KR101659062B1 - Pediococcus parvulus against pathogen of American Foulbrood microbial agent comprising the same and method for preventing pathogen using the same - Google Patents
Pediococcus parvulus against pathogen of American Foulbrood microbial agent comprising the same and method for preventing pathogen using the same Download PDFInfo
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Classifications
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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
-
- 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
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/41—Pediococcus
Abstract
본 발명은 페디오코커스 파버러스 YML 002(Pediococcus parvulus YML 002)에 관한 것으로, 보다 상세하게는 미국부저병(American Foulbrood)의 원인균인 패니바실러스 유충의 생육을 효과적으로 억제하는 페디오코커스 파버러스 YML 002에 관한 것이다.
본 발명에 따른 페디오코커스 파버러스 YML 002는 양봉산업에 있어 큰 질병인 미국 부저병으로 인한 봉군 붕괴 및 수확되는 벌꿀의 품질의 저하 현상을 예방하고 치료할 수 있는 미생물제제로서 사용가능하며, 특히 항생제 남용으로 인한 항생제 잔류 벌꿀 제품에 대한 문제를 해결할 수 있는 항생제 대체물로서 양봉 사료의 첨가물, 양봉 약품으로 유용하게 사용될 수 있다.The present invention relates to Pediococcus parvulus YML 002, and more particularly to Pediococcus parvulus YML 002, which effectively inhibits the growth of Fanny Cassia larva, a causative organism of American Foulbrood. .
The Pediococcus fulvirus YML 002 according to the present invention can be used as a microbial preparation capable of preventing and treating the degradation of the quality of honey harvested and the collapse of the swine caused by the American buzzing disease which is a serious disease in the beekeeping industry, Antibiotic residues due to abuse As an antibiotic substitute that can solve problems with honey products, it can be usefully used as an additive for beekeeping feeds, bee-keeping drugs.
Description
본 발명은 미국부저병(American Foulbrood)의 원인균에 대한 항균활성을 가지는 페디오코커스 파버러스 YML 002, 상기 미생물을 함유하는 항균성 미생물제제 및 상기 미생물을 이용한 방제방법에 관한 것이다.The present invention relates to Pediococcus fulvirus YML 002 having antimicrobial activity against a causative microorganism of American Foulbrood, an antimicrobial microorganism preparation containing the microorganism, and a method of controlling the microorganism using the microorganism.
국내·외 양봉 사육 현장에서 발생하는 중요한 전염병인 미국부저병은 패니바실러스 유충(Paenibacillus larvae subsp. larvae)에 의해서 일어나며, 발생 시기는 주로 봉군 이동기이다. 우리나라 양봉업의 기반이 되고 있는 아까시 꽃이 기후의 변동으로 더워지는 기후에 꽃은 피는데 꿀 생산량이 감소함에 따라서 이것을 보충하려고 업자들이 설탕을 대량으로 급여하고 있고 이에 따라서 벌의 건강상태가 허약해져 질병에 쉽게 노출된다. 뿐만 아니라 애벌레와 성충의 질병이 많고, 진드기의 항생제 내성이 증가하여 뚜렷한 대책이 없는 상태이다. American buzzers, an important infectious disease occurring in both domestic and overseas beekeeping sites, are caused by Paenibacillus larvae subsp. Larvae, and are mainly populated. As the production of honey from the flowers in the climate that warms up due to the fluctuation of the climate, which is the base of the beekeeping industry in Korea, the honey is being supplied in large quantities by the workers to supplement it as the production of honey decreases, It is easily exposed. In addition, there are many diseases of larvae and adults, and there is no clear countermeasure due to increased antibiotic resistance of ticks.
주요 병적 증상으로는 유개봉 유충만이 발병하며 죽은 유충은 액화가 된다. 미국 부저병의 원인균은 주로 유충에 기생하며 유충의 폐사율이 증가함에 따라 봉군 자체의 붕괴를 야기하는 심각한 질병이다. The main pathological symptoms are only open larvae, and dead larvae are liquefied. The causative organism of American buzzers is largely parasitic on larvae and is a serious disease causing the collapse of the progeny itself as the larval mortality rate increases.
