KR102662543B1 - Microbial agent for preventing fungal diseases of plant and manufacturing method thereof - Google Patents
Microbial agent for preventing fungal diseases of plant and manufacturing method thereof Download PDFInfo
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- KR102662543B1 KR102662543B1 KR1020210190550A KR20210190550A KR102662543B1 KR 102662543 B1 KR102662543 B1 KR 102662543B1 KR 1020210190550 A KR1020210190550 A KR 1020210190550A KR 20210190550 A KR20210190550 A KR 20210190550A KR 102662543 B1 KR102662543 B1 KR 102662543B1
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- microbial
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- fungal diseases
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- manufacturing
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Classifications
-
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
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- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Environmental Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 발명은 국산 소재를 이용한 저가 및 고효율의 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법에 관한 것으로, 담체 및 배지의 멸균공정; 멸균 후 포자생산과 균주 배양공정; 포자 분쇄공정; 분쇄 후 계면활성제와의 교반공정; 및 포장공정;으로 이루어지는 작물 곰팡이병 방제용 미생물 농약 제조방법 및 이 제조방법에 의하여 제조된 작물 곰팡이병 방제용 미생물 농약을 제공한다. The present invention relates to a low-cost and highly efficient microbial pesticide for controlling fungal diseases in crops using domestic materials and a method for producing the same, including a sterilization process for the carrier and medium; Spore production and strain culture process after sterilization; Spore crushing process; Stirring process with surfactant after grinding; and a packaging process; and a microbial pesticide for controlling crop fungal diseases produced by the manufacturing method.
Description
본 발명은 살균제 대체 미생물농약 및 생화학 농약에 관한 것으로서, 특히 국산 소재를 이용한 저가 및 고효율의 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법에 관한 것이다.The present invention relates to alternative microbial pesticides and biochemical pesticides for fungicides, and in particular to a low-cost and highly efficient microbial pesticide for controlling fungal diseases in crops using domestic materials and a method for producing the same.
본 발명은 농림축산식품부의 재원으로 농림식품기술기획평가원의 핵심농자재국산화기술개발사업(321055051HD030)의 지원으로 수행된 연구결과이다.This invention is the result of research conducted with the support of the Core Agricultural Materials Localization Technology Development Project (321055051HD030) of the Korea Institute of Agriculture, Food and Rural Affairs, 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, in the global crop protection market, biological pesticides account for 9.6% of the total market compared to chemical pesticides, but it is expected to grow significantly to 17.7% in 2023 at an average annual growth rate of 6.6% due to the policy to reduce chemical pesticides and promote organic farming.
특히 아시아 태평양 국가들은 인구증가로 인해 작물보호제 수요가 급증하고 있어 2023년에는 생물농약의 비중이 23.4%가 될 것으로 예측된다.In particular, demand for crop protection agents in Asia-Pacific countries is rapidly increasing due to population growth, and the proportion of biological pesticides is expected to reach 23.4% in 2023.
따라서 작물보호제 시장의 다국적 기업과 중소기업에게는 생물농약이 새로운 블루오션으로 떠오르고 있는 실정이다. Therefore, biological pesticides are emerging as a new blue ocean for multinational companies and small and medium-sized companies 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, plans are being promoted at the government level to expand the practice of eco-friendly agriculture, reduce the use of chemical fertilizers, and improve soil strength according to the eco-friendly agriculture development plan, canceling registration of highly toxic pesticides, reducing the use of chemical pesticides, and using eco-friendly biological pesticides such as microorganisms and natural extracts. The development and use of is expanding, and the demand for domestic eco-friendly agricultural products and the market size of organic products are increasing in accordance with consumer demand for eco-friendly and safe agricultural products and the increase in social value. With the creation of new organic complexes, the current pesticide usage is increasing. A plan is being promoted to reduce pesticide-free crops by 3% every year to raise the cultivation area of pesticide-free or higher crops to 15% of the total cultivation area.
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 effect due to diluted spraying of small-unit microbial products is minimal, the concentration of active ingredients contained in microbial products is low, the number of active bacterial cells is unstable over time, and the substrate or host specificity of the bacterial cells is weak, making them difficult to farm. Because of the difficulty in establishing microbial pesticides, there is an urgent need for innovative low-cost, high-efficiency formulation technology to increase microbial pesticides and registration of Korea's first biochemical disinfectant pesticide.
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 liquid-cultured using expensive media and incubators, dried using methods such as freeze-drying, spray-drying, and ventilation-drying, then mixed with excipients and thickeners, crushed, and packaged. Because 2 billion worth of incubators, dryers, and grinders are used, 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, in order to develop internationally competitive microbial pesticides, there is a need to develop innovative microbial pesticides that are inexpensive and have pest control capabilities comparable to those of chemical pesticides. Domestic registered disinfectant pesticides are a group that destroys the cell membrane of plant pathogens, so they have a variety of sterilizing effects. The development of various biological pesticides with different 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 the problem of being ineffective because the number of microorganisms and the content of antibacterial substances are insufficient, and since there are no registered plant-derived biochemical fungicide pesticide products, the high price and unstable effects of biological pesticides are driving the growth of the biological pesticide market. It is becoming a limitation.
그러므로 가격이 저렴하고 방제능력이 화학농약과 동일한 혁신적인 생물농약 개발이 필요한 상황이다.Therefore, there is a need 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 난방제병해인 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.Accordingly, the present inventor comprehensively considered the above-mentioned matters and focused on solving the limitations and problems of existing microbial pesticides, and developed domestic materials for controlling crop heating diseases such as pepper anthracnose, potato borer disease, and tomato gray mold disease. The present invention was created as a result of continuous research with great efforts to develop a low-cost, highly efficient microbial pesticide using microbial pesticides.
따라서 본 발명이 해결하고자 하는 기술적 과제 및 목적은 국산 소재를 이용한 저가 및 고효율의 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법을 제공하는 데 있는 것이다.Therefore, the technical problem and purpose to be solved by the present invention is to provide a low-cost and highly efficient microbial pesticide for controlling crop fungal diseases using domestic materials and a method for producing the same.
따라서 본 발명이 해결하고자 하는 상세한 기술적 과제 및 목적은 Bacillus velezensis CE100 균주와 고체배양 기술을 이용하여 밀기울, 규조토, 제올라이트와 같은 담체를 사용한 포자 생산기술을 활용한 저가 및 고효율의 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법을 제공하는 데 있는 것이다.Therefore, the detailed technical problem and purpose to be solved by the present invention is to develop low-cost and highly efficient microorganisms for controlling fungal diseases in crops using spore production technology using carriers such as wheat bran, diatomaceous earth, and zeolite using the Bacillus velezensis CE100 strain and solid culture technology. The purpose is to provide pesticides and their manufacturing methods.
또한, 본 발명이 해결하고자 하는 다른 상세한 기술적 과제 및 목적은 대사물질을 생산해 포자와 함께 배양하고 계면활성제를 활용한 제형 연구 및 제조공정 별 조건변화에 따른 생산효율을 조사하여 고효율의 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법을 제공하는 데 있는 것이다.In addition, other detailed technical problems and objectives to be solved by the present invention are to produce metabolites and cultivate them with spores, study formulations using surfactants, and investigate production efficiency according to changes in conditions for each manufacturing process to control crop fungal diseases with high efficiency. The purpose is to provide microbial pesticides and methods for producing them.
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 objectives to be solved by the present invention are not limited to the technical problems and objectives mentioned above, and other technical problems and objectives not mentioned will be clearly understood by those skilled in the art from the description below.
