 수분함량은 최적 배양조건 확립을 위한 것으로, 배지 3g을 채취하여 수분측정기(OHAUS, USA)에서 120℃에서 반응시켜 고체 배지상의 수분함유량을 측정하였다. The medium moisture content is for establishing optimal culture conditions, and 3 g of the medium was collected and reacted at 120 ° C. in a moisture meter (OHAUS, USA) to measure the water content on the solid medium.
포자 형성율은 선발된 균주의 안정적인 생산과 유통을 위한 미생물의 최적 형태인 포자의 형성정도를 배양 후 생균과 포자의 비율로 나타내는 것으로, 위상차 현미경(Leica, DEU)을 활용한 관찰법과 80℃에서 15분 처리 후 생균수 변화를 측정하는 방법을 병용하여 조사하였다. The sporulation rate indicates the degree of formation of spores, the optimal form of microorganisms for the stable production and distribution of selected strains, as the ratio of viable cells and spores after cultivation. After 15 minutes of treatment, a method of measuring the change in the number of viable cells was investigated in combination.
전술한 바와 같이, 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법은 국산 소재를 이용한 저가 및 고효율의 작물 곰팡이병 방제용 미생물 농약을 제조하기 위한 포자 생산기술을 제공한다. As described above, the method for producing spores for microbial pesticides for fungal disease control of crops provides a spore production technology for producing low-cost and high-efficiency microbial pesticides for fungal disease control in crops using domestic materials.
한편, 본 발명의 상세한 설명에서는 첨부된 도면에 의해 참조되는 바람직한 실시 예를 중심으로 구체적으로 기술되었으나, 본 발명의 범위에서 벗어나지 않는 한도 내에서 여러 가지 변형이 가능함은 물론이다. 그러므로 본 발명의 범위는 설명된 실시 예에 국한되어 정해져서는 안되며 후술하는 특허청구의 범위뿐 아니라 이 특허청구의 범위와 균등한 것들에 의해서 정해져야 한다.Meanwhile, in the detailed description of the present invention, although it has been specifically described with reference to the preferred embodiments referenced by the accompanying drawings, various modifications are possible without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the described embodiments and should not be defined, but should be defined by not only the scope of the claims to be described later, but also those equivalent to the scope of these claims.
Claims (4)
밀기울을 담체로 하는 고체배지를 멸균하는 단계; 및
상기 액체배양된 균주를 상기 고체배지에서 고체배양하는 단계;로 이루어지는 것을 특징으로 하는 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법.
liquid culture of the strain Bacillus velezensis (hereinafter referred to as strain) in MS medium;
Sterilizing a solid medium containing wheat bran as a carrier; and
A method for producing spores for microbial pesticides for fungal disease control of crops, characterized in that comprising: solid-cultivating the liquid-cultured strain in the solid medium.
글루코스(Glucose) 20g/L, 효모추출물(Yeast extract) 15g/L, K2HPO4 2.5g/L, MgSO4 1g/L, MnSO4 0.1g/L 및 NaCl 1g/L를 포함하는 것을 특징으로 하는 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법.
The method of claim 1, wherein the MS medium,
Glucose (Glucose) 20g / L, yeast extract (Yeast extract) 15g / L, K 2 HPO 4 2.5g / L, MgSO 4 1g / L, MnSO 4 0.1g / L and NaCl 1g / L characterized by including A method for producing spores for microbial pesticides for the control of fungal diseases in crops.
고체발효기를 이용하여 1차 저온멸균(90℃, 4시간) 및 6시간 경과 후 2차 저온멸균을 실시하는 것을 특징으로 하는 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법.
The method of claim 1, wherein the sterilization of the solid medium,
A method for producing spores for microbial pesticides for the control of fungal diseases in crops, characterized by performing primary pasteurization (90 ° C, 4 hours) and secondary pasteurization after 6 hours using a solid fermenter.
상기 균주의 접종량은 상기 담체 대비 액체배양액 10중량%이고,
상기 담체에 대한 수분 접종량은 담체 대비 수분 100중량%인 것을 특징으로 하는 작물의 곰팡이병 방제를 위한 미생물 농약용 포자 생산방법.According to claim 1,
The inoculation amount of the strain is 10% by weight of the liquid culture medium compared to the carrier,
A method for producing spores for microbial pesticides for fungal disease control of crops, characterized in that the amount of water inoculation for the carrier is 100% by weight of water compared to the carrier.
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| KR101971017B1 (en) | 2017-12-19 | 2019-04-23 | 전남대학교산학협력단 | Bacillus methylotrophicus 8-2 strain producing natural volatile compound and having antimicrobial activity |
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