미국 부저병은 미생물성 질병으로 주로 항생제를 투여하여 치료를 하는데 패니바실러스 유충의 항생제 내성 균주 생성 및 양봉 부산물내 항생제 잔존의 문제가 발생하게 된다. 양봉에서 얻어지는 생산물로서 꿀, 프로폴리스 등은 대개 식품으로 이용되는 양봉 부산물 내 항생제 잔존 문제가 발생하게 된다. 미국 부저병의 감염 및 치료를 위해 항생제가 사용되고 있으나 항생제의 사용에 의한 부작용 또한 발생할 수 있다는 문제점이 있다. The American buzzers disease is a microbial disease mainly treated with antibiotics, which causes the generation of antibiotic resistant strains of Fanny Bacillus larvae and the remaining antibiotics in the beekeeping byproducts. Honey, propolis, etc., as a product obtained from beekeeping, usually cause the problem of residual antibiotics in beekeeping by-products used as food. Although antibiotics are used for infection and treatment of American buzzers, side effects due to the use of antibiotics may also occur.
또한 현재는 항생제 잔존량에 대한 규제가 갈수록 엄격해지고 있으므로 계속적인 항생제의 사용 및 남용은 제고해야 할 문제로 이미 대두되어 왔다. 이에 대한 대안으로 현재 미생물제제(Probiotics)에 대한 관심이 증대되고 있다.Also, since the regulations on the residual amount of antibiotics are becoming increasingly strict, current use of antibiotics and abuse have been raised as problems. As an alternative to this, interest in microbiological agents (probiotics) is increasing.
인체 및 동물체의 장내에는 다양한 미생물들이 존재하면서 장내의 미생물 균총을 이루고 있다. 이러한 미생물 중에는 숙주 동물에 유용한 것으로 알려진 유산균 등의 유익한 미생물들이 있는가 하면 반대로 숙주에 직접적 또는 잠재적인 유해성을 가지는 대장균, 살모넬라, 포도상구균 등의 미생물들이 또한 포함된다. 인체 및 동물체의 경우 스트레스의 증가, 유해 세균의 감염 등의 외부 환경의 변화에 의해서 장내 미생물의 균총이 파괴될 경우 유해 미생물들의 급격한 성장으로 이어져 숙주동물의 건강상태를 악화시키거나 설사에서부터 심지어는 폐사의 위험으로까지 진행될 수 있다.There are various microorganisms in the intestines of human and animal, and they form microorganisms in the intestines. Among these microorganisms are beneficial microorganisms such as lactic acid bacteria known to be useful in host animals and microorganisms such as Escherichia coli, Salmonella and Staphylococcus which directly or indirectly have potential harm to the host. In the case of human and animal, when the intestinal microorganisms are destroyed by changes in the external environment such as increased stress and infection of harmful bacteria, it leads to rapid growth of harmful microorganisms, which deteriorates the health condition of the host animal, The risk can be advanced to.
이러한 일이 발생하였을 경우 치료의 목적으로 항생제를 투여하게 되나 이러한 항생제는 체외로 완전히 배설이 되지 않고 숙주 내에 잔존하게 되며 지속적인 투여 시 유해 미생물들이 내성을 가지게 되므로 효과적인 치료를 할 수 없게 되는 상황이 발생하게 된다.When this happens, antibiotics are administered for the purpose of treatment. However, these antibiotics are not completely excreted in the body and remain in the host. In case of continuous administration, the harmful microorganisms become resistant, .
인체 및 동물체와 마찬가지로 곤충인 꿀벌 또한 비슷한 결과를 초래할 것으로 예상하여 유산균을 생산하는 미생물제제를 이용하여 양봉에 무분별하게 사용되는 항생제의 대체제 개발이 중요하다.It is important to develop an antibiotic substitute that is used indiscriminately in beekeeping by using a microbial agent that produces lactic acid bacteria, as the insect bee, as well as humans and animals, expects similar results.
미생물제제는 인체나 동물체의 장내에 서식하는 유익한 미생물을 분리하여 제제화한 것으로, 호기성균군, 혐기성균군, 유산균, 효모균들이 사용되고 있으나 주로 유산균들이 주요 균주로 이용되고 있다. 이러한 유산균 프로바이오틱스는 항생제 남용으로 인해 발생할 수 있는 부작용을 일으키지 않고 유해 미생물의 감염으로 인한 질병을 사전에 억제할 수 있다. Microorganism preparations are prepared by separating beneficial microorganisms living in the intestines of humans and animals. Aerobic bacteria, anaerobic bacteria, lactic acid bacteria and yeast bacteria are used, but lactic acid bacteria are mainly used as a major strain. These lactic acid probiotics can prevent diseases caused by infection with harmful microorganisms without causing side effects that may be caused by antibiotic abuse.