상술한 목적을 달성하기 위한 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약 제조방법은, 담체 및 배지의 멸균공정; 멸균 후 포자생산과 균주 배양공정; 포자 분쇄공정; 분쇄 후 계면활성제와의 교반공정; 및 포장공정;으로 이루어지는 것을 특징으로 한다.The method for producing microbial pesticides for controlling crop fungal diseases according to the present invention to achieve the above-described object includes a sterilization process of the carrier and medium; Spore production and strain culture process after sterilization; Spore crushing process; Stirring process with surfactant after grinding; And a packaging process.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약 제조방법에 있어서, 상기 멸균공정은 90-95℃에서 10-15시간 가열한 후 6-8시간동안 40-55℃에서 보관하며 다시 90-95℃에서 6-8시간 반응하여 멸균하는 간헐적 멸균공정인 것을 특징으로 한다.In addition, in the method for producing microbial pesticides for controlling crop fungal diseases according to the present invention, the sterilization process is performed by heating at 90-95°C for 10-15 hours, then storing at 40-55°C for 6-8 hours, and then sterilizing at 90-95°C. It is characterized as an intermittent sterilization process that sterilizes by reacting at ℃ for 6-8 hours.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약 제조방법에 있어서, 상기 배양공정은 균주를 배지에 고체배양하는 1차 배양 및 고체배양으로 생산된 균주(내생포자)를 미생물농약으로 제조하는 대사물질을 투입하여 진행하는 2차 배양이 순차적으로 이루어지는 것을 특징으로 한다.In addition, in the method for producing microbial pesticides for controlling crop fungal diseases according to the present invention, the culturing process includes primary culture of the strain on solid culture medium and metabolic process of producing the strain (endospores) produced by solid culture into microbial pesticide. It is characterized by sequentially performing secondary culture by adding substances.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약 제조방법에 있어서, 상기 대사물질은 골분제, 효모 추출물, 랑베나이트, 아미노산, 게껍질 파우더 및 미생물 균주를 약 6개월 동안 고농축 장기 액체배양하여 만들어진 장기액제배양액과 유기영양분을 건조발효기에서 50-60℃로 건조발효한 후 분쇄하여 파우더 형태로 제형화한 것을 특징으로 한다.In addition, in the method for producing microbial pesticides for controlling crop fungal diseases according to the present invention, the metabolites are produced by high-concentration long-term liquid culture of bone meal, yeast extract, langbenite, amino acids, crab shell powder, and microbial strains for about 6 months. It is characterized by dry-fermenting the organ liquid culture medium and organic nutrients at 50-60℃ in a dry fermenter, then pulverizing them, and formulating them into powder form.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약은 제 1항 내지 제 4항 중 어느 한 항의 제조방법에 의하여 제조된 것을 특징으로 한다.In addition, the microbial pesticide for controlling crop fungal diseases according to the present invention is characterized in that it is manufactured by the production method of any one of claims 1 to 4.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약은 상기 포자 31중량%와, 상기 대사물질 20중량%와, 상기 담체 34중량%와, 상기 계면활성제 15중량%로 구성된 것을 특징으로 한다. In addition, the microbial pesticide for controlling crop fungal diseases according to the present invention is characterized by being composed of 31% by weight of the spores, 20% by weight of the metabolites, 34% by weight of the carrier, and 15% by weight of the surfactant.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약은 상기 담체 34중량%는, 이산화규소 함량 80%인 규조토 건조분말 10중량%와, 유기물을 제거한 이산화규소 함량 80%인 규조토 건조분말 24중량%로 구성된 것을 특징으로 한다.In addition, the microbial pesticide for controlling fungal diseases in crops according to the present invention includes 34% by weight of the carrier, 10% by weight of diatomaceous earth dry powder with a silicon dioxide content of 80%, and 24% by weight of diatomaceous earth dry powder with a silicon dioxide content of 80% from which organic matter has been removed. It is characterized by being composed of.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약에 있어서, 상기 계면활성제 15중량%는 Sodium bis(2-ethylhexyl)와 Sulfosuccinate 및 Polyethylene glycol을 주성분으로 하는 제1 계면활성제 5중량%와, Ethoxylated octyl Phenol 및 Isopropyl alcohol을 주성분으로 하는 제2 계면활성제 10중량%로 구성된 것을 특징으로 한다.In addition, in the microbial pesticide for controlling crop fungal diseases according to the present invention, 15% by weight of the surfactant is 5% by weight of a first surfactant mainly composed of Sodium bis (2-ethylhexyl), Sulfosuccinate, and Polyethylene glycol, and Ethoxylated octyl. It is characterized by being composed of 10% by weight of a second surfactant containing phenol and isopropyl alcohol as main ingredients.
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 has the effect of contributing to the revitalization of domestic eco-friendly agriculture by being used as a manual for controlling major crop diseases and pests in eco-friendly large areas in Korea.
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 mixing methods with existing organic materials, which has the effect of contributing to increasing farm 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 has been commercialized through packaging verification by overseas certified organizations (USA, Israel, etc.), and through registration of low-cost, high-efficiency microbial pesticides, import substitution of imported microbial disinfectants, which accounts for more than half of the domestic microbial disinfectant market, and export of domestically produced eco-friendly agricultural materials. It has the effect of contributing to revitalizing 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 significantly 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 products and manuals for replacing chemical pesticides. It has the effect of contributing to increasing farm income and improving 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,
도 9는 본 발명에 따른 미생물 농약에 사용되는 대사물질의 생산공정을 도식화한 도면,
도 10은 본 발명에 따른 미생물 농약에 사용되는 대사물질의 원료인 국내산 홍게와 베트남산 갈색에의 키틴함량 분석 그래프,
도 11 및 12는 국내산 홍게를 원료로 하여 만든 대사물질 파우더의 여과 및 비여과 희석액의 각 효소활성 시험 사진.
도 13은 본 발명에 따른 미생물 농약의 균주 배양을 위한 제조 공정에 의한 균수 변화 조사 결과를 나타낸 그래프,
도 14는 본 발명에 따른 미생물 농약의 균주 제조공정을 도식화한 도면,
도 15는 본 발명에 따른 미생물 농약 제품의 포자 안정성 시험 결과 그래프,
도 16은 본 발명에 따른 미생물 농약 제품의 항균활성 시험 사진,
도 17은 본 발명에 따른 미생물 농약 제품의 소포제 시험 결과 그래프,
도 18은 본 발명에 따른 미생물 농약 제품의 밀기울 분말도에 따른 균수 변화 조사 그래프,
도 19는 본 발명에 따른 미생물 농약 제품의 담체 분쇄 공정에 의한 포자안정성 조사 그래프.Figure 1 is a graph showing the results of a viable cell count survey according to liquid medium components for cultivating strains of microbial pesticides according to the present invention;
Figure 2 is a graph showing the results of a survey on the number of viable cells according to the sugar content of the solid medium for culturing the microbial pesticide strain according to the present invention;
Figure 3 is a graph showing the results of a survey on the number of viable cells according to the protein content of the solid medium for culturing the microbial pesticide strain according to the present invention;
Figure 4 is a graph showing the results of a viable cell count survey according to the solid culture carrier for cultivating the microbial pesticide strain according to the present invention;
Figure 5 is a graph showing the results of a bacterial count investigation according to the medium sterilization method for cultivating strains of microbial pesticides according to the present invention;
Figure 6 is a graph showing the results of a bacterial count survey according to the initial strain inoculum for culturing the microbial pesticide strain according to the present invention;
Figure 7 is a graph showing the results of a bacterial count survey according to the moisture inoculum amount for culturing the microbial pesticide strain according to the present invention;
Figure 8 is a graph showing the results of investigation of spores and moisture changes according to solid culture for cultivating strains of microbial pesticides according to the present invention;
Figure 9 is a schematic diagram of the production process of metabolites used in microbial pesticides according to the present invention;
Figure 10 is a graph of chitin content analysis of domestic red crab and Vietnamese brown crab, which are raw materials for metabolites used in microbial pesticides according to the present invention;
Figures 11 and 12 are photos of each enzyme activity test of filtered and non-filtered dilutions of metabolite powder made from domestic red crab as a raw material.
