JP2015513910A - New Bacillus subtilis {NOVELBACILLUSSUBTILIS} - Google Patents
New Bacillus subtilis {NOVELBACILLUSSUBTILIS} Download PDFInfo
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- JP2015513910A JP2015513910A JP2015504498A JP2015504498A JP2015513910A JP 2015513910 A JP2015513910 A JP 2015513910A JP 2015504498 A JP2015504498 A JP 2015504498A JP 2015504498 A JP2015504498 A JP 2015504498A JP 2015513910 A JP2015513910 A JP 2015513910A
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Abstract
本発明は、新たなバチルス・サブチルス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株及びこれを含むプロバイオティクス製剤と、これを活用した家畜疾病の予防又は治療方法に関するものである。The present invention relates to a new Bacillus subtilis CJMPB957 (KCCM11271P) strain, a probiotic preparation containing the same, and a method for preventing or treating livestock diseases using the same.
Description
本発明は、新たなバチルス・サブチルス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株及びそのプロバイオティクス製剤と、これを活用した家畜疾病の予防又は治療方法に関するものである。 The present invention relates to a new Bacillus subtilis CJMPB957 (KCCM11271P) strain and a probiotic preparation thereof, and a method for preventing or treating livestock diseases using the same.
家畜の感染症は、家畜の体重を減少させたり色々な病状を起こさせるなど、商品価値を著しく低下させる。家畜の感染症を起こさせる細菌性疾病には、腸毒性大腸菌（ＥＴＥＣ、Ｅｎｔｅｒｏｔｏｘｉｇｅｎｉｃ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）による下痢病、鳥類病原性大腸菌（ＡＰＥＣ、ａｖｉａｎ ｐａｔｈｏｇｅｎｉｃ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）による大腸菌症（Ｃｏｌｉｂａｉｌｏｓｉｓ）、敗血症、急慢性腸炎を起こさせるサルモネラ症（Ｓａｌｍｏｎｅｌｌｏｓｉｓ）、クロストリジウム属（Ｃｌｏｓｔｒｉｄｉｕｍ ｓｐ．）による壊疽性腸炎などがある。 Livestock infectious diseases significantly reduce commercial value, such as reducing the weight of livestock and causing various medical conditions. Bacterial diseases that cause infectious diseases in livestock include diarrhea caused by enterotoxic Escherichia coli (ETEC), avian pathogenic Escherichia coli, acute septicemia, sepsis Examples include salmonellosis causing enteritis, gangrenous enteritis due to Clostridium sp.
また、飼料の原料である穀類に増殖するアスペルギルス属（Ａｓｐｅｒｇｉｌｌｕｓ ｓｐ．）によるアフラトキシン中毒症（Ａｓｐｅｒｇｉｌｌｏｓｉｓ）は、急性の場合には食欲低下、発熱、呼吸困難、下痢、痙攣などの神経症を起こさせる真菌性の疾病である。 In addition, aspergillosis caused by Aspergillus sp., Which grows in cereals that are feed ingredients, causes neuropathies such as decreased appetite, fever, dyspnea, diarrhea, and convulsions in the acute case. It is a fungal disease.
このような疾病を治療と予防するために多くの抗生剤が開発されてきた。しかし、抗生剤の無分別な使用で耐性菌の出現頻度が増加することにより、抗生剤の使用において疾病治療の目的以外には、疾病予防と生長促進を目的とする飼料への添加は、全面禁止又は規制されている。抗生剤使用の規制によって家畜の生産性低下と斃死率増加の恐れがあり、このような問題点を補える抗生剤の代替剤の開発が非常に急を要している。
最近、このような問題を反映し抗生剤を代替することができる生菌剤の開発研究が行われている。生菌剤に関する先行技術は下記に示す。
Many antibiotics have been developed to treat and prevent such diseases. However, due to an increase in the frequency of the appearance of resistant bacteria due to the indiscriminate use of antibiotics, in addition to the purpose of disease treatment in the use of antibiotics, addition to feed for the purpose of disease prevention and growth promotion is Prohibited or regulated. Antibiotic use regulations may reduce livestock productivity and increase the death rate, and the development of alternatives to antibiotics that can compensate for these problems is extremely urgent.
Recently, research on the development of viable bacteria that can replace antibiotics reflecting such problems has been conducted. Prior art relating to viable agents is shown below.
韓国公開特許第２０１０−００７６２６３号は、子牛の下痢病を予防するための大腸菌、サルモネラ抑制に効果があるラクトバチルス・ファーメンタム（Ｌａｃｔｏｂａｃｉｌｌｕｓ Ｆｅｒｍｅｎｔｕｍ）ＢＬＡＢ１９を開示している。 Korean Published Patent No. 2010-0076263 discloses Lactobacillus Fermentum BLAB19, which is effective in suppressing Escherichia coli and Salmonella to prevent calf diarrhea.
韓国登録特許第１０−０８７８７９９号は、抗生剤に耐性を有する病原性微生物、又は腸内有害微生物であるエドワードシエラ・タルダ（Ｅｄｗａｒｄｓｉｅｌｌａ ｔａｒｄａ）、大腸菌と表皮ブドウ球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｅｐｉｄｅｒｍｉｄｉｓ）の生長を抑制するバチルス・アミロリケファシエンス（Ｂａｃｉｌｌｕｓ ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ）Ｋ３７を開示している。 Korean Registered Patent No. 10-0878799 suppresses the growth of pathogenic microorganisms resistant to antibiotics or enteric harmful microorganisms Edwardsiella tarda, Escherichia coli and Staphylococcus epidermidis Bacillus amyloliquefaciens K37 is disclosed.
韓国公開特許第２００６−００２６３７１号には、子豚の下痢を誘発する大腸菌を抑制する乳酸菌（Ｌａｃｔｏｂａｃｉｌｌｕｓ Ｆｅｒｍｅｎｔｕｍ １２−１、ＫＡＣＣ９１１３５）を開示されている。 Korean Patent No. 2006-0026371 discloses a lactic acid bacterium (Lactobacillus Fermentum 12-1, KACC91135) that suppresses Escherichia coli that induces diarrhea in piglets.
しかし、上述の先行技術は、養鶏又は養豚において問題となっている大腸菌、サルモネラ又はクロストリジウムに対する抗菌力を有する菌株を開示するだけで、飼料の材料である穀類で増殖し、これを摂取した家畜にアフラトキシン中毒症などを誘発するアスペルギルスに対する抗真菌力を共に有する菌株に関しては報告されたことがない。 However, the above-described prior art only discloses a strain having antibacterial activity against Escherichia coli, Salmonella or Clostridium, which is a problem in poultry or pig farming. No strain has been reported that has both antifungal activity against Aspergillus that induces aflatoxin poisoning and the like.
本発明は、抗菌及び抗真菌活性に優れ、複合消化酵素の生産能、耐酸性及び耐胆汁性が優秀な、バチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）の提供を目的とする。 An object of the present invention is to provide Bacillus subtilis CJMPB957 (KCCM11271P) which is excellent in antibacterial and antifungal activities and has excellent ability to produce complex digestive enzymes, acid resistance and bile resistance.
また、本発明は、前記バチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株の培養物の提供を目的とする。 Another object of the present invention is to provide a culture of the Bacillus subtilis CJMPB957 (KCCM11271P) strain.
また、本発明は、前記バチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株又はその培養物を含む、プロバイオティクス製剤、飼料添加剤及び飼料の提供を目的とする。 Another object of the present invention is to provide a probiotic preparation, a feed additive, and a feed containing the Bacillus subtilis CJMPB957 (KCCM11271P) strain or a culture thereof.
また、本発明は、前記バチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株、又はその培養物を家畜に投与することで、家畜の細菌性、又は真菌性の疾病を予防又は治療する方法の提供を目的とする。 Another object of the present invention is to provide a method for preventing or treating livestock bacterial or fungal diseases by administering the Bacillus subtilis CJMPB957 (KCCM11271P) strain or a culture thereof to livestock. .