지금까지 미생물제제 프로바이오틱스로 다양한 종류의 유산균이 연구되어 왔으며, 특히 꿀벌의 장내 유용균인 비피더스균, 유산간균 및 유산구균을 혼합한 양봉사료 및 그 사용방법이 보고된 바 있다(대한민국 특허 제67946호).Various types of lactic acid bacteria have been studied with the microbiological preparation probiotics, and in particular, bee-bred diets mixed with bifidobacteria, lactic acid bacterium, and lactic acid bacteria, and methods of using them have been reported (Korean Patent No. 67946) .
이에 본 발명자들은 기존의 발표된 비피더스균 등을 혼합한 양봉사료를 이용하여 꿀벌의 면역기능을 증진시키는 것보다 꿀벌에 질병을 일으키는 직접적인 미국부저병의 원인균인 패니바실러스 유충을 억제하는 항균활성을 지닌 신규 미생물을 발견함으로써 본 발명을 완성하였다.Accordingly, the present inventors have found that the use of bee-bait mixed with existing bifidobacteria has an antimicrobial activity that inhibits Fanny Bacillus larva, which is a causative agent of direct American buzzers causing diseases in bees, rather than promoting the immune function of bees The present invention has been accomplished by discovering new microorganisms.
본 발명의 목적은 기존 항생제의 대체용으로 꿀벌에게 투여하는 양봉용 사료 첨가제, 양봉 약품 등에 사용하여 미국부저병(American Foulbrood)의 원인균인 패니바실러스 유충(Paenibacillus larvae subsp. larvae)을 억제할 수 있는 미생물, 이를 함유하는 미생물제제 및 이를 이용한 방제방법을 제공하는 데에 있다.An object of the present invention is to provide a microorganism capable of inhibiting Paenibacillus larvae subsp . Larvae, which is a causative organism of American Foulbrood, for use in feed additives, beekeeping drugs and the like for bees to be administered to bees for replacing existing antibiotics , A microbial preparation containing the same, and a control method using the same.
상기 목적을 달성하기 위하여, 본 발명은 미국부저병(American Foulbrood)의 원인균에 대한 항균활성을 가지는 페디오코커스 파버러스 YML 002(Pediococcus parvulus YML 002)를 제공한다.In order to achieve the above object, the present invention provides Pediococcus parvulus YML 002, which has an antibacterial activity against a causative organism of American Foulbrood.
상기 원인균은 패니바실러스 유충(Paenibacillus larvae subsp. larvae)이며, 상기 페디오코커스 파버러스 YML 002는 최적온도 30℃-37℃, 바람직하게는 30℃에서 배양될 수 있다.The causative organism is Paenibacillus larvae subsp . Larvae, and the Pediococcus paverus YML 002 can be cultured at an optimal temperature of 30 ° C to 37 ° C, preferably 30 ° C.
상기 페디오코커스 파버러스 YML 002의 16S rRNA 유전자의 염기 서열은 서열번호 1로 표시되며, 수탁번호 KCTC11628BP 로 기탁된 것이다.The nucleotide sequence of the 16S rRNA gene of Pediococcus pseudorabies YML 002 is shown in SEQ ID NO: 1 and deposited with accession number KCTC11628BP.
또한, 본 발명은 상기 페디오코커스 파버러스 YML 002를 함유하는 항균성 미생물제제를 제공한다. 상기 미생물제제는 양봉 사료 첨가제나 양봉 약품의 형태로 제공될 수 있다.In addition, the present invention provides an antibacterial microorganism preparation containing the Pediococcus fulvirus YML 002. The microbial formulation may be provided in the form of a bee feed additive or a bee-keeping drug.
또한, 본 발명은 상기 항균성 미생물제제을 이용한 미국부저병(American Foulbrood)의 방제방법을 제공한다.The present invention also provides a method for controlling American Foulbrood using the above-mentioned antibacterial microorganism preparation.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에서는 미국부저병(American Foulbrood)의 원인균인 패니바실러스 유충을 억제할 수 있는 신규 미생물 페디오코커스 파버러스 YML 002를 제공한다.The present invention provides a novel microorganism, Pediococcus albumus YML 002, capable of inhibiting Fanny Bacillus larva, a causative organism of American Foulbrood.