Figure 13 is a graph showing the results of an investigation of changes in the number of bacteria due to the manufacturing process for cultivating strains of the microbial pesticide according to the present invention;
Figure 14 is a schematic diagram of the manufacturing process of a microbial pesticide strain according to the present invention;
Figure 15 is a graph showing the spore stability test results of the microbial pesticide product according to the present invention;
16 is a photograph of an antibacterial activity test of a microbial pesticide product according to the present invention;
Figure 17 is a graph of the antifoam test results of the microbial pesticide product according to the present invention;
Figure 18 is a graph investigating changes in the number of bacteria according to the bran fineness of the microbial pesticide product according to the present invention;
Figure 19 is a graph showing the spore stability of the microbial pesticide product according to the present invention by the carrier grinding process.
이하에서는 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법에 대한 실시 예를 도면을 참조하여 상세히 설명한다. 본 발명에 따른 작물 곰팡이병은 탄저병, 더뎅이병, 잿빛곰팡이병을 포함한다. Hereinafter, examples of the microbial pesticide for controlling crop fungal diseases and the method for producing the same 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, armyworm, 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 is not limited to the embodiments disclosed below and may be implemented in various forms.
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. Identical elements in the drawings are denoted by identical symbols wherever possible. Specific details appear in the following description, which are provided only to facilitate 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. , should be understood to include equivalents or substitutes. Also, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.
제1, 제2 등의 용어는 다양한 구성요소들을 설명하는데 사용될 수 있지만, 상기 구성요소들은 상기 용어들에 의해 한정되지 않는다. 상기 용어들은 하나의 구성요소를 다른 구성요소로부터 구별하는 목적으로만 사용된다. 예를 들어, 본 발명의 권리범위를 벗어나지 않으면서 제1 구성요소는 제2 구성요소로 명명될 수 있고, 유사하게 제2 구성요소도 제1 구성요소로 명명될 수 있다. Terms such as first, second, etc. may be used to describe various components, but the components are not limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, a first component may be named a second component without departing from the scope of the present invention, and similarly, the second component may also be named a first component.
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, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.
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 otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms such as those defined in commonly used dictionaries are defined in consideration of their functions in the present invention, and should be interpreted as concepts consistent with the technical idea of the present invention and meanings commonly used or commonly recognized in the technical field. And, unless clearly defined in this application, it is not to be interpreted in an ideal or excessively formal sense.
본 발명에 따른 작물 곰팡이병 방제용 미생물 농약에 사용되는 Bacillus velezensis는 많은 식물들에 대한 직간접적인 성장개선효과와 식물병원균에 대한 항균활성효과로 인해 많이 연구되고 사용되는 바실러스 종이다. Bacillus velezensis , used in the microbial pesticide for controlling crop fungal diseases according to the present invention, is a Bacillus species that is widely studied and used due to its direct and indirect growth improvement effects on many plants and 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 these, Bacillus velezensis CE100 is a strain isolated from Chonnam National University and has been reported in various papers in 2020 and 2021 to have a nematode inhibitory effect, promote the growth of tomatoes and cypress trees, and kill strawberry anthracnose ( Colletotrichum gloeosporioides ) and methyl bacterium by its secondary metabolite, Cyclic Tetrapeptide. The antibacterial activity of hippurate against gray mold ( Botrytis cinerea ) 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 fields such as food, medicine, and agriculture. Spore formation, one of the major characteristics of Bacillus species, can respond to nutrient fluctuations and various stresses and is a strategy used advantageously to increase population by protecting genes. This spore formation is industrially useful in the production, storage, and use of Bacillus species.
우선, 본 발명에 따른 작물 곰팡이병 방제용 미생물의 포자 및 대사산물 생산을 위한 기계적 및 환경적 설정 조건을 하기와 같이 도출하였다. First, the mechanical and environmental settings for the production of spores and metabolites of microorganisms for controlling crop fungal diseases according to the present invention were derived as follows.
1. 포자 생산 기술1. Spore production technology
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 endospore production of Bacillus velezensis strain was established, and based on the composition, carriers for solid culture were selected, and culture composition, sterilization, and culture methods were established.
1-1. 액체배지 조성1-1. 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% of the medium according to the type of medium shown in Table 1 below and cultured at 30°C at 120 rpm for 3 days, and the culture medium was incubated at 0.1%. ml was treated and diluted to 10 1 to 10 9 in TSA (Tryptic soy agar) medium, and then the number of microorganisms was measured using the 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 cultivating strains for inoculation on solid media. Based on this, CN1 (Glucose modified) was prepared by modifying the amount of trace components, including sugar and protein components, using TSB medium as a control. , MS (Glucose, Yeast extract modified) medium compositions 1 to 2 were cultured at 30°C at 120 rpm and the number of bacteria was measured. As a result, the TSB treatment group showed a bacterial count of 2.7 This is shown in Figure 1. Figure 1 shows the number of viable bacteria according to liquid medium components.
1-2. 고체배지 조성1-2. 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.Figures 2 and 3 show the number of viable bacteria according to the sugar and protein content of the solid medium, respectively. For solid culture using vermiculite, which has a trace amount of microbial nutrients, as a carrier, sugar (1-4%) and protein (1-2%) were used. ) The change in the number of viable bacteria according to the amount of ingredients was confirmed by culturing at 34°C for 3 days. As shown in Figure 2 , the sugar content was highest in the glucose (1%) treatment group at 5.1 The bacterial count was shown. As shown in Figure 3, the protein content was confirmed to be 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 wet carrier without flowing water. Unlike tank culture, microorganisms grow in sufficient contact with oxygen in the air because the moisture present in the solid matrix is used and sufficient oxygen delivery is easy. , enzymes and production can be increased. These solid cultures can be produced with low energy input using relatively inexpensive equipment and have the advantage of increasing the value of cheap materials such as agricultural by-products.
1-3. 고체배양 담체1-3. 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 medium conditions established to find a suitable carrier for solid culture, the strain was solid-cultured at 34°C for 3 days using zeolite, vermiculite, diatomaceous earth, and wheat bran as carriers. As a result, 9.3 It showed the highest number of bacteria in g. Figure 4 shows the number of viable bacteria according to solid culture carriers such as zeolite, vermiculite, diatomaceous earth, and wheat bran. It is believed that the pores secured due to the certain amount of carbon nutrient source and large particle amount that the bran medium basically has served as better conditions for strains to grow, resulting in the highest number of bacteria.