本発明は、新たに分離したバチルス・サブチルス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株及びこれを含むプロバイオティクス製剤と、これを活用した家畜疾病の予防又は治療方法に関するものである。 The present invention relates to a newly isolated Bacillus subtilis CJMPB957 (KCCM11271P) strain, a probiotic preparation containing the same, and a method for preventing or treating livestock diseases using the same.
本発明のある実施様態において、抗菌及び抗真菌活性を有し、複合消化酵素の生産能、耐酸性及び耐胆汁性を有する、新たなバチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株が提供される。 In one embodiment of the present invention, a new Bacillus subtilis CJMPB957 (KCCM11271P) strain is provided that has antibacterial and antifungal activities and has the ability to produce complex digestive enzymes, acid resistance and bile resistance.
本発明のさらなる実施様態において、前記バチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株の培養物が提供される。 In a further embodiment of the invention there is provided a culture of said Bacillus subtilis CJMPB957 (KCCM11271P) strain.
本発明のさらなる実施様態において、前記バチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株、又は前記菌株の培養物を含む、プロバイオティクス（Ｐｒｏｂｉｏｔｉｃｓ）製剤が提供される。 In a further embodiment of the invention there is provided a Probiotic formulation comprising said Bacillus subtilis CJMPB957 (KCCM11271P) strain, or a culture of said strain.
本発明のさらなる実施様態において、前記プロバイオティクス製剤を含む飼料添加剤が提供される。
本発明のさらなる実施様態において、前記飼料添加剤を含む飼料が提供される。
In a further embodiment of the invention, a feed additive comprising the probiotic formulation is provided.
In a further embodiment of the invention, a feed comprising the feed additive is provided.
本発明のさらなる実施様態において、前記プロバイオティクス製剤、飼料添加剤、及び飼料からなる群より選択される少なくとも一つを家畜に投与する段階を含む、家畜の細菌性又は真菌性の疾病を予防又は治療する方法が提供される。 In a further embodiment of the present invention, prevention of bacterial or fungal diseases in livestock, comprising the step of administering to livestock at least one selected from the group consisting of the probiotic preparation, feed additive, and feed Alternatively, a method of treatment is provided.
本発明は、抗菌及び抗真菌活性に優れ、複合消化酵素の生産能、耐酸性及び耐胆汁性が優秀なバチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株、又はその培養物を含む、プロバイオティクス、飼料添加剤及び飼料を提供する。これを家畜に投与し家畜の細菌性又は真菌性の疾病を予防又は治療する方法を提供することで、家畜の疾病管理及び消化力向上と腸内微生物に均衡維持を助け、家畜用飼料を効率を増進することにより畜産業発展に寄与する効果がある。 The present invention relates to a Bacillus subtilis CJMPB957 (KCCM11271P) strain excellent in antibacterial and antifungal activities, and capable of producing a complex digestive enzyme, excellent in acid resistance and bile resistance, or a culture thereof, including a probiotic and feed addition Provide food and feed. By providing this to livestock and providing a method for preventing or treating bacterial or fungal diseases in livestock, it will help improve the disease management and digestibility of livestock and maintain a balance with intestinal microorganisms, and improve livestock feed efficiency. It has the effect of contributing to livestock industry development.
下記において本発明に関してより詳しく説明する。本明細書に記載のない内容は、本発明の技術分野又は類似分野の熟練者なら十分に認識と類推できるものであり、その説明を省略する。 In the following, the present invention will be described in more detail. The contents not described in this specification can be sufficiently recognized and analogized by those skilled in the technical field of the present invention or similar fields, and the description thereof will be omitted.
本発明のある実施様態において、新たに分離したバチルス・サブチルス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）（以下、‘ＣＪＭＰＢ９５７’）菌株が提供される。 In one embodiment of the present invention, a newly isolated Bacillus subtilis CJMPB957 (KCCM11271P) (hereinafter 'CJMPB957') strain is provided.
前記ＣＪＭＰＢ９５７は、形態学的にグラム陽性桿菌に該当し（図１参照）、１６ｓ ｒＤＮＡ塩基配列（配列番号１、図８）の分析結果、バチルス・サブチルスと９８％の相同性を有する菌株である。 The CJMPB957 is morphologically a Gram-positive bacilli (see FIG. 1) and is a strain having 98% homology with Bacillus subtilis as a result of analysis of the 16s rDNA base sequence (SEQ ID NO: 1, FIG. 8). .
前記ＣＪＭＰＢ９５７は２０１２年３月２２日に、韓国微生物保存センター（Ｋｏｒｅａｎ Ｃｕｌｔｕｒｅ Ｃｅｎｔｅｒ ｏｆ Ｍｉｃｒｏｏｒｇａｎｉｓｍｓ、ソウル市西大門区弘濟１洞３６１−２２１）に寄託番号ＫＣＣＭ１１２７１Ｐとして寄託された。 The CJMPB957 was deposited on March 22, 2012 at the Korean Culture Center of Microorganisms (Korean Culture Center of Microorganisms, Seoul, Dongdaemun-gu, Hongdae-dong 1-361-221) under the deposit number KCCM11271P.
前記ＣＪＭＰＢ９５７は、複合抗菌及び抗真菌活性がある。
前記ＣＪＭＰＢ９５７は、抗菌又は抗真菌活性の対象菌として、アスペルギルス属（Ａｓｐｅｒｇｉｌｌｕｓ ｓｐ．）を含み、腸毒性大腸菌（ＥＴＥＣ、Ｅｎｔｅｒｏｔｏｘｉｇｅｎｉｃ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）、鳥類病原性大腸菌（ＡＰＥＣ、ａｖｉａｎ ｐａｔｈｏｇｅｎｉｃ Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）、サルモネラ属（Ｓａｌｍｏｎｅｌｌａ ｓｐ．）及びクロストリジウム属（Ｃｌｏｓｔｒｉｄｉｕｍ ｓｐ．）からなる群より選ばれる少なくとも一つを含む。
The CJMPB957 has combined antibacterial and antifungal activities.
The CJMPB957 includes Aspergillus sp. As a target bacterium having antibacterial or antifungal activity, enterotoxic Escherichia coli (ETEC, Enterotoxigenic Escherichia coli), avian pathogenic E. coli (APEC), avian pathogenic Escherichia coli (Salmonella sp.) And at least one selected from the group consisting of Clostridium sp.
前記ＣＪＭＰＢ９５７は、好ましくはアスペルギルス属、腸毒性大腸菌、鳥類病原性大腸菌、サルモネラ属及びクロストリジウム属の全てに対する抗菌又は抗真菌活性を表すことができる。 Said CJMPB957 can preferably exhibit antibacterial or antifungal activity against all of the genus Aspergillus, enterotoxic E. coli, avian pathogenic E. coli, Salmonella and Clostridium.
前記ＣＪＭＰＢ９５７は、複合消化酵素の生産能を有する。
前記ＣＪＭＰＢ９５７の生産できる消化酵素は、プロテアーゼ（Ｐｒｏｔｅａｓｅ）、セルラーゼ（Ｃｅｌｌｕｌａｓｅ）、アミラーゼ（Ａｍｙｌａｓｅ）、キシラナーゼ（Ｘｙｌａｎａｓｅ）、マンナナーゼ（Ｍａｎｎａｎａｓｅ）、リパーゼ（Ｌｉｐａｓｅ）及びフィターゼ（Ｐｈｙｔａｓｅ）からなる群より選ばれる少なくとも一つである。
The CJMPB957 has the ability to produce complex digestive enzymes.
The digestive enzyme capable of producing CJMPB957 is at least selected from the group consisting of protease, cellulase, amylase, xylanase, mannanase, lipase, and phytase. One.
前記ＣＪＭＰＢ９５７は、より好ましくは、プロテアーゼ、セルラーゼ、アミラーゼ、キシラナーゼ、マンナナーゼ、リパーゼ及びフィターゼを全て生産することができる。 More preferably, the CJMPB957 can produce all of protease, cellulase, amylase, xylanase, mannanase, lipase and phytase.