상기 균주는 패니바실러스 유충을 억제하는 미생물을 탐색한 결과, 얻어진 새로운 페디오코커스 파버러스 균주로서 패니바실러스 유충의 억제 능력이 우수한 것으로 나타났다.As a result of searching for a microorganism inhibiting the Fanny Bacillus larva, the strain was found to be excellent in the ability to inhibit the Fanny Bacillus larva as a new strain of Pediococcus faecalis.
구체적으로, 본 발명의 균주 페디오코커스 파버러스 YML 002가 원인균인 패니바실러스 유충의 생장을 억제하는 억제환의 지름(단위는 mm)은 약 16mm로서, 대조군으로 100ppm 농도의 테트라사이클린 50㎕을 사용한 억제환의 지름(약 17mm)과 유사한 항균 활성을 나타내었다. Specifically, the diameter (unit: mm) of the inhibitory ring that inhibits the growth of Fanny Bacillus larvae causing the strain Pediococcus pseudobacter YELL 002 of the present invention was about 16 mm, and the inhibition using 50 μl of tetracycline at a concentration of 100 ppm as a control group And exhibited antibacterial activity similar to the diameter of the ring (about 17 mm).
본 발명에 따른 페디오코커스 파버러스 YML 002가 미국부저병의 원인균의 생장을 억제하기 위한 최적 배양온도는 30℃이었다.The optimal culture temperature for inhibiting the growth of causative microorganisms of US bee venom caused by Pediococcus fulvirus YML 002 according to the present invention was 30 ° C.
본 발명에 따른 페디오코커스 파버러스 YML 002의 형태 및 생리학적 특성은 다음과 같다.The morphology and physiological characteristics of the Pediococcus fulvirus YML 002 according to the present invention are as follows.
페디오코커스 파버러스 YML 002는 그람양성의 세균으로, 호기적인 조건과 혐기적인 조건에서 동시에 성장하며 포자를 형성하지 않고 운동성이 없으며, 세포의 형태는 구균이다. 상기 균주의 성장을 위한 최적온도는 30℃인 것이 바람직하다. Pediococcus flurbus YML 002 is a gram - positive bacterium that grows simultaneously under aerobic conditions and anaerobic conditions, does not form spores, has no motility, and the form of cells is staphylococci. The optimum temperature for the growth of the strain is preferably 30 < 0 > C.
본 발명의 페디오코커스 파버러스 YML 002의 16S rRNA 유전자의 염기 서열은 서열번호 1로 기재되며, 16S rDNA 염기서열에 기초한 분자계통분류학적 분석을 통하여 본 발명의 균주는 페디오코커스(Pediococcus) 속에 속하는 균주로서 99%의 16S rDNA 상동성으로 페디오코커스 파버러스(Pediococcus parvulus) 균주 2.6(NCBI Blast 동정결과)에 가장 높은 분자계통학적 유연관계를 보여주는 균주로 동정되었다.The nucleotide sequence of the 16S rRNA gene of Pediococcus pylori YML 002 of the present invention is shown in SEQ ID NO: 1. Through the molecular phylogenetic analysis based on the 16S rDNA nucleotide sequence, the strain of the present invention is found in Pediococcus The strains belonging to the genus Pseudomonas were identified as the strain showing the highest molecular phylogenetic relationship to Pediococcus parvulus strain 2.6 (result of identification of NCBI Blast) with 99% 16S rDNA homology.
따라서, 상기의 결과에 기초하여 본 발명의 균주를 페디오코커스 파버러스(Pediococcus parvulus)의 새로운 균주로 동정하고, 생물자원센터에 2010년 2월 1일자로 기탁하였다(수탁번호: KCTC11628BP).Therefore, based on the above results, the strain of the present invention was identified as a new strain of Pediococcus parvulus and deposited on February 1, 2010 (Accession No .: KCTC11628BP) in the BRC.
또한, 본 발명은 페디오코커스 파버러스 YML 002를 함유하는 미생물제제를 제공한다. 상기 미생물제제는 양봉사료 첨가제 또는 양봉약품 등의 형태로 제공될 수 있다.In addition, the present invention provides a microorganism preparation containing Pediococcus flavus YML 002. The microbial agent may be provided in the form of a bee-growing feed additive or a bee-making drug.
본 발명의 미생물은 장기간 안정적으로 보존하기 위해 글리세롤 성분을 포함하여 -80℃에 보관하거나 멸균된 10% 탈지유에 현탁하여 동결건조하는 것이 바람직하다.The microorganism of the present invention is preferably stored at -80 DEG C containing glycerol component or suspended in sterilized 10% skim milk for freeze-drying for long-term stable storage.