1-4. 배지 멸균1-4. 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 media and carriers is an important culture factor. In order to establish an efficient sterilization method, existing sterilization (121℃, 15 minutes) was used as a control, followed by intermittent sterilization (121℃, 15 minutes, re-sterilization after 6 hours), low-temperature sterilization (90℃, 4 hours), and intermittent low-temperature sterilization. The degree of sterilization of wheat bran, a solid culture carrier, was confirmed using methods such as (sterilization at 90°C for 4 hours, 6 hours, and then again for 4 hours). As a result, the number of bacteria was confirmed to be 2.3×10 5 CFU/g when non-sterilized, and when sterilized at low temperature, about 2.3×10 5 bacteria were found to remain. Intermittent sterilization (121℃, sterilization for 15 minutes and then re-sterilization after 6 hours) was about 6.7 ), when low-temperature sterilization was performed again 6 hours later, approximately 2.1 × 10 3 CFU/g of bacteria were confirmed. Figure 5 shows the number of bacteria according to the medium sterilization method.
1-5. 고체 배양 조건 및 포자변화1-5. Solid culture conditions and spore changes
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 medium was established to establish spore production and optimal carrier and culture conditions. Based on this, the optimal amount of inoculant and moisture content were investigated, and the degree of spore production was confirmed by culturing the strain on various carriers 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 must be done considering the total amount of moisture for the inoculation culture medium and the entire carrier. The initial inoculation dose test showed the highest number of bacteria at 11.1×10 9 CFU/g when the liquid culture 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% of the water inoculation amount compared to the carrier. Figures 6 and 7 show the number of bacteria according to the initial bacterial inoculum amount and the number of bacteria according to the moisture inoculum amount, 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 cultivating the strain of microbial pesticide according to the present invention. The date of inoculation when the culture temperature was 34°C, initial inoculation amount was 10%, and moisture inoculation amount was 100% during solid culture. The number of bacteria, spore rate, and moisture content were measured from start to drying. The bacterial count was confirmed to be 6.3×10 7 CFU/g on the day of inoculation and 4.8×10 9 CFU/g on the 3rd day of inoculation. The moisture content ranged from 57.8% on the day of inoculation to 7.3% on the third day of incubation. The spore formation rate was 42.6% on day 1 of culture and 90% on day 3 of culture.
배지 수분함량은 최적 배양조건 확립을 위한 것으로, 배지 3g을 채취하여 수분측정기(OHAUS, USA)에서 120℃에서 반응시켜 고체 배지상의 수분함유량을 측정하였다. The moisture content of the medium was determined to establish optimal culture conditions. 3 g of the medium was collected and reacted at 120°C in a moisture meter (OHAUS, USA) to measure the moisture content of 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 spore formation rate indicates the degree of spore formation, which is the optimal form of microorganisms for stable production and distribution of the selected strain, as the ratio of live cells and spores after culture. Observation method using a phase contrast microscope (Leica, DEU) and temperature at 80°C. After 15 minutes of treatment, a method of measuring changes in the number of viable bacteria was used in combination to investigate.
2. 대사물질 생산 기술2. Metabolite production technology
본 발명에서는 고체배양으로 생산된 포자와 2차 배양을 위한 대사물질 생산 방법을 개발 및 개량하였다. 대사물질은 다양한 배지성분들을 장기간 고농축액체배양을 통해 생산되며 이를 파우더 제형화한 후 내생포자와 2차배양을 하여 미생물농약을 제조하는데 사용된다. 본 발명에서는 대사산물의 주요성분인 게껍질의 경제적 생산을 위한 대체물질의 품질검사와 대사산물의 다양한 효소활성을 조사하였다.In the present invention, we developed and improved a method for producing spores produced in solid culture and metabolites for secondary culture. Metabolites are produced through long-term, highly concentrated liquid culture of various media components, and are used to manufacture microbial pesticides by formulating them into powder and then performing secondary culture with endospores. In the present invention, the quality of substitute materials for economical production of crab shell, which is the main component of metabolites, was tested and various enzyme activities of metabolites were investigated.
2-1. 대사물질 생산공정2-1. Metabolite production process
여기서, 본 발명에 따른 미생물농약을 구성하는 대사산물은 대사물질 원료를 장기간 고농축액체배양하여 제조한 것으로, 하기에서 도 9 내지 12 및 표 2를 참조하여 상세히 설명한다.Here, the metabolites constituting the microbial pesticide according to the present invention are manufactured by long-term highly concentrated liquid culture of metabolite raw materials, and are described in detail below with reference to FIGS. 9 to 12 and Table 2.
도 9에 본 발명에 따른 미생물 농약에 사용되는 대사물질의 생산공정을 도식화하여 나타내었다.Figure 9 schematically shows the production process of metabolites used in microbial pesticides according to the present invention.
도 9에 도시된 바와 같이, 대사물질 생산공정은 원료인 골분제, 효모 추출물, 랑베나이트, 아미노산, 게껍질 파우더 및 미생물 균주를 약 6개월 동안 고농축 장기 액체배양(S111)하여 만들어진 장기액제배양액과 유기영양분을 사료건조발효기에서 50-60℃로 건조발효(S113)한 후 분쇄하여(S115) 포장하는 공정(S117)으로 이루어진다.As shown in Figure 9, the metabolite production process involves high-concentration long-term liquid culture (S111) of the raw materials bone meal, yeast extract, langbenite, amino acids, crab shell powder, and microbial strains, and It consists of the process of drying and fermenting organic nutrients at 50-60°C in a feed dry fermenter (S113), grinding them (S115), and packaging them (S117).
대사물질 원료인 게껍질은 국내산 홍게를 100~120mesh로 분쇄한 제품과 베트남산 갈색게를 60~80mesh로 분쇄한 제품이 있으며, 두 제품의 키틴함량 분석 시험 결과를 하기의 표 2에 나타냈다.Crab shell, which is a raw material for metabolites, includes products made by pulverizing domestic red crabs to 100-120 mesh and products made by pulverizing Vietnamese brown crabs to 60-80 mesh. The chitin content analysis test results of the two products are shown in Table 2 below.
게껍질 내 키틴분석은 게껍질 파우더를 50℃ 오븐에서 24시간 건조시킨 후 게껍질 파우더 5g을 1M HCl 500ml(55~65℃)에 넣고 2시간동안 교반시켜 미네랄과 카테콜을 제거시킨다. 필터페이퍼로 내용물을 걸러 증류수로 잘 헹궈낸다. 걸러진 잔여물의 단백질 제거를 위해 1M NaOH 500ml(75~80℃)에 넣고 23시간동안 교반시킨다. 다시 필터페이퍼로 잔여물(키틴)을 걸러 증류수로 잘 씻어준 뒤, 60℃ 오븐에서 24시간동안 잘 건조시킨 후 무게를 잰다.For chitin analysis in crab shell, crab shell powder is dried in an oven at 50℃ for 24 hours, then 5g of crab shell powder is added to 500ml of 1M HCl (55~65℃) and stirred for 2 hours to remove minerals and catechol. Filter the contents with filter paper and rinse well with distilled water. To remove protein from the filtered residue, place it in 500ml of 1M NaOH (75-80℃) and stir for 23 hours. Filter the residue (chitin) again with filter paper, wash it well with distilled water, dry it well in an oven at 60°C for 24 hours, and then weigh it.