前記ＣＪＭＰＢ９５７に耐熱性に非常に優れている。
より具体的に、前記ＣＪＭＰＢ９５７は、３７℃、２００ｒｐｍの条件で２４時間培養した後、９５℃で１０分間熱処理した後の内生胞子の形成率が、好ましくは９５％以上、より好ましくは１００％である。
The CJMPB957 is very excellent in heat resistance.
More specifically, the CJMPB957 has an endospore formation rate of preferably 95% or more, more preferably 100% after culturing at 37 ° C. and 200 rpm for 24 hours and then heat treating at 95 ° C. for 10 minutes. It is.
前記内生胞子は、高温条件の環境だけでなく、紫外線、低音、乾燥、及び／又は、高圧などの極限環境に抵抗性を有し、内生胞子形成率が高いほど菌株の生存率が高くなる。 The endospores are resistant not only to high-temperature conditions but also to extreme environments such as ultraviolet rays, bass, dryness, and / or high pressure. The higher the endospore formation rate, the higher the survival rate of the strain. Become.
前記ＣＪＭＰＢ９５７は、耐酸性に非常に優れている。
より具体的に、前記ＣＪＭＰＢ９５７は、ｐＨ２．５に調整した溶液にペプシン（Ｐｅｐｓｉｎ）を１％（ｗ／ｖ）で添加して製造された人工胃液を含む培地で、３時間培養した後の生存率が、好ましくは９５％以上、より好ましくは１００％である。
The CJMPB957 is very excellent in acid resistance.
More specifically, the CJMPB957 is a survival medium after being cultured for 3 hours in a medium containing artificial gastric juice prepared by adding 1% (w / v) pepsin to a solution adjusted to pH 2.5. The rate is preferably 95% or more, more preferably 100%.
前記ＣＪＭＰＢ９５７は、耐胆汁性に非常に優れている。
より具体的に、前記ＣＪＭＰＢ９５７は１％（ｗ／ｖ）のパンクレアチン（Ｐａｎｃｒｅａｔｉｎ）が含まれた人工胆汁を含有する培地で、３時間培養した後の生存率が、好ましくは９５％以上、より好ましくは１００％である。
The CJMPB957 is very excellent in bile resistance.
More specifically, the CJMPB957 is a medium containing artificial bile containing 1% (w / v) pancreatin, and the survival rate after culturing for 3 hours is preferably 95% or more. Preferably it is 100%.
本発明の新たに分離した菌株ＣＪＭＰＢ９５７は、バチルス菌株の通常的な培養方法により培養することができる。前記ＣＪＭＰＢ９５７は、好ましくは２０ないし４０℃の培養温度で、１２時間ないし４日間培養して得ることができる。 The newly isolated strain CJMPB957 of the present invention can be cultured by a conventional method for culturing Bacillus strains. The CJMPB957 can be obtained by culturing at a culture temperature of preferably 20 to 40 ° C. for 12 hours to 4 days.
本発明のさらなる実施様態において、前記ＣＪＭＰＢ９５７の培養物が提供される。
前記培養物は、前記ＣＪＭＰＢ９５７菌株を培養した培養培地又は培養液、及び前記培養培地又は培養液で培養したその結果物、を含む概念であり、前記培養物は前記ＣＪＭＰＢ９５７菌株の含有有無は適宜選択できる。
In a further embodiment of the invention, a culture of said CJMPB957 is provided.
The culture is a concept including a culture medium or a culture solution obtained by culturing the CJMPB957 strain and a result obtained by culturing the culture medium or the culture solution, and the culture is appropriately selected for the presence or absence of the CJMPB957 strain. it can.
前記培養物の剤形は特に限定されず、当該技術分野において通常的に用いられる剤形であり得る。例えば、前記培養物は、液体又は固体であり、培養物の原形状態又はその濃縮形態・乾燥形態であり得る。 The dosage form of the culture is not particularly limited, and may be a dosage form commonly used in the art. For example, the culture may be liquid or solid, and may be in the original form of the culture or in a concentrated / dried form thereof.
培養培地
本発明のＣＪＭＰＢ９５７を培養する培地は、天然培地、又は合成培地であり得る。
Culture Medium The medium for culturing CJMPB957 of the present invention can be a natural medium or a synthetic medium.
前記培地の炭素原は、特に限りはなく、当該技術分野において公知のものが用いられる。前記炭素原の非制限的例として、グルコース、スクロース、デキストリン、グリセロール、又はデンプンなどを挙げることができる。これらは一つ、又は二つ以上を混合して用いることができる。 The carbon source of the medium is not particularly limited, and those known in the art can be used. Non-limiting examples of the carbon source include glucose, sucrose, dextrin, glycerol, starch and the like. These can be used alone or in combination of two or more.
前記培地の窒素原は、特に限りはなく、当該技術分野において公知のものを用いることができる。前記窒素原の非制限的例としてペプトン、肉類抽出物、酵母抽出物、乾燥された酵母、大豆、アンモニウム塩、硝酸塩、及びその他の有機・無機窒素含有化合物などを挙げることができる。これらは一つ、又は二つ以上を混合して用いることができる。 The nitrogen source of the medium is not particularly limited, and those known in the art can be used. Non-limiting examples of the nitrogen source include peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate, and other organic / inorganic nitrogen-containing compounds. These can be used alone or in combination of two or more.
前記培地に含まれる無機塩は、特に限りはなく、当該技術分野において公知のものを用いることができる。前記無機塩の非制限的例として、マグネシウム、マンガン、カルシウム、鉄、カルシウムなどを挙げることができる。これらは一つ、又は二つ以上を混合して用いることができる。 The inorganic salt contained in the medium is not particularly limited, and those known in the art can be used. Non-limiting examples of the inorganic salt include magnesium, manganese, calcium, iron, calcium and the like. These can be used alone or in combination of two or more.
本発明のＣＪＭＰＢ９５７の培養培地には、前記炭素原、窒素原及び無機塩成分の他に、アミノ酸、ビタミン、核酸、及び／又は、その他一般的に培養培地に含まれる成分を添加することができる。 In addition to the carbon source, nitrogen source and inorganic salt components, amino acids, vitamins, nucleic acids and / or other components generally contained in the culture media can be added to the CJMPB957 culture medium of the present invention. .
培養液
本発明のＣＪＭＰＢ９５７菌株の培養液は、培養原液であることもできるし、前記培養原液から培養上澄液を除去した液、及び／又は、それの濃縮液であり得る。前記培養液には前記ＣＪＭＰＢ９５７菌株を含むこともできる。
Culture Solution The culture solution of the CJMPB957 strain of the present invention can be a culture stock solution, a solution obtained by removing the culture supernatant from the culture stock solution, and / or a concentrated solution thereof. The culture solution may contain the CJMPB957 strain.
前記培養液の組成は、特に限りはなく、通常的にバチルス菌株の培養において必要であると知られた成分だけでなく、バチルスの生長に上昇的に作用する成分をされに含むこともでき、それによる組成は当該技術分野において通常の知識を有する者により容易に選択できる。 The composition of the culture solution is not particularly limited, and not only components that are usually known to be necessary in the culture of Bacillus strains, but also components that act to increase the growth of Bacillus can be included. The composition thereby can be easily selected by those having ordinary knowledge in the art.
前記培養液は、液状状態、又は乾燥状態であり得る。
前記培養液の乾燥方法は、特に限りはなく、当該技術分野で通常的に用いる方法が利用できる。前記乾燥方法の非制限的例として、通風乾燥、自然乾燥、噴霧乾燥、又は凍結乾燥などを挙げることができる。これらは一つ、又は二つ以上の方法を併合して用いることができる。
The culture solution may be in a liquid state or a dry state.
The method for drying the culture solution is not particularly limited, and a method commonly used in the art can be used. Non-limiting examples of the drying method include ventilation drying, natural drying, spray drying, freeze drying, and the like. These can be used singly or in combination of two or more methods.