또한, 본 발명은 페디오코커스 파버러스 YML 002를 이용하여 미국부저병의 원인균인 패니바실러스 유충을 억제할 수 있는 미국부저병의 방제방법을 제공한다.In addition, the present invention provides a method for controlling American buzzers that can inhibit Fanny Bacillus larva, a causative organism of American buzzers, using Pediococcus pearlus YML 002.
상기 방제방법에서, 페디오코커스 파버러스 YML 002가 패니바실러스 유충을 억제하기 위한 최적 온도는 30℃인 것이 바람직하다.In the above-mentioned control method, it is preferable that the optimum temperature for suppressing the Pediococcus larvae of Pediococcus parvulus YML 002 is 30 ° C.
본 발명에 따른 페디오코커스 파버러스 YML 002는 미국 부저병의 원인균인 패니바실러스 유충을 효과적으로 억제하여 기존 항생제의 잔류, 오염 등의 문제를 대체 수 있는 환경친화적인 생물농약으로서의 고부가가치산물로 경제적 활용성이 증대될 것이다.The Pediococcus flurbus YML 002 according to the present invention is a high value-added product as an environmentally friendly biological pesticide capable of effectively suppressing the problem of residual antibiotic, pollution, etc. of Fani Bacillus larva, Sex will increase.
또한, 병원성 생물의 증식 억제 효과를 갖는 신기능 프로바이오틱스인 본 발명에 따른 미생물제제를 사용함으로써 항생제 내성균의 출현을 효율적으로 차단하고 봉군의 꿀벌의 질병을 치료하거나 사전에 예방할 수 있다. In addition, by using the microorganism preparation according to the present invention, which is a new-function probiotics having an effect of inhibiting the growth of pathogenic organisms, the appearance of antibiotic resistant bacteria can be effectively blocked and the disease of bee bees in the bud can be treated or prevented in advance.
이하, 하기 실시예에 의해 본 발명을 보다 상세히 설명한다. 다만, 이러한 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 이러한 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are illustrative of the present invention, and the contents of the present invention are not limited by these examples.
<실시예 1> 산을 생성하는 성질을 가지는 미생물의 분리Example 1: Isolation of microorganisms having acid-producing properties
대구ㅇ경산 일대의 50여 곳에서 채집한 김치 1g을 0.85% 생리식염수(0.85g/100ml)에 십진희석하고 BCP 한천[2.5ｇ 건조 효모 추출물, 5.0ｇ 펩톤, 1.0ｇ 글루코오스, 0.1ｇ L-시스테인, 1.0ｇ 트윈80, 0.04ｇ 브로모크레졸 퍼플(Bromcresol purple: BCP), 15.0ｇ 한천 파우더, pH6.9±, EIKEN CHEMICAL]에 도말하여 30℃에서 48시간 동안 배양하였다. 1 g of kimchi collected from 50 sites in Gyeongsan, Daegu was decanted into 0.85% physiological saline (0.85 g / 100 ml), and the BCP agar [2.5 g dried yeast extract, 5.0 g peptone, 1.0 g glucose, 0.1 g L- cysteine , 1.0 g Tween 80, 0.04 g Bromocresol purple (BCP), 15.0 g agar powder, pH 6.9, EIKEN CHEMICAL) and cultured at 30 ° C for 48 hours.
그 중 산을 생성한 콜로니를 다시 MRS 한천(10g 프로테오스 펩톤 No.3, 10g 쇠고기 추출물, 5g 효모 추출물, 20g 덱스트로오스, 5g 폴리솔베이트80, 5g 초산나트륨, 2g 구연산암모늄, 0.1g MnSO4, 0.05g MnSO4, 2g 제2인산칼륨)을 사용하여 약 1,800 여종의 산 생성균을 순수분리 하였고 보존은 30% 글리세롤(H2O:글리세롤＝7:3) 300㎕와 배양액 500㎕를 현탁하여 -70℃에 보존하였다. The acid-producing colonies were again suspended in MRS agar (10 g protease peptone No. 3, 10 g beef extract, 5 g yeast extract, 20 g dextrose, 5 g polysorbate 80, 5 g sodium acetate, 2 g ammonium citrate, MnSO 4 , 0.05 g MnSO 4 , 2 g potassium phosphate) were used to purify about 1,800 acid-producing bacteria. To store, 300 μl of 30% glycerol (H 2 O: glycerol = 7: 3) Suspended and stored at -70 ° C.