국내산 홍게 게껍질과 베트남산 갈색게 게껍질의 키틴함량 분석을 3차에 걸쳐 진행한 결과, 국내산 홍게는 23%, 베트남산 갈색게는 12%의 키틴함량을 나타내어, 약 2배의 키틴함량 차이를 보인 것을 확인하였다. 국내산 홍게와 베트남산 갈색에의 키틴함량 분석결과를 도 10에 나타내었다.As a result of analyzing the chitin content of domestic red crab crab shell and Vietnamese brown crab shell three times, domestic red crab showed a chitin content of 23% and Vietnamese brown crab showed a chitin content of 12%, a difference in chitin content of about two times. It was confirmed that it was shown. The results of chitin content analysis of domestic red crab and Vietnamese brown crab are shown in Figure 10.
2-2. 대사물질 효소활성 시험2-2. Metabolite enzyme activity test
효소활성 테스트를 진행하기 위해 키틴, 젤라틴, 셀룰로오스, 지질, 인, 단백질, 아밀라아제, 글루카나아제 8종의 배지를 제작하였다. 제작방법은 다음과 같다. To conduct enzyme activity tests, eight types of media were prepared: chitin, gelatin, cellulose, lipid, phosphorus, protein, amylase, and glucanase. The production method is as follows.
키틴: Na2HPO4 6g, KH2PO4 3g, NH4Cl 1g, NaCl 0.5g, yeast extract 0.05g, colloidal chitin 10ml, agar 20g, D.W 1LChitin: Na 2 HPO 4 6g, KH 2 PO 4 3g, NH 4 Cl 1g, NaCl 0.5g, yeast extract 0.05g, colloidal chitin 10ml, agar 20g, DW 1L
셀룰로오스: Yeast extract 1g, peptone 0.5g, cellulose 0.5g, congo red 0.1g, agar 20g, D.W 1LCellulose: Yeast extract 1g, peptone 0.5g, cellulose 0.5g, congo red 0.1g, agar 20g, D.W 1L
지질: pH 7.3, CaCl2 1g, phenol red 0.1g, olive oil 20ml, agar 20g, D.W 1LLipids: pH 7.3, CaCl 2 1g, phenol red 0.1g, olive oil 20ml, agar 20g, DW 1L
인가용화(PVK): Glucose 10g, Ca3(PO4)2 5g, (NH4)2SO4 0.5g, NaCl 0.2g, MgSO4W7H2O 0.1g, KCl 0.2g, yeast extract 0.5g, MnSO4WH2O 0.002g, FeSO4W7H2O 0.002g, D.W 1L Solubilization (PVK): Glucose 10g, Ca 3 (PO 4 ) 2 5g, (NH 4 ) 2 SO 4 0.5g, NaCl 0.2g, MgSO 4 W7H 2 O 0.1g, KCl 0.2g, yeast extract 0.5g, MnSO 4 WH 2 O 0.002 g, FeSO 4 W 7 H 2 O 0.002 g, DW 1 L
단백질: TSB 0.3g, agar 20g, casein 20g, D.W 1L, Protein: TSB 0.3g, agar 20g, casein 20g, D.W 1L,
아밀라아제: starch 20g, D.W 1L Amylase: starch 20g, D.W 1L
젤라틴: Gelatin 20g, D.W 1L Gelatin: Gelatin 20g, D.W 1L
글루카나아제: Laminarin 1g, D.W 1LGlucanase: Laminarin 1g, D.W 1L
각 배지를 제작 후 대사물질을 1000배 희석하여 1ml씩 배지 중앙에 접종하였다. 1일 뒤 관찰을 용이하게 하기 위해 아이오딘 용액(Iodine 1g, potassium iodide 5g, D.W 330ml)을 제작 후 배지에 접종하여 효소 활성을 확인하였다.After preparing each medium, the metabolites were diluted 1000 times and inoculated into the center of the medium at 1 ml each. To facilitate observation 1 day later, an iodine solution (Iodine 1g, potassium iodide 5g, D.W. 330ml) was prepared and inoculated into the medium to confirm enzyme activity.
국내산 홍게를 원료로 하여 만든 대사물질 파우더의 희석액을 가지고 효소활성 시험을 진행한 결과 도 11 및 도 12와 같이 비여과액(no filtrate) 및 여과액(filtrate) 각각에 대하여, 지질, 키틴, 젤라틴, 단백질, 녹말, 셀룰로오스, 글루칸 분해와 인가용화 효소의 활성을 확인하였다.As a result of conducting an enzyme activity test with a diluted solution of metabolite powder made from domestic red crab, lipid, chitin, and gelatin were observed for the no filtrate and filtrate, respectively, as shown in Figures 11 and 12. , protein, starch, cellulose, and glucan decomposition and solubilization enzyme activity were confirmed.
3. 제조공정 및 생산공정3. Manufacturing process and production process
3-1. 제조공정3-1. Manufacture process
본 발명에 따른 미생물 농약 제조공정에서 포자의 제형화 중 포자의 안정성을 확인하기 위해 각 제조공정별 변화를 확인하고, 본 발명에 따른 미생물의 고체배양에 최적화된 고체배양기의 배양조건을 확립하여 고체배양기를 개량 및 제작하였다.In order to confirm the stability of spores during formulation of spores in the microbial pesticide manufacturing process according to the present invention, changes in each manufacturing process were confirmed, and culture conditions of a solid culture incubator optimized for solid culture of microorganisms according to the present invention were established. The incubator was improved and manufactured.
3-1-1. 포자 안정성3-1-1. Spore Stability
포자 생산 후 공정에서 분쇄에 의한 포자 안정성의 변화를 확인하고, 이를 도 13에 나타내었다. 도 13은 본 발명에 따른 미생물 농약의 균주 배양을 위한 제조 공정에 의한 균수 변화 조사 결과를 나타낸 그래프이다.Changes in spore stability due to grinding in the post-spore production process were confirmed, and this is shown in Figure 13. Figure 13 is a graph showing the results of an investigation of changes in the number of bacteria due to the manufacturing process for cultivating strains of the microbial pesticide according to the present invention.
포자 생산 후 공정에서 분쇄에 의한 포자 안정성의 변화를 확인한 결과, 포자 분쇄 전 7.6×109 CFU/g이었던 균수는 분쇄 후 5.8×109 CFU/g이였으며 포자율은 100%에서 96%로 낮아진 것을 확인할 수 있었다. 분쇄 전 80℃의 열처리를 할 경우 균수는 6.5×109 CFU/g에서 10.4×109 CFU/g으로 증가하는 것으로 확인되었으며 포자율 역시 65.1%에서 99.8%로 증가하는 것으로 조사되었다. 80℃의 열처리 후 분쇄할 경우 포자수는 6.4×109 CFU/g이었으며 포자율은 99%로 분쇄전과 비슷한 균수가 확인되었다.As a result of confirming the change in spore stability due to grinding in the post-spore production process, the bacterial count , which was 7.6 could be confirmed. When heat treatment was performed at 80°C before grinding, the bacterial count was found to increase from 6.5×10 9 CFU/g to 10.4×10 9 CFU/g, and the spore rate was also found to increase from 65.1% to 99.8%. When pulverized after heat treatment at 80°C, the number of spores was 6.4
3-1-2 고체배양기 배양조건 및 제작3-1-2 Solid culture culture conditions and production
하기의 표 3와 같이, 고체배양기에 포자생산을 위해 접종량과 수분, 교반 회전수, 주입공기량, 멸균 및 배양온도 등을 조절하여 최적의 배양조건을 확립하기 위한 실험을 실시하였다. 담체인 밀기울의 뭉침 현상을 방지하기 위해 최소의 수분함량으로 60%를 설정하였으며 멸균을 위해 90-95
3-2. 생산공정3-2. production process
3-2-1. 고체배양기 제조 및 개량3-2-1. Manufacture and improvement of solid culture equipment
자체 제작한 고체배양기에 다양한 조건으로 밀기울 배양 결과를 바탕으로 회전과 열 가열방법, 수분 보급 등에 관한 장치들을 개량하여 새로운 고체배양기로 제작하였다. 이에 따라, 제작된 고체배양기에 대한 배양조건을 새롭게 확립하였다.Based on the results of cultivating wheat bran under various conditions in a self-produced solid culture culture, devices related to rotation, heating method, moisture supply, etc. were improved and a new solid culture culture was manufactured. Accordingly, new culture conditions for the manufactured solid culture medium were established.