本発明のさらなる実施様態において、前記ＣＪＭＰＢ９５７菌株、又は前記菌株の培養物を含む、プロバイオティクス製剤が提供される。 In a further embodiment of the invention there is provided a probiotic formulation comprising said CJMPB957 strain or a culture of said strain.
前記プロバイオティクス（Ｐｒｏｂｉｏｔｉｃｓ）は、人間や動物などの宿主の健康に有益な効果を表す微生物、又はその成分の意味であり、宿主の腸内の消化管の壁に定着し他の有害菌の定着、及び病原菌の増殖を抑制し、プロバイオティクスが生産する有益な消化酵素は栄養素の吸収と利用を容易にすることと知られている。 Probiotics means microorganisms that exhibit beneficial effects on the health of the host, such as humans and animals, or components thereof, and colonize the walls of the digestive tract in the intestine of the host to prevent other harmful bacteria. The beneficial digestive enzymes produced by probiotics that suppress colonization and pathogen growth are known to facilitate the absorption and utilization of nutrients.
本発明のプロバイオティクス製剤は、前記ＣＪＭＰＢ９５７菌株、及び／又は、前記菌株の培養物を含むものであり得る。 The probiotic preparation of the present invention may contain the CJMPB957 strain and / or a culture of the strain.
本発明のプロバイオティクスは、好ましくは前記ＣＪＭＰＢ９５７菌株を５×１０４ないし５×１０１０ＣＦＵ／ｍｌ、より好ましくは１×１０６ないし１×１０９ＣＦＵ／ｍｌ含むことができる。 The probiotic of the present invention may preferably contain 5 × 10 4 to 5 × 10 10 CFU / ml, more preferably 1 × 10 6 to 1 × 10 9 CFU / ml of the CJMPB957 strain.
前記プロバイオティクス製剤は、薬学的に許容される担体をさらに含むことができ、前記担体と共に製剤化されて提供できる。 The probiotic preparation can further include a pharmaceutically acceptable carrier, and can be provided by being formulated with the carrier.
前記薬学的に許容される担体は、生物体を刺激せず投与化合物の生物学的活性・特性を阻害しない担体、又は希釈剤を意味する。 The pharmaceutically acceptable carrier means a carrier or diluent that does not irritate the organism and does not inhibit the biological activity / characteristics of the administered compound.
前記担体において、液状溶液として製剤化されるプロバイオティクス製剤に利用できる担体として、滅菌と生体に適合するものは、食塩水、滅菌水、緩衝食塩水、アルブミン注射溶液、デキストロース溶液、マルトデキストリン溶液、又はグリセロールなどを挙げることができる。これらは一つ、又は二つ以上を混合して用いることもできるし、必要に応じて抗酸化剤、緩衝液、及び／又は、静菌剤などの他の通常の添加剤を添加することもできる。 Among the above carriers, those that are compatible with sterilization and living body can be used for probiotic preparations formulated as liquid solutions, including saline, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution. Or glycerol. These can be used alone or in combination of two or more, and other usual additives such as antioxidants, buffers and / or bacteriostatic agents can be added as necessary. it can.
また、希釈剤、分散剤、界面活性剤、結合剤、及び／又は、潤滑剤を付加的に添加し、水溶液、懸濁液、乳濁液などの注射用剤形、丸薬、カプセル、顆粒、又は錠剤に製剤化することができる。 In addition, diluents, dispersants, surfactants, binders, and / or lubricants are additionally added, and dosage forms for injection such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, Or it can be formulated into tablets.
本発明のプロバイオティクス製剤を有効成分として含む経口投与用の剤形は、特に限りはなく、当該技術分野で知られている剤形を用いることができる。前記経口投与用剤形の非制限的例として、錠剤、トローチ、ローゼンジ（ｌｏｚｅｎｇｅ）、水溶性又は油性懸濁液、調剤粉末、又は顆粒、エマルション、ハード又はソフトカプセル、シロップ、又はエリキシル剤などを挙げることができる。 The dosage form for oral administration containing the probiotic preparation of the present invention as an active ingredient is not particularly limited, and a dosage form known in the art can be used. Non-limiting examples of dosage forms for oral administration include tablets, troches, lozenges, water-soluble or oily suspensions, dispensing powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. be able to.
前記錠剤又はカプセルなどの剤形として製剤化するため、ラクトース、サッカロース、ソルビトール、マンニトール、澱粉、アミロペクチン、セルロース、又はゼラチンなどの結合剤、ジカルシウムホスフェートなどの賦形剤、トウモロコシ澱粉、又はサツマイモ澱粉などの崩壊剤、ステアリン酸マグネシウム、ステアリン酸カルシウム、ナトリウムステアリルフマラート、又はポリエチレングリコールワックスなどの潤滑油などを含むことができ、カプセル剤形の場合は上述した物質以外にも脂肪油などの液体担体をさらに含むことができる。 Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin, excipients such as dicalcium phosphate, corn starch, or sweet potato starch for formulation as a dosage form such as the tablet or capsule Disintegrating agents such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or a lubricating oil such as polyethylene glycol wax. In the case of capsule dosage forms, liquid carriers such as fatty oils in addition to the substances mentioned above Can further be included.
本発明のさらなる実施様態において、前記プロバイオティクス製剤を含む飼料添加剤が提供される。 In a further embodiment of the invention, a feed additive comprising the probiotic formulation is provided.
本発明のＣＪＭＰＢ９５７菌株、及び／又は、その培養物が含まれたプロバイオティクス製剤は、飼料添加剤の形態で別途に製造して家畜飼料に混合すること、又は飼料製造のとき前記プロバイオティクス製剤を直接添加する方法で混合することができる。 The probiotic preparation containing the CJMPB957 strain and / or culture thereof of the present invention is separately produced in the form of a feed additive and mixed with livestock feed, or the probiotic at the time of feed production It can mix by the method of adding a formulation directly.
前記飼料添加剤の形態は、特に限りはなく、好ましくは液状、又は乾燥状態であり、より好ましくは乾燥粉末形態であり得る。 The form of the feed additive is not particularly limited, and is preferably in a liquid or dry state, and more preferably in a dry powder form.
前記乾燥粉末形のプロバイオティクス製剤の乾燥方法は、特に限りはなく、当該技術分野での公知方法を利用することができる。前記乾燥方法の非制限的例として、通風乾燥、自然乾燥、噴霧乾燥、又は凍結乾燥などを挙げることができる。これらは一つ、又は二つ以上の方法を合わせて利用することができる。 The drying method of the dry powder type probiotic preparation is not particularly limited, and a known method in this technical field can be used. Non-limiting examples of the drying method include ventilation drying, natural drying, spray drying, freeze drying, and the like. These can be used singly or in combination of two or more methods.
前記飼料添加剤は、本発明のＣＪＭＰＢ９５７、及び／又は、その培養物を含む、プロバイオティクス製剤の他に飼料の保存性を高められる他の添加剤をさらに含むことができる。 In addition to the probiotic preparation, the feed additive may further include other additives capable of enhancing the storage stability of the feed, including the CJMPB957 of the present invention and / or its culture.
本発明の飼料添加剤にさらに含められる添加剤は、特に限りはなく、当該技術分野で知られたもとを利用することができる。前記添加剤の非制限的例として、飼料添加剤の品質低下を防止する決着剤、乳化剤、保存剤など；効用を増大させるアミノ酸剤、ビタミン剤、酵素剤、香味剤、非タンパク質態窒素化合物、ケイ酸塩剤、緩衝剤、抽出剤、オリゴ糖などがあり、その他にも飼料混合剤などをさらに含むことができる。これらは一つ、又は二つ以上を混合して用いることができる。 The additive further included in the feed additive of the present invention is not particularly limited, and those known in the art can be used. Non-limiting examples of the additives include deciding agents, emulsifiers, preservatives, etc. that prevent the quality of feed additives from degrading; amino acid agents, vitamin agents, enzyme agents, flavoring agents, non-protein nitrogen compounds that increase their utility, There are a silicate agent, a buffer agent, an extractant, an oligosaccharide, and the like, and a feed mixture can be further included. These can be used alone or in combination of two or more.