<실시예 2> 분리된 미생물의 항균 활성 검토<Example 2> Examination of antibacterial activity of isolated microorganisms
본 발명에 사용된 미국부저병의 원인균인 패니바실러스 유충은 생물 자원센터에서 분양 받은 균으로, 기탁번호 KCTC3744 균주를 사용하였다.The Fanny Bacillus larva, which is a causative organism of the US buzzer disease used in the present invention, was purchased from the BRC and the strain No. KCTC3744 was used.
1% 접종한 패니바실러스 유충을 BHI broth에서 24시간 동안 37℃, 호기적 조건에서 배양하고, 실시예 1에서 얻어진 실험에 사용된 분리균은 MRS broth에 48시간 동안 30℃, 호기적 조건에서 배양하였다. 분리균의 배양액은 12,000rpm에서 15분 동안 원심분리를 수행하여 상등액과 균체를 분리하였고, 상등액을 항균활성 시험에 사용하였다.The 1% inoculated Fanny Bacillus larvae were cultured in BHI broth for 24 hours at 37 ° C under aerobic conditions. The isolates used in the experiment obtained in Example 1 were cultured in MRS broth for 48 hours at 30 ° C under aerobic conditions Respectively. The culture broth of the isolate was centrifuged at 12,000 rpm for 15 minutes to separate the supernatant and cells, and the supernatant was used for the antimicrobial activity test.
1차 선별 단계로 96-well plate에 BHI broth를 100㎕씩 각 well에 분주하고 패니바실러스 유충 배양액을 50㎕를 분주하였다. 그 위에 분리균의 상등액 50㎕를 처리하였다. 양성대조군으로 100ppm 테트라사이클린(Generay Biotech)을 상등액과 동량으로 사용하였다. 테트라사이클린은 내성 균주가 생기기 전까지 양봉 항균제로 사용되었으며 아직도 몇몇 양봉업에서는 사용하고 있다. 본 발명에서 사용한 패니바실러스 유충은 내성이 생기지 않은 균주이기에 테트라사이클린을 사용하였다. 100 μl of BHI broth was dispensed into each well in a 96-well plate and 50 μl of Fanny Bacillus larva culture was dispensed. And 50 占 퐇 of the supernatant of the isolated bacteria was treated. As a positive control, 100 ppm tetracycline (Generay Biotech) was used in the same amount as the supernatant. Tetracycline has been used as a bee - tanning antibiotic until a resistant strain has emerged and is still used by some beekeeping companies. Tetracycline was used because the Fanny Bacillus larva used in the present invention was a strain that did not develop resistance.
처리가 끝난 96-well plate를 37℃에서 24시간 동안 배양하고 분광광도계(NanoBometer OPTIZEN 3220UV, Mecasys Co.)를 이용하여 OD 600nm에서 흡광도 값을 측정하였다.The treated 96-well plate was incubated at 37 ° C for 24 hours and absorbance was measured at OD 600nm using a spectrophotometer (NanoBometer OPTIZEN 3220UV, Mecasys Co.).
1차 시험에서 선발된 후보 균주는 2차 시험으로 페이퍼 디스크 항균활성 시험을 수행하였다. 앞선 시험과 같이 패니바실러스 유충을 BHI broth에서 24시간 동안 37℃, 호기적 조건에서 배양하고, 테스트에 사용된 분리균은 MRS broth에 48시간 동안 30℃, 호기적 조건에서 배양하였다. 패니바실러스 유충 배양액 400㎕을 BHI 한천 배지에 도말하고, 페이퍼 디스크에 분리균의 상등액 500㎕을 10배 농축하여 처리하였고 양성대조군으로 100ppm 테트라사이클린(100ppm) 50㎕을 사용하였다. 모든 처리가 끝난 플레이트를 37℃에서 24시간 동안 배양하고 클리어존(clear zone)의 직경을 측정하였다.Candidate strains selected in the first test were subjected to a paper disk antimicrobial activity test as a secondary test. Fanny Bacillus larvae were cultured in BHI broth for 24 hours at 37 ℃ under aerobic conditions. The isolates used for the test were cultured in MRS broth for 48 hours at 30 ℃ under aerobic conditions. 400 μl of Fanny Bacillus larvae culture was plated on BHI agar medium and 500 μl of the supernatant of the isolate was concentrated on a paper disk by 10 times and treated with 50 μl of 100 ppm tetracycline (100 ppm) as a positive control. All treated plates were incubated at 37 ° C for 24 hours and the diameter of the clear zone was measured.