3-2-2. 생산공정3-2-2. production process
멸균 후 포자생산과 균주의 배양에 최적화된 고체배양기를 제작하였으며, 생산된 포자의 건조조건을 확립하였다. 포자 분쇄를 위한 분쇄장치의 최적화와 분쇄 후 계면활성제와의 교반기 사용조건 및 포장의 제조공정을 확립하였다. 도 14에 본 발명에 따른 미생물 농약의 균주 제조공정을 도식화하여 나타내었다. 도 14에 도시된 바와 같이, 본 발명에 따른 미생물 농약의 균주 제조공정은 담체 및 배지의 멸균공정(S101), 멸균 후 포자생산과 균주 배양공정(S103), 포자 분쇄공정(S105), 분쇄 후 계면활성제와의 교반공정(S107) 및 포장공정(S109)으로 이루어진다. 여기서, 상기 배양공정(S103)은 균주를 배지에 고체배양하는 1차 배양 및 고체배양으로 생산된 균주(내생포자)를 미생물농약으로 제조하는 대사물질을 투입하여 진행하는 2차 배양이 순차적으로 이루어진다.After sterilization, a solid culture medium optimized for spore production and strain culture was manufactured, and drying conditions for the produced spores were established. Optimization of the grinding equipment for spore grinding, conditions for using a stirrer with surfactant after grinding, and manufacturing process for packaging were established. Figure 14 schematically shows the manufacturing process of the microbial pesticide strain according to the present invention. As shown in Figure 14, the microbial pesticide strain manufacturing process according to the present invention includes the sterilization process of the carrier and medium (S101), the spore production and strain culture process after sterilization (S103), the spore grinding process (S105), and the post-grinding process. It consists of a stirring process with surfactant (S107) and a packaging process (S109). Here, the culture process (S103) sequentially involves primary culture in which the strain is cultured on a solid medium and secondary culture in which metabolites produced as microbial pesticides are added to the strain (endospores) produced through solid culture. .
4. 항균활성 및 안정성 시험4. Antibacterial activity and stability test
전술한 본 발명에 따른 미생물농약의 안정성을 극대화하기 위하여 안정제, 증진제 첨가에 따른 항균활성 및 제형에 따른 안정성을 조사하고 확인하였다.In order to maximize the stability of the microbial pesticide according to the present invention described above, antibacterial activity according to the addition of stabilizers and enhancers and stability according to formulation were investigated and confirmed.
안정성 조사 및 확인을 위해 사용된 규조토와 계면활성제는 하기의 표 4와 같다. Diatomaceous earth and surfactants used for stability investigation and confirmation are shown in Table 4 below.
4-1. 최적 제형4-1. optimal formulation
내생포자와 대사산물을 이용한 미생물 농약을 제조하기 위해 포자안정성, 분산성, 수화성, 현수성 등을 확립하기 위해 다양한 규조토와 계면활성제들을 조합하여 실험하였다.In order to manufacture microbial pesticides using endospores and metabolites, various combinations of diatomaceous earth and surfactants were tested to establish spore stability, dispersibility, hydration, and suspension properties.
4-1-1. 계면활성제 안정성4-1-1. Surfactant Stability
먼저, 제품의 품질관리를 위해 계면활성제의 수분안정성을 조사하였다. 계면활성제의 수분안정성은 드라이오븐에서 60℃에서 120시간 건조하여 중량변화를 측정하였다. 하기의 표 5는 규조토와 계면활성제의 수분안정성 결과를 정리한 표로서, 표 5와 같이, 계면활성제 A의 경우 26.3%의 중량 변화가 확인되었으며 계면활성제 B의 경우 5%의 중량변화를 확인할 수 있었다. First, the moisture stability of the surfactant was investigated for product quality control. The moisture stability of the surfactant was measured by drying it in a dry oven at 60°C for 120 hours and measuring the weight change. Table 5 below summarizes the moisture stability results of diatomaceous earth and surfactant. As shown in Table 5, in the case of surfactant A, a weight change of 26.3% was confirmed, and in the case of surfactant B, a weight change of 5% was confirmed. there was.
다음으로, 계면활성제 제형 시험으로, 액체형 계면활성제와 분말제 계면활성제의 혼합 시 제형의 안정성을 확인하였다. 모든 계면활성제에서 뭉침 현상이 발생하였지만 계면활성제 C, D가 보다 안정성이 있음을 확인할 수 있다(표 6 참조).Next, through a surfactant formulation test, the stability of the formulation was confirmed when mixing a liquid surfactant and a powdered surfactant. Although agglomeration occurred in all surfactants, it can be seen that surfactants C and D are more stable (see Table 6).
4-1-2. 계면활성제 수화성4-1-2. Surfactant hydration
규조토와 계면활성제의 조합을 통해 물에 혼합 시 신속하게 젖어드는지를 평가하기 위해 규조토와 계면활성제 A, B를 혼합하여 수화성을 조사하였다. 조사결과, 규조토의 양 증가 시 수화성이 증가하는 것을 확인할 수 있다(표 7 참조).In order to evaluate whether the combination of diatomaceous earth and surfactant becomes wet quickly when mixed with water, the hydration property was investigated by mixing diatomaceous earth with surfactants A and B. As a result of the investigation, it was confirmed that hydration increased as the amount of diatomaceous earth increased (see Table 7).
또한, 규조토와 계면활성제 분산력 조사를 위해, 규조토 3종류와 계면활성제 3종류의 조합을 통해 분산성과 제품 안정성을 조사하였다. 액체계면활성제와 혼합공정에서는 규조토 B가 우수했으며 계면활성제 A, B와 규조토 B의 조합이 분산성이 높았다(표 8 참조).In addition, to investigate the dispersion power of diatomaceous earth and surfactant, the dispersibility and product stability were investigated through a combination of 3 types of diatomaceous earth and 3 types of surfactants. In the mixing process with liquid surfactants, diatomaceous earth B was superior, and the combination of surfactants A and B with diatomaceous earth B had high dispersibility (see Table 8).
그리고 포자의 담체인 밀기울의 수분친화도를 높이기 위해 담체와 계면활성제 F, G의 조합을 통해 현수성을 알아 보았다. 밀기울의 지방 성분으로 인해 수분친화도가 낮아 이에 적합한 계면활성제를 조사하여 5~10%함량으로 시험한 결과, 계면활성제 F가 습윤성이 높은 것을 확인할 수 있었다(표 9 참조).In order to increase the water affinity of wheat bran, which is the carrier of spores, the suspension properties were investigated through a combination of carrier and surfactant F and G. Due to the fat content of wheat bran, water affinity is low, so a suitable surfactant was investigated and tested at a content of 5 to 10%, and it was confirmed that surfactant F had high wettability (see Table 9).