本発明のプロバイオティクス製剤、又は飼料添加剤は、動物に単独で投与されることもできるし、食用担体の中に他の飼料添加剤を組み合わせて投与されることもできる。また、トップ・ドレス、家畜飼料への直接混合、飼料とは別に経口剤形での投与、又は他成分と組合わせて投与することもできる。 The probiotic formulation or feed additive of the present invention can be administered to an animal alone, or can be administered in combination with other feed additives in an edible carrier. It can also be administered in a top dress, directly mixed with livestock feed, administered in an oral dosage form separately from the feed, or in combination with other ingredients.
本発明のさらなる実施様態において、前記飼料添加剤を含む飼料が提供される。
本発明の前記飼料は、前記飼料添加剤を、好ましくは飼料１００重量部に対して０．０５ないし１０重量部、より好ましくは０．１ないし１重量部で含むことができる。前記の範囲内で家畜の消化力を効果的に増進させ飼料の効率を高めることができる。
In a further embodiment of the invention, a feed comprising the feed additive is provided.
The feed of the present invention may contain the feed additive in an amount of preferably 0.05 to 10 parts by weight, more preferably 0.1 to 1 part by weight with respect to 100 parts by weight of the feed. Within the above range, the digestive power of livestock can be effectively increased and the efficiency of feed can be increased.
本発明の前記飼料の成分は、特に限りはなく、当該技術分野で知られたものであり得る。前記飼料成分の非制限的例として、植物性として、穀物類、根果類、食品加工副産物類、藻類、繊維質類、油脂類、澱粉類、フクベ類、穀物副産物類など；動物性として、タンパク質類、無機物類、油脂類、鉱物性類、油脂類、単細胞タンパク質、動物性プランクトン類、魚粉などを挙げることができる。これらは一つ、又は二つ以上を混合して用いることができる。 The ingredients of the feed of the present invention are not particularly limited, and may be those known in the art. Non-limiting examples of the feed ingredients include plants, cereals, roots, food processing by-products, algae, fibers, fats, starches, fuchsia, cereal by-products, etc .; Examples include proteins, inorganic substances, fats and oils, minerals, fats and oils, single-cell proteins, zooplanktons, and fish meal. These can be used alone or in combination of two or more.
本発明のプロバイオティクス製剤、飼料添加剤、又は前記飼料添加剤が含まれた飼料が使用できる家畜は、例えば、食用牛、乳牛、子牛、豚、子豚、羊、ヤギ、馬、ウサギ、犬、猫などの家畜、及びひよこ、卵鶏、家庭用ニワトリ、雄鶏、アヒル、ガチョウ、七面鳥、ウズラなどの家禽類が含まれるが、これらに限らない。 Examples of livestock that can use the probiotic preparation, feed additive, or feed containing the feed additive of the present invention include, for example, edible cows, dairy cows, calves, pigs, piglets, sheep, goats, horses, and rabbits. , Domestic animals such as dogs and cats, and poultry such as but not limited to chicks, hens, domestic chickens, roosters, ducks, geese, turkeys and quails.
本発明のさらなる実施様態において、
本発明のＣＪＭＰＢ９５７菌株、及び／又は、前記菌株の培養物を含むプロバイオティクス製剤、前記プロバイオティクス製剤を含む飼料添加剤、及び前記飼料添加剤を含む飼料からなる群より選ばれる少なくとも一つを家畜に投与する段階を含む、家畜の細菌性、又は真菌性の疾病を予防と治療する方法が提供される。
In a further embodiment of the invention,
CJMPB957 strain of the present invention and / or at least one selected from the group consisting of a probiotic preparation containing a culture of the strain, a feed additive containing the probiotic preparation, and a feed containing the feed additive A method of preventing and treating bacterial or fungal diseases in livestock is provided, comprising the step of administering to a livestock.
前記“予防”は、前記ＣＪＭＰＢ９５７菌株、及び／又は、前記菌株の培養物を対象動物に薬学的に製剤形態で、又は飼料添加剤などの摂食できる形態で提供し、当該疾病を抑制させたり発病を遅延させる全ての行為をを意味する。 The “prevention” provides the CJMPB957 strain and / or a culture of the strain to a target animal in a pharmaceutical form or in a form that can be consumed such as a feed additive to suppress the disease. It means all actions that delay the onset.
前記、“治療”は、前記ＣＪＭＰＢ９５７菌株、及び／又は、前記菌株の培養物を対象動物に薬学的に製剤形態で、又は飼料添加剤などの摂食できる形態で提供し、既に感染された当該疾病の病的状態を改善する全ての行為を意味する。 The “treatment” provides the CJMPB957 strain and / or a culture of the strain to a target animal in a pharmaceutical preparation form or a feedable form such as a feed additive. It means all actions that improve the morbidity of the disease.
前記細菌性疾病は、例えば、腸毒性大腸菌による下痢病、鳥類病原性大腸菌による大腸菌症（Ｃｏｌｉｂａｉｌｏｓｉｓ）、敗血症、急慢性腸炎を起こさせるサルモネラ症（Ｓａｌｍｏｎｅｌｌｏｓｉｓ）、クロストリジウム属（Ｃｌｏｓｔｒｉｄｉｕｍ ｓｐ．）による壊疽性腸炎などがあるが、これらに限らない。 Examples of the bacterial diseases include diarrhea caused by enterotoxic Escherichia coli, colitis caused by avian pathogenic Escherichia coli, sepsis, salmonellosis causing acute enterocolitis, and gangrenous caused by Clostridium sp. Examples include, but are not limited to enteritis.
前記真菌性の疾病は、例えば、アスペルギルス属による食欲低下、発熱、呼吸困難、下痢、痙攣などを起こさせるアフラトキシン中毒症（Ａｓｐｅｒｇｉｌｌｏｓｉｓ）があるが、これらに限らない。 Examples of the fungal disease include, but are not limited to, aspergillosis that causes decreased appetite, fever, dyspnea, diarrhea, convulsions and the like due to Aspergillus.
前記プロバイオティクス製剤は、薬学的製剤の形態で動物に投与するか、家畜の飼料、又は飲み水に混合され摂食させる方法により投与することができ、より好ましくは飼料添加剤に形態で飼料に混合されて投与することができる。 The probiotic preparation can be administered to an animal in the form of a pharmaceutical preparation, or can be administered by a method of feeding a livestock feed or drinking water, more preferably a feed additive in the form of a feed additive. Can be mixed and administered.
前記プロバイオティクス製剤の投与経路は、目的組織まで達することができる限り特に限定されず、経口・非経口の多様な経路を通じて投与することができ、具体的に、口腔、直腸、局所、静脈内、腹腔内、筋肉内、動脈内、経皮、鼻側内、吸入などの通常的方法により投与することができる。 The route of administration of the probiotic preparation is not particularly limited as long as it can reach the target tissue, and can be administered through various oral and parenteral routes, specifically, oral, rectal, topical, intravenous Intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, and inhalation can be used for administration.
下記で、実施例を記述することにより、本発明をより詳しく説明する。ただし、下記の実施例は本発明の一つの例示に過ぎなく、本発明の内容がこれらに限定して解釈されることではない。 In the following, the present invention will be described in more detail by describing examples. However, the following embodiment is merely an example of the present invention, and the content of the present invention is not construed as being limited thereto.
実施例１
バチルス・サブチルス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＣＪＭＢＰ９５７菌株の分離
（１）試料の用意と菌株の分離
伝統発酵食品であるメジュと各種醤類の試料を用意し、前記試料を段階的に希釈して３％塩化ナトリウムが添加されたＢＨＩ固体培地（Ｄｉｆｃｏ、ＵＳＡ）に塗抹した後、３７℃の条件で２４時間培養した。
Example 1
Isolation of Bacillus subtilis CJMBP957 strain (1) Preparation of sample and isolation of strain Prepare a sample of traditional fermented foods, mash and various soy sauces, and gradually dilute the sample with 3% sodium chloride. After smearing on a BHI solid medium (Difco, USA) to which was added, the cells were cultured at 37 ° C. for 24 hours.