상기 분리균 1,800개 중에서 가장 항균활성이 우수한 것을 YML 002로 명명하였으며, YML 002의 패니바실러스 유충에 대한 억제 활성을 시간별로 측정하기 위하여 3시간 단위로 배양액을 취하여 항균활성 시험을 하였다.YML 002 was named as the most antimicrobial active among the 1,800 isolates. In order to measure the inhibitory activity of YML 002 against Fanny Bacillus larvae over time, the culture broth was taken every 3 hours for antibacterial activity test.
병원균의 생육 억제를 확인하기 위하여 OD 600nm에서 흡광도를 측정하였고, 21시간째에 항균활성이 가장 우수하였다. 그 결과를 하기 표 1 에 나타내었다.The absorbance at OD 600nm was measured to confirm inhibition of pathogen growth, and the antimicrobial activity was the best at 21 hours. The results are shown in Table 1 below.
<실시예 3> 선발한 미생물 YML002의 동정≪ Example 3 > Identification of the selected microorganism YML002
상기 실시예 2에서 선발된 YML 002 균주를 MRS 배지(Difco사 제품)에서 30℃ 조건으로 배양하였다. YML 002 균주를 동정하기 위하여, 16S rDNA 유전자의 염기서열 결정 및 분석은 1492R과 27F를 프라이머로 사용하였다.The YML 002 strain selected in Example 2 was cultured in an MRS medium (manufactured by Difco) at 30 ° C. In order to identify the YML 002 strain, the nucleotide sequence of the 16S rDNA gene was determined and analyzed using 1492R and 27F as primers.
YML002 16S rRNA 유전자의 염기 서열은 서열번호 1로 기재되며, 16S rDNA 염기서열에 기초한 분자계통분류학적 분석을 통하여 본 발명의 균주는 페디오코커스(Pediococcus) 속에 속하는 균주로서 99%의 16S rDNA 상동성으로 페디오코커스 파버러스(Pediococcus parvulus)의 표준균주에 가장 높은 분자계통학적 유연관계를 보여주는 균주로 동정되었다.The nucleotide sequence of the YML002 16S rRNA gene is shown in SEQ ID NO: 1, and through the molecular phylogenetic analysis based on the 16S rDNA nucleotide sequence, the strain of the present invention is a strain belonging to the genus Pediococcus , and has 99% 16S rDNA homology Was identified as the strain showing the highest molecular phylogenetic relationship to the standard strain of Pediococcus parvulus .
따라서, 상기의 결과에 기초하여 YML 002는 기존의 미생물제제와는 다른 페디오코커스 파버러스(Pediococcus parvulus)의 균주로 동정하고, 한국미생물보존센터에 2010년 2월 1일자로 기탁하였다(수탁번호: KCTC11628BP).Based on the above results, YML 002 was identified as a strain of Pediococcus parvulus different from the existing microorganism preparation and deposited on Feb. 1, 2010 at the Korean Microorganism Conservation Center : KCTC11628BP).