4-1-3. 제형 최적 조합4-1-3. Optimal combination of formulations
먼저, 제품 제형 구성에 따른 공정과 분산성을 조사한 결과, 규조토의 함량을 10% 올렸을 때 분산성이 향상되는 것을 확인할 수 있었다(표 10 참조).First, as a result of investigating the process and dispersibility according to the product formulation composition, it was confirmed that the dispersibility improved when the content of diatomaceous earth was increased by 10% (see Table 10).
다음으로, 포자 및 계면활성제 조합에 따른 습윤성 시험을 통해 포자와 대사산물 함량 고정(51%)시 규조토 3종과 계면활성제 4종의 조합에 따른 수분에 대한 친화도를 조사한 결과, 규조토 A 4%와 규조토 B 27% 그리고 계면활성제 A 5%, B 10% 조합이 15초로 가장 빠른 습윤 시간을 보였으며 계면활성제 D를 19% 처리한 시험구는 50초로 가장 늦은 습윤 시간을 보였다(표 11 참조).Next, through a wettability test according to the combination of spores and surfactants, the affinity for moisture according to the combination of 3 types of diatomaceous earth and 4 types of surfactants was investigated when the spore and metabolite content was fixed (51%). Diatomaceous earth A was 4%. The combination of 27% diatomaceous earth B and 5% surfactant A and 10% B showed the fastest wetting time at 15 seconds, and the test group treated with 19% surfactant D showed the slowest wetting time at 50 seconds (see Table 11).
포자 및 계면활성제 조합에 따른 수화성 시험을 위해, 포자와 대사산물과 규조토 3종, 계면활성제 4종의 조합에 따른 제형이 물에 젖어드는 시간을 측정하여 수화성을 확인한 결과, 다양한 계면활성제의 조합으로 제품이 물에 젖어드는 시간을 1분 30초~2분 이내로 수분 친화도를 확인할 수 있었다(표 12 참조).To test the hydrability according to the combination of spores and surfactants, the hydration time of the formulation according to the combination of spores, metabolites, 3 types of diatomaceous earth, and 4 types of surfactants was measured to confirm the hydration property of various surfactants. With the combination, the moisture affinity was confirmed as the time the product was soaked in water was within 1 minute and 30 seconds to 2 minutes (see Table 12).
제품 제작을 위한 제형 조건을 확립하기 위해 선정된 규조토 2종과 계면활성제 4종을 3가지 조합으로 하여 침전 속도를 측정한 결과, 9-1과 9-2는 15초, 9-3은 30초의 침전속도를 보였다(표 13 참조).In order to establish the formulation conditions for product production, the sedimentation speed was measured using three combinations of two types of diatomaceous earth and four types of surfactants selected, and the results showed that 9-1 and 9-2 were 15 seconds and 9-3 was 30 seconds. Sedimentation speed was shown (see Table 13).
4-2. 제품 제조 및 항균활성4-2. Product manufacturing and antibacterial activity
4-2-1. 제품 제조4-2-1. product manufacturing
전술한 실험결과를 바탕으로 총 3개의 제품을 제조하였으며, 그에 따른 수화성, 현수성, 침강성을 종합하여 측정하였다(표 14 참조). A total of three products were manufactured based on the above-mentioned experimental results, and the resulting hydration, suspension, and settling properties were comprehensively measured (see Table 14).
4-2-2. 안정성 및 항균활성4-2-2. Stability and antibacterial activity
각 제품의 균수를 조사하여 포자 안정성을 확인하고 항균활성 그리고 자체 약해시험 등을 통해 제품의 안정성과 항균활성을 확인하였다. 제작된 제품의 항균활성을 확인하기 위하여 제품을 500배, 1000배, 2000배 희석한 후 0.2μm의 필터를 이용해 배양액만을 회수한 다음 식물병원균이 접종된 PDA배지에 paper disk를 올려두어 20μl를 접종하고 대치 배양하여 항균활성을 확인하였다.The bacterial count of each product was investigated to confirm spore stability, and the safety and antibacterial activity of the product were confirmed through antibacterial activity and self-toxicity tests. In order to check the antibacterial activity of the manufactured product, the product was diluted 500-, 1000-, and 2000-fold, and only the culture solution was recovered using a 0.2μm filter. Then, a paper disk was placed on the PDA medium inoculated with plant pathogens and inoculated with 20μl. Then, the antibacterial activity was confirmed by replacement culture.
그 결과, MF-01보다 MF-02와 MF-03의 제형이 더 좋았으며 MF-01이 제조 후 안정성 조사에서 가장 높은 2.8×109 CFU/g를 보였다. 도 15는 본 발명에 따른 미생물 농약 제품의 포자 안정성 시험 결과 그래프이다.As a result, the formulations of MF-02 and MF-03 were better than MF-01, and MF-01 showed the highest level of 2.8×10 9 CFU/g in the stability study after manufacturing. Figure 15 is a graph of the spore stability test results of the microbial pesticide product according to the present invention.
다음으로, 제조된 제품의 약효보증기간 동안 기간의 경과에 따른 물리적·화학적으로 주성분을 잘 유지하는지를 분석하기 위하여 제품 제작 후 매월 샘플을 채취한 후 TSA배지에 도말하여 미생물 개체수를 측정하였다.Next, in order to analyze whether the manufactured product maintains its main ingredients physically and chemically over time during the efficacy guarantee period, samples were collected every month after the product was manufactured and smeared on TSA medium to measure the number of microorganisms.
분말형상의 제품의 자체 약해시험을 진행하기 위하여 준비된 공시토양에 배추 및 상추 유묘를 이식하고, 제품MF-01과 MF-03을 기준량과 배량으로 조제하여 식물체의 엽면에 살포 처리 후 3일, 5일, 7일째에 걸쳐 유묘의 생육양상을 3회 달관 조사하였다.In order to conduct an in-house toxicity test for the powder-type product, cabbage and lettuce seedlings were transplanted into the prepared test soil, products MF-01 and MF-03 were prepared in standard and double amounts, and sprayed on the leaves of the plants for 3 days and 5 days. The growth patterns of seedlings were examined three times a month over the first and seventh days.
조사결과, 모든 처리구에서 이상 증상이 보이지 않아 상추와 배추에 약해는 없는 것으로 확인되었다. 하기의 표 15에 약해시험을 위한 처리구에 따른 처리방법을 정리하였다.As a result of the investigation, it was confirmed that no abnormal symptoms were observed in all treatments, so there was no damage to lettuce and cabbage. Table 15 below summarizes the treatment methods according to the treatment group for the drug toxicity test.
도 16에 나타난 바와 같이, 분말제품 MF01, 02, 03을 500배 희석하여 고추 탄저병과 잿빛곰팡이병 균주들에 대한 항균활성을 확인한 결과, 모든 처리구에서 항균활성이 확인되었다. 도 16은 본 발명에 따른 미생물 농약 제품의 항균활성 시험 사진이다.As shown in Figure 16, the powder products MF01, 02, and 03 were diluted 500 times and the antibacterial activity against pepper anthracnose and gray mold strains was confirmed. As a result, antibacterial activity was confirmed in all treatments. Figure 16 is a photograph of an antibacterial activity test of a microbial pesticide product according to the present invention.
제품의 계면활성제의 영향으로 생기는 거품을 제거하기 위해 소포제(CLS500)를 0~50ppm 농도로 사용하여 제품의 거품 생성을 억제하였다. 소포제 농도에 따른 시험 결과를 도 17에 나타내었다.To remove foam caused by the product's surfactant, an antifoaming agent (CLS500) was used at a concentration of 0 to 50 ppm to suppress foam generation in the product. Test results according to defoamer concentration are shown in Figure 17.