前記各試料から分離された菌株はコロニー観察によりグループ化し、菌株を分離した。選別されたコロニーは３回にわたって新しい培地に移し培養する方法により純粋分離し、純粋培養された菌を２０％グリセロール（ｇｌｙｃｅｒｏｌ）が添加された培地に入れ、−７０℃以下で保存した。 The strains separated from each sample were grouped by colony observation, and the strains were separated. The selected colonies were transferred to a new medium three times and purified by a method of culturing. The purely cultured bacteria were placed in a medium supplemented with 20% glycerol and stored at −70 ° C. or lower.
（２）複合抗菌活性の優秀な菌株を選抜
養鶏と養豚において問題となる代表的病原性細菌である大腸菌とサルモネラに対する抗菌活性がある菌株を１次選抜するために分離された醤類由来菌に対し、腸毒性大腸菌（ＥＴＥＣ）、鳥類病原性大腸菌（ＡＰＥＣ）、サルモネラ・ティフィムリウム（ＳＴ、Ｓａｌｍｏｎｅｌｌａ ｔｙｐｈｉｍｕｒｉｕｍ）の３種に対する抗菌評価を行った。前記抗菌評価は病原性菌に対する生育阻害環（ｃｌｅａｒ ｚｏｎｅ）分析により評価した。
(2) Selection of excellent strains with combined antibacterial activity In order to primarily select strains with antibacterial activity against Escherichia coli and Salmonella, which are typical pathogenic bacteria that are problematic in poultry and pig farming, On the other hand, antibacterial evaluation was carried out against three types of enterotoxic Escherichia coli (ETEC), avian pathogenic Escherichia coli (APEC), and Salmonella typhimurium (ST). The antibacterial evaluation was evaluated by a clear zone analysis for pathogenic bacteria.
ＹＭ（Ｄｉｆｃｏ、ＵＳＡ）固体培地製造のとき、前記病原性細菌３種の振盪培養液を各々０．４％混合し、抗菌評価培地を製造した。製造された前記評価培地上に醤類由来菌の培養液１．５μｌを滴下し３７℃で１８時間培養したあと、選抜菌株の周囲に形成される生育阻害環の有無をを観察した。醤類由来菌の中で前記病原性細菌３種に対して抗菌性を表す菌株１３種を１次的に選抜した。 When producing a solid medium of YM (Difco, USA), 0.4% of each of the three pathogenic bacteria shaking cultures was mixed to prepare an antibacterial evaluation medium. After 1.5 μl of a culture solution of soy sauce-derived bacteria was dropped onto the produced evaluation medium and cultured at 37 ° C. for 18 hours, the presence or absence of a growth inhibition ring formed around the selected strain was observed. Among the soy-derived bacteria, 13 strains exhibiting antibacterial activity against the three pathogenic bacteria were primarily selected.
複合抗菌活性が優秀な、１次選抜された１３種の菌株に対し、腸毒性大腸菌３種とクロストリジウム・パーフリンゲンス（Ｃｌｏｓｔｒｉｄｉｕｍ ｐｅｒｆｒｉｎｇｅｎｓ）に対する抗菌活性を評価した。腸毒性大腸菌は、Ｋ８８吸着繊毛（ａｄｈｅｒｅｎｃｅ ｐｉｌｉ）を生産するＥＴＥＣＯ１４９、Ｋ８８と９８７ｐを生産するＥＴＥＣＯ１２２、Ｋ９９を生産するＥＴＥＣ２６１７を用いた。クロストリジウム・パーフリンゲンスを評価するときに選抜菌株の培養上澄液を使用し、３７℃、嫌気条件で培養したことを除いて、上記の方法と同様に行った。 The antimicrobial activity against three types of enterotoxic Escherichia coli and Clostridium perfringens was evaluated against 13 primary selected strains with excellent combined antimicrobial activity. Enterotoxic Escherichia coli used ETECO149 that produces K88 adsorption cilia, ETECO122 that produces K88 and 987p, and ETEC2617 that produces K99. When evaluating Clostridium perfringens, the culture supernatant of the selected strain was used, and the culture was performed under the anaerobic condition at 37 ° C. in the same manner as described above.
その結果、表１に示したとおり、複合抗菌活性の優秀な菌株３種（７３９、８２３、９５７）を候補菌株として選抜した。
実施例２
腸毒性大腸菌に対する生体外（ｉｎ ｖｉｔｒｏ）抗菌活性の評価
前記複合抗菌活性の優秀な三つの候補菌株を用いて、腸毒性大腸菌２種に対する活性抑制効果を生体外で測定した。
Example 2
Evaluation of in vitro antibacterial activity against enterotoxic Escherichia coli Using the above three candidate strains having excellent combined antibacterial activity, the activity inhibitory effect against two types of enterotoxic Escherichia coli was measured in vitro.
ＢＨＩ液体培地に、前記候補菌株３種とＥＴＥＣＯ１２２、ＥＴＥＣＯ１４９を各々０．１％ずつ接種し、３７℃、２００ｒｐｍで１５時間培養した。１／２ＢＨＩ液体培地に候補菌株の一種とＥＴＥＣ一種を各々約１×１０５ＣＦＵ／ｍｌになるよう同時接種し、３７℃、２００ｒｐｍで培養した。各時間帯別に培養液をＢＨＩ固体培地とマッコンキー（ＭａｃＣｏｎｋｅｙ、Ｄｉｆｃｏ、ＵＳＡ）固体培地に塗抹して候補菌株とＥＴＥＣの菌数を測定した。 BHI liquid medium was inoculated with 0.1% of each of the three candidate strains, ETECO122 and ETECO149, and cultured at 37 ° C. and 200 rpm for 15 hours. One type of candidate strain and one type of ETEC were co-inoculated in a 1/2 BHI liquid medium so that each was about 1 × 10 5 CFU / ml, and cultured at 37 ° C. and 200 rpm. The culture solution was smeared on a BHI solid medium and a MacConkey (MacConkey, Difco, USA) solid medium for each time period, and the numbers of candidate strains and ETEC were measured.
前記結果は、下表２と図２ａ、２ｂに示し、２４時間以上培養したとき、９５７菌株がＥＴＥＣ Ｏ１２２とＥＴＥＣ Ｏ１４９に対し生長抑制効果がより優秀であることを確認した。よって、抗菌活性の優秀な菌株として９５７最終選抜し、これをＣＪＭＰＢ９５７と命名した。
最終選抜されたＣＪＭＰＢ９５７は、腸毒性大腸菌、鳥類病原性大腸菌、サルモネラ・ティフィムリウム、クロストリジウム・パーフリンゲンスに対して抗菌活性が優秀であり、前記抗菌活性結果は図３示した。 The final selected CJMPB957 has excellent antibacterial activity against enterotoxic Escherichia coli, avian pathogenic Escherichia coli, Salmonella typhimurium, Clostridium perfringens, and the antibacterial activity results are shown in FIG.
実施例３
ＣＪＭＰＢ９５７菌株の形態学的、生化学的特性の調査と同定
（１）形態学的、生化学的特性の調査
複合消化酵素活性の優秀な菌株として最終選抜されたＣＪＭＰＢ９５７菌株を同定するため、１次的に形態学的、生化学的調査を行った。形態的な特徴はグラム陽性桿菌であることを確認し（図１）、生化学的特性を分析するためＡＰＩ５０ＣＨＢシステム（ｂｉｏｍｅｒｉｅｕｘ、フランス）を用いてＣＪＭＰＢ９５７菌株の糖発酵パターンを分析した。
Example 3
Investigation and identification of morphological and biochemical characteristics of CJMPB957 strain (1) Investigation of morphological and biochemical characteristics In order to identify CJMPB957 strain finally selected as an excellent strain of complex digestive enzyme activity, Morphological and biochemical investigations were conducted. The morphological characteristics were confirmed to be Gram-positive bacilli (FIG. 1) and the sugar fermentation pattern of CJMPB957 strain was analyzed using API50CHB system (biomerieux, France) to analyze biochemical properties.