<110> Industry-Academic Cooperation Foundation, Yeungnam University <120> Lactobacillus plantarum YML 002 against pathogen of American Foulbrood, microbial agent comprising the same, and method for preventing pathogen using the same <130> DP-2010-0066 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1480 <212> DNA <213> Artificial Sequence <220> <223> YML002 16S RNA gene <400> 1 nnnnnnnngn ncagctataa tgcagtcgaa cgcactttcg ttgaatgaat tagaggtgct 60 tgcaccgaag atgattttag ctataaagtg agtggcgaac gggtgagtaa cacgtgggta 120 acctgcccag aagtggggga taacacctgg aaacagatgc taataccgca taacaaagta 180 aaccgcatgg tttactttta aaagatggct tcggctatca cttctggatg gacccgcggc 240 gtattagcta gttggtgaga taaaggctca ccaaggcagt gatacgtagc cgacctgaga 300 gggtaatcgg ccacattggg actgagacac ggcccagact cctacgggag gcagcagtag 360 ggaatcttcc acaatggacg aaagtctgat ggagcaacgc cgcgtgagtg atgaaggctt 420 tagggtcgta aaactctgtt gttggagaag aacgtgtgtg agagtaactg ctcatgcagt 480 gacggtatcc aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttatccggat ttattgggcg taaagcgagc gcaggcggtc ttttaagtct 600 aatgtgaaag ccttcggctt aaccgaagaa gtgcattgga aactggaaga cttgagtgca 660 gaagaggaca gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc 720 agtggcgaag gcggctgtct ggtctgtaac tgacgctgag gctcgaaagc atgggtagcg 780 aacaggatta gataccctgg tagtccatgc cgtaaacgat gaatgctaag tgttggaggg 840 tttccgccct tcagtgctgc agctaacgca ttaagcattc cgcctgggga gtacgaccgc 900 aaggttgaaa ctcaaaagaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcta cgcgaagaac cttaccaggt cttgacatct tctgccaatc tagagattag 1020 acgttccctt cgggacagaa tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1080 atgttggggt taagtcccgc acgagcgcaa cccttattat tagttgccag cattaagttg 1140 ggcactctag tgagactgcc ggtgacaaac cggaggaagg tggggacgac gtcaaatcat 1200 catgcccctt atgacctggg ctacacacgt gctacaatgg acggtacaac gagttgcgag 1260 accgcgaggt ttagctaatc tcttaaaacc gttctcagtt cggactgcag gctgcaactc 1320 gcctgcacga agttggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1380 ccgggccttg tacacaccgc ccgtcacacc atgagagttt gtaacaccca aagccggtgg 1440 agtaaccttc gggagctagc cgtntatgtg ncngngnnnn 1480 <110> Industry-Academic Cooperation Foundation, Yeungnam University <120> Lactobacillus plantarum YML 002 against pathogen of American Foulbrood, microbial agent comprising the same, and method for preventing pathogen using the same <130> DP-2010-0066 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1480 <212> DNA <213> Artificial Sequence <220> <223> YML002 16S RNA gene <400> 1 tgcagtcgaa tgcaccgaag atgattttag ctataaagtg agtggcgaac gggtgagtaa cacgtgggta 120 acctgcccag aagtggggga taacacctgg aaacagatgc taataccgca taacaaagta 180 aaccgcatgg tttactttta aaagatggct tcggctatca cttctggatg gacccgcggc 240 gtattagcta gttggtgaga taaaggctca ccaaggcagt gatacgtagc cgacctgaga 300 gggtaatcgg ccacattggg actgagacac ggcccagact cctacgggag gcagcagtag 360 ggaatcttcc acaatggacg aaagtctgat ggagcaacgc cgcgtgagtg atgaaggctt 420 tagggtcgta aaactctgtt gttggagaag aacgtgtgtg agagtaactg ctcatgcagt 480 gacggtatcc aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttatccggat ttattgggcg taaagcgagc gcaggcggtc ttttaagtct 600 aatgtgaaag ccttcggctt aaccgaagaa gtgcattgga aactggaaga cttgagtgca 660 gaagaggaca gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc 720 agtggcgaag gcggctgtct ggtctgtaac tgacgctgag gctcgaaagc atgggtagcg 780 aacaggatta gataccctgg tagtccatgc cgtaaacgat gaatgctaag tgttggaggg 840 tttccgccct tcagtgctgc agctaacgca ttaagcattc cgcctgggga gtacgaccgc 900 aaggttgaaa ctcaaaagaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcta cgcgaagaac cttaccaggt cttgacatct tctgccaatc tagagattag 1020 acgttccctt cgggacagaa tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1080 atgttggggt taagtcccgc acgagcgcaa cccttattat tagttgccag cattaagttg 1140 ggcactctag tgagactgcc ggtgacaaac cggaggaagg tggggacgac gtcaaatcat 1200 catgcccctt atgacctggg ctacacacgt gctacaatgg acggtacaac gagttgcgag 1260 accgcgaggt ttagctaatc tcttaaaacc gttctcagtt cggactgcag gctgcaactc 1320 gcctgcacga agttggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1380 ccgggccttg tacacaccgc ccgtcacacc atgagagttt gtaacaccca aagccggtgg 1440 agtaaccttc gggagctagc cgtntatgtg ncngngnnnn 1480
Claims (8)
A method for controlling American Foulbrood, which comprises adding an antimicrobial microbial preparation according to claim 7 to bees in a beekeeping feed additive or beekeeping medicine to inhibit the Fanny cilius larva, which is a causative microorganism of American buzzers.
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