5. 제품의 분말도에 따른 효율 평가5. Efficiency evaluation according to product powder level
제품의 분말도별 효능 및 안정성을 조사하였다. 밀기울 분말화에 대한 안정성을 확인하고자 밀기울 포자 분쇄 전후의 균수와 포자율을 확인하였고 분쇄공정 후 경시적 변화를 확인한 결과, 분쇄 전에 3.7×109 CFU/g이였던 포자가 분쇄 후 3.9×109 CFU/g으로 거의 변동이 없는 것을 확인 할 수 있었으며, 분쇄 2주 후부터 4.5×109 CFU/g으로 안정적인 균수를 보였다. 도 18 및 도 19에 본 발명에 따른 미생물 농약 제품의 밀기울 분말도에 따른 균수 변화 및 담체 분쇄 공정에 의한 포자안정성을 조사하여 그래프로 나타내었다.The efficacy and stability of each product was investigated by powder level. To check the stability of wheat bran powder, the number of bacteria and spore rate were checked before and after grinding of wheat bran spores, and as a result of checking the change over time after the grinding process, the spores that were 3.7 × 10 9 CFU/g before grinding decreased to 3.9 × 10 9 CFU / g after grinding. It was confirmed that there was almost no change in CFU/g, and the bacterial count was stable at 4.5×10 9 CFU/g after 2 weeks of grinding. Figures 18 and 19 show graphs showing changes in the number of bacteria according to the bran fineness of the microbial pesticide product according to the present invention and spore stability by the carrier grinding process.
전술한 바와 같이, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법은 국산 소재를 이용한 저가 및 고효율의 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법을 제공한다.As described above, the microbial pesticide for controlling crop fungal diseases and its manufacturing method according to the present invention provides a low-cost and highly efficient microbial pesticide for controlling crop fungal diseases using domestic materials and a manufacturing method thereof.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법은 Bacillus velezensis CE100 균주와 고체배양 기술을 이용하여 밀기울, 규조토, 제올라이트와 같은 담체를 사용한 포자 생산기술을 활용한 저가 및 고효율의 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법을 제공한다.In addition, the microbial pesticide for controlling crop fungal diseases according to the present invention and its manufacturing method are low-cost and highly efficient crops using spore production technology using carriers such as wheat bran, diatomaceous earth, and zeolite using Bacillus velezensis CE100 strain and solid culture technology. Provides microbial pesticides for controlling fungal diseases and methods for producing them.
또한, 본 발명에 따른 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법은 대사물질을 생산해 포자와 함께 배양하고 계면활성제를 활용한 제형 연구 및 제조공정 별 조건변화에 따른 생산효율을 조사하여 고효율의 작물 곰팡이병 방제용 미생물 농약 및 그 제조방법을 제공한다.In addition, the microbial pesticide for controlling crop fungal diseases and its manufacturing method according to the present invention produce metabolites and culture them with spores, study formulations using surfactants, and investigate production efficiency according to changes in conditions for each manufacturing process to produce highly efficient crops. Provides microbial pesticides for controlling fungal diseases and methods for producing them.
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, the preferred embodiments are specifically described with reference to the accompanying drawings, but of course, 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, but should be determined not only by the scope of the patent claims described later, but also by the scope of this patent claim and equivalents.
Claims (8)
담체 및 배지의 멸균공정;
멸균 후 포자생산과 균주 배양공정;
포자 분쇄공정;
분쇄 후 계면활성제와의 교반공정; 및
포장공정;을 포함하고,
상기 담체는 이산화규소 함량 80%인 규조토 건조분말이고,
상기 균주 배양공정에서 사용한 균주는 Bacillus velezensis CE100이고,
상기 균주 배양공정은, 상기 균주를 배지에 고체배양하는 1차 배양 단계 및 상기 고체배양으로 생산된 균주(내생포자)를 미생물농약으로 제조하는 대사물질을 투입하여 진행하는 2차 배양 단계를 포함하고,
상기 계면활성제는 Sodium bis(2-ethylhexyl)와 Sulfosuccinate 및 Polyethylene glycol을 포함하는 제1 계면활성제 및 Ethoxylated octyl Phenol 및 Isopropyl alcohol를 포함하는 제2 계면활성제가 혼합된 것이고,
상기 방법으로 제조한 작물 곰팡이병 방제용 미생물 농약은, 상기 미생물 농약 전체 중량을 기준으로, 상기 포자 20중량%, 상기 대사물질 20중량%, 상기 담체 35중량% 및 상기 계면활성제 25중량%를 포함하는,
작물 곰팡이병 방제용 미생물 농약 제조방법.In the method for manufacturing microbial pesticides for controlling crop fungal diseases,
Sterilization process of carrier and medium;
Spore production and strain culture process after sterilization;
Spore crushing process;
Stirring process with surfactant after grinding; and
Including packaging process;
The carrier is diatomaceous earth dry powder with a silicon dioxide content of 80%,
The strain used in the strain culture process is Bacillus velezensis CE100,
The strain culture process includes a primary culture step of culturing the strain on a solid medium and a secondary culture step of introducing metabolites produced as microbial pesticides into the strain (endospores) produced by the solid culture, ,
The surfactant is a mixture of a first surfactant containing Sodium bis(2-ethylhexyl), Sulfosuccinate, and Polyethylene glycol, and a second surfactant containing Ethoxylated octyl Phenol and Isopropyl alcohol,
The microbial pesticide for controlling crop fungal diseases prepared by the above method includes 20% by weight of the spores, 20% by weight of the metabolites, 35% by weight of the carrier, and 25% by weight of the surfactant, based on the total weight of the microbial pesticide. doing,
Method for manufacturing microbial pesticides for controlling crop fungal diseases.
90-95℃에서 10-15시간 가열한 후 6-8시간동안 40-55℃에서 보관하며 다시 90-95℃에서 6-8시간 반응하여 멸균하는 간헐적 멸균공정인 것을 특징으로 하는 작물 곰팡이병 방제용 미생물 농약 제조방법.
The method of claim 1, wherein the sterilization process is:
Crop fungal disease control characterized by an intermittent sterilization process of heating at 90-95℃ for 10-15 hours, storing at 40-55℃ for 6-8 hours, and then sterilizing by reacting at 90-95℃ for 6-8 hours. Method for manufacturing microbial pesticides.
골분제, 효모 추출물, 랑베나이트, 아미노산, 게껍질 파우더 및 미생물 균주를 6개월 동안 고농축 장기 액체배양하여 만들어진 장기액제배양액과 유기영양분을 건조발효기에서 50-60℃로 건조발효한 후 분쇄하여 파우더 형태로 제형화한 것을 특징으로 하는 작물 곰팡이병 방제용 미생물 농약 제조방법.The method of claim 1, wherein the metabolite is:
Bone powder, yeast extract, langbenite, amino acids, crab shell powder, and microbial strains are cultured in highly concentrated long-term liquid culture for 6 months. The organ liquid culture medium and organic nutrients are dried and fermented at 50-60℃ in a dry fermenter, and then pulverized into powder form. A method for manufacturing a microbial pesticide for controlling crop fungal diseases, characterized in that it is formulated with .
A microbial pesticide for controlling fungal diseases in crops, characterized in that it is manufactured by the manufacturing method of claim 1.
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