下表３は、ＣＪＭＰＢ９５７菌株の糖発酵パターンを分析した結果を示す。
上記表３において、‘＋’は陽性、‘−’は陰性を表し、対照区は基質のない試験群である。
上記表３の結果をまとめて、ＣＪＭＰＢ９５７菌株は、バチルス・サブチルス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）と９２．２％の相同性があることを確認した。
In Table 3 above, “+” represents positive, “−” represents negative, and the control group is a test group having no substrate.
Summarizing the results in Table 3 above, it was confirmed that the CJMPB957 strain had 92.2% homology with Bacillus subtilis.
（２）ＣＪＭＰＢ９５７菌株の同定
ＣＪＭＰＢ９５７菌株のより正確な同定のため、ＤＮＡ塩基配列による分子系統分類学的方法を行った。
(2) Identification of CJMPB957 strain In order to more accurately identify the CJMPB957 strain, a molecular phylogenetic method using a DNA base sequence was performed.
塩基配列分析はＰＣＲ ｐｒｅｍｉｘ（バイオニア、韓国）とユニバーサルプライマー２７Ｆ（５‘ＡＧＡＧＴＴＴＧＡＴＣＣＴＧＧＣＴＣＡＧ３’：配列番号２）、及び１４９２Ｒ（５‘ＧＧＴＴＡＣＣＴＴＧＴＴＡＣＧＡＣＴＴ ３’： 配列番号３）を用いて、１６ｓ ｒＤＮＡの遺伝子を増幅した。遺伝子増幅の際、総反応液は２０μｌに合わせて、９４℃で１分、５６℃で１分、７２℃で１分を３０回繰り返し、増幅されたＤＮＡ塩基配列を分析した。ＣＪＭＰＢ９５７菌株の分析された１６ｓ ｒＤＮＡの塩基配列は配列番号１（図８）に示した。 For the nucleotide sequence analysis, 16 s rDNA gene was amplified using PCR premix (Bionia, Korea) and universal primer 27F (5'AGAGTTGATCCTGCTCAG3 ': SEQ ID NO: 2) and 1492R (5'GGTTACCTTTTACGACTTT 3': SEQ ID NO: 3). . At the time of gene amplification, the total reaction solution was adjusted to 20 μl, and the amplified DNA base sequence was analyzed by repeating 1 minute at 94 ° C., 1 minute at 56 ° C. and 1 minute at 72 ° C. 30 times. The analyzed base sequence of 16s rDNA of CJMPB957 strain is shown in SEQ ID NO: 1 (FIG. 8).
前記の分析結果、バチルス・サブチルスと９８％の相同性を有する微生物として同定された。
上述した方法により同定された本発明の新たな微生物であるバチルス・サブチルス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＣＪＭＰＢ９５７は、２０１２年３月２２日に、韓国微生物保存センター（Ｋｏｒｅａｎ Ｃｕｌｔｕｒｅ Ｃｅｎｔｅｒ ｏｆ Ｍｉｃｒｏｏｒｇａｎｉｓｍｓ、ソウル市西大門区弘濟１洞３６１−２２１）に寄託番号ＫＣＣＭ１１２７１Ｐとして寄託された。
As a result of the above analysis, the microorganism was identified as a microorganism having 98% homology with Bacillus subtilis.
Bacillus subtilis CJMPB957, a new microorganism of the present invention identified by the above-described method, was introduced on March 22, 2012 in Korean Culture Center of Microorganisms, Hiroshi Seodaemun-gu, Seoul. It was deposited under the deposit number KCCM11271P.
実施例４
ＣＪＭＰＢ９５７菌株の抗真菌活性
抗菌活性の優秀な菌株であるＣＪＭＰＢ９５７の抗真菌活性を調べるため飼料において問題となっているアスペルギルス・フラブス（Ａｓｐｅｒｇｉｌｌｕｓ ｆｌａｖｕｓ）に対する抗真菌活性を評価した。
Example 4
Antifungal activity of CJMPB957 strain To examine the antifungal activity of CJMPB957, which is an excellent antibacterial activity, the antifungal activity against Aspergillus flavus, which is a problem in feed, was evaluated.
アスペルギルスをＰＤＡ（Ｄｉｆｃｏ、ＵＳＡ）固体培地で、３０℃、７２時間静置培養して使用した。固体培地で培養されたアスペルギルスを５ｍｍ×５ｍｍ（横幅）のサイズで切断して、新しいＰＤＡ固体培地の中央に接種し２４時間培養した。 Aspergillus was used after stationary culture at 30 ° C. for 72 hours in a PDA (Difco, USA) solid medium. Aspergillus cultured in a solid medium was cut to a size of 5 mm × 5 mm (width), inoculated into the center of a new PDA solid medium, and cultured for 24 hours.
活性化されたＣＪＭＰＢ９５７をアスペルギルスの培養されている固体培地に１０μｌ分株し、３０℃で４８時間培養して形成されるアスペルギルスの生育阻害環の形成有無を観察した。 10 μl of activated CJMPB957 was cultivated in a solid medium in which Aspergillus was cultured and cultured at 30 ° C. for 48 hours to observe whether or not an Aspergillus growth inhibitory ring was formed.
図４に示したとおり、バチルス・サブチルスＣＪＭＰＢ９５７がアスペルギルスの生育を阻害することを確認し、前記菌株が抗真菌活性を有することを確認した。 As shown in FIG. 4, it was confirmed that Bacillus subtilis CJMPB957 inhibits the growth of Aspergillus, and it was confirmed that the strain has antifungal activity.
実施例５
ＣＪＭＰＢ９５７菌株の消化酵素活性
前記バチルス・サブチルスＣＪＭＰＢ９５７の消化酵素生産能を調べるため、プロテアーゼ、セルラーゼ、アミラーゼ、キシラナーゼ、マンナナーゼ、リパーゼ、及び、フィターゼに対して、消化酵素活性評価を行った。
Example 5
Digestive enzyme activity of CJMPB957 strain In order to examine the digestive enzyme production ability of the Bacillus subtilis CJMPB957, digestive enzyme activity was evaluated for protease, cellulase, amylase, xylanase, mannanase, lipase, and phytase.
前記酵素活性評価は、各酵素に対する基質が入っている培地を用いて、透明環の形成程度により酵素活性能力を測定した。 In the enzyme activity evaluation, the enzyme activity ability was measured based on the degree of formation of a transparent ring using a medium containing a substrate for each enzyme.
１）助酵素液の採取
ＣＪＭＰＢ９５７菌株をＢＨＩ液体培地で、２４時間、４８時間、培養した培養上澄液を採取し、４℃、１３，０００ｒｐｍで５分間遠心分離した後、最終上層液を酵素活性分析用の助酵素液として、下記の通り、各酵素別各々の基質が含まれている培地を用いて、基質分解の程度を判定した。
1) Collection of coenzyme solution Culture supernatant obtained by culturing CJMPB957 strain in BHI liquid medium for 24 hours and 48 hours is collected and centrifuged at 13,000 rpm for 5 minutes at 4 ° C. As a co-enzyme solution for activity analysis, as described below, the degree of substrate degradation was determined using a medium containing each enzyme for each enzyme.
２）プロテアーゼ活性
脱脂粉乳（ｓｋｉｍ ｍｉｌｋ、Ｓｉｇｍａ、ＵＳＡ）を２％添加したＹＭ（Ｙｅａｓｔ ｅｘｔｒａｃｔ ３ｇ／ｌ、Ｍａｌｔ ｅｘｔｒａｃｔ ３ｇ／ｌ、Ｐｅｐｔｏｎｅ ５ｇ／ｌ、Ｄｅｘｔｒｏｓｅ １０ｇ／ｌ、Ａｇａｒ ２０ｇ／ｌ； Ｄｉｆｃｏ、ＵＳＡ、以下、‘ＹＭ培地’）培地を製造した。上記で採取した助酵素液を１．５μｌずつ基質培地に分株した後、３０℃で１５時間反応した後、透明環の形成程度により酵素活性能力を測定した。
2) Protease activity YM (Yeast extract 3 g / l, Malt extract 3 g / l, Peptone 5 g / l, Dextrose 10 g / l, Agar 20 gf / l, supplemented with 2% skim milk powder (skim milk, Sigma, USA); USA, hereinafter “YM medium”) medium. The coenzyme solution collected above was divided into 1.5 μl each in a substrate medium, reacted at 30 ° C. for 15 hours, and then the enzyme activity ability was measured by the degree of formation of a transparent ring.
３）セルラーゼ活性
１％ＣＭＣ（ｃａｒｂｏｘｙｌ ｍｅｔｈｙｌ ｃｅｌｌｕｌｏｓｅ）基質が添加されたＹＭ培地を製造した。上記で採取した助酵素液を１．５μｌずつ基質培地に分株し、３７℃で１５時間反応した後、０．２％のコンゴーレッド（Ｃｏｎｇｏ ｒｅｄ）水溶液で３０分間染色し、１Ｍ ＮａＣｌ水溶液で脱色した。助酵素液周囲の基質が分解されて生じる透明環の形成程度により酵素の活性能力を測定した。
3) Cellulase activity A YM medium supplemented with 1% CMC (carboxyl methyl cellulose) substrate was produced. 1.5 μl of the coenzyme solution collected above was divided into a substrate medium, reacted at 37 ° C. for 15 hours, stained with 0.2% Congo red aqueous solution for 30 minutes, and washed with 1M NaCl aqueous solution. Decolorized. The activity of the enzyme was measured based on the degree of formation of a transparent ring formed by decomposition of the substrate around the coenzyme solution.
４）アミラーゼ活性
１％の可溶性デンプン（ｓｏｌｕｂｌｅ ｓｔａｒｃｈ）基質が添加されたＹＭ培地を製造した。上記で採取した助酵素液を１．５μｌずつ基質培地に分株した後、３７℃で１５時間反応した。Ｉ２とＫＩが各々０．１％、２％添加された水溶液で染色した後、助酵素液周囲の基質が分解されて生じる透明環の形成程度により酵素の活性能力を測定した。
4) Amylase activity A YM medium supplemented with 1% soluble starch substrate was prepared. The coenzyme solution collected above was divided into 1.5 μl each of the substrate medium, and then reacted at 37 ° C. for 15 hours. After staining with aqueous solutions to which I 2 and KI were added at 0.1% and 2%, respectively, the activity ability of the enzyme was measured by the degree of formation of a transparent ring formed by decomposition of the substrate around the coenzyme solution.
５）キシラナーゼ活性
１％キシラン（ｘｙｌａｎ）が添加されたＹＭ培地を製造した。上記助酵素液１．５μｌを基質培地に分株して、３７℃で１５時間反応した後、０．２％コンゴーレッド水溶液で３０分染色し、１Ｍ ＮａＣｌ水溶液で脱色した。その後、透明環の形成程度により酵素活性能力を測定した。
5) Xylanase activity A YM medium supplemented with 1% xylan was produced. The coenzyme solution (1.5 μl) was divided into a substrate medium, reacted at 37 ° C. for 15 hours, stained with 0.2% Congo red aqueous solution for 30 minutes, and decolorized with 1M NaCl aqueous solution. Thereafter, the enzyme activity ability was measured by the degree of formation of a transparent ring.
６）マンナナーゼ活性
１％マンナン（ｌｏｃｕｓｔ ｂｅａｎ ｇｕｍ、ｓｉｇｍａ、ＵＳＡ）が添加された基質培地（Ｙｅａｓｔ ｅｘｔｒａｃｔ ３ｇ／ｌ、Ｐｅｐｔｏｎｅ ５ｇ／ｌ、ＫＨ２ＰＯ４ １ｇ／ｌ、Ａｇａｒ ２０ｇ／ｌ 、ｐＨ５）を製造した。上記助酵素液１．５μｌを基質培地に分株した後、３７℃で２４時間反応した後、透明環の形成程度により酵素活性能力を測定した。
6) Mannanase activity A substrate medium (Yeast extract 3 g / l, Peptone 5 g / l, KH 2 PO 4 1 g / l, Agar 20 g / l, pH 5) supplemented with 1% mannan (locust bean gum, sigma, USA) Manufactured. After dividing 1.5 μl of the coenzyme solution into the substrate medium and reacting at 37 ° C. for 24 hours, the enzyme activity ability was measured by the degree of formation of a transparent ring.
７）リパーゼ活性
１％トリカプリリン（ｔｒｉｃａｐｒｙｌｉｎ）が添加されたＹＭ培地を製造した。上記助酵素液１．５μｌを基質培地に分株し、３７℃で１５時間反応した後、透明環の形成程度により酵素活性能力を測定した。
7) Lipase activity A YM medium supplemented with 1% tricaprylin was produced. 1.5 μl of the coenzyme solution was divided into a substrate medium, reacted at 37 ° C. for 15 hours, and then the enzyme activity ability was measured by the degree of formation of a transparent ring.
８）フィターゼ活性
１％フィチン酸（Ｐｈｙｔｉｃ ａｃｉｄ、ｓｉｇｍａ、ＵＳＡ）が添加された基質培地（グルコース（Ｇｌｕｃｏｓｅ） １５ｇ／ｌ、ＮＨ４ＮＯ３ ５ｇ／ｌ、ＭｇＳＯ４７Ｈ２Ｏ ０．５ｇ／ｌ 、ＫＣｌ ０．５ ｇ／ｌ 、ＦｅＳＯ４７Ｈ２Ｏ ０．０１ｇ／ｌ、ＭｎＳＯ４４Ｈ２Ｏ ０．０１ ｇ／ｌ 、Ａｇａｒ ２０μｌ、ｐＨ５．５）を製造した。上記助酵素液１．５μｌを基質培地に分株して、３７℃で２４時間反応した後、透明環の形成程度により酵素活性能力を測定した。
8) Phytase activity Substrate medium (glucose) 15 g / l, NH 4 NO 3 5 g / l, MgSO 47 H 2 O 0.5 g / l, supplemented with 1% phytic acid (Physic acid, sigma, USA) KCl 0.5 g / l, FeSO 47 H 2 O 0.01 g / l, MnSO 44 H 2 O 0.01 g / l, Agar 20 μl, pH 5.5) were prepared. After dividing 1.5 μl of the coenzyme solution into a substrate medium and reacting at 37 ° C. for 24 hours, the enzyme activity ability was measured by the degree of formation of a transparent ring.
前記ＣＪＭＰＢ９５７の消化酵素活性は、下表４、及び図５に示した。
実施例６
ＣＪＭＰＢ９５７菌株の内生胞子形成能
バチルスは、必須栄養源の中で一つ以上の栄養源が枯渇するなどのストレスを受けると生存のため内生胞子を形成する。内生胞子は、紫外線、高温、低温、乾燥、及び高圧などの極限環境に抵抗性を有するため、内生胞子の形成はバチルスの生存率を維持することにおいて非常に重要である。このようなことで、バチルス・サブチルスＣＪＭＰＢ９５７を長時間培養し、内生胞子の形成能を確認した。
Example 6
The endospore-forming ability of the CJMPB957 strain Bacillus forms endospores for survival when subjected to stress such as the depletion of one or more nutrient sources among the essential nutrient sources. Since endospores are resistant to extreme environments such as ultraviolet light, high temperature, low temperature, drying, and high pressure, the formation of endospores is very important in maintaining the viability of Bacillus. In this way, Bacillus subtilis CJMPB957 was cultured for a long time, and the ability to form endospores was confirmed.
ＢＨＩ液体培地に菌を０．１％接種し、３７℃、２００ｒｐｍで２４時間、４８時間培養した。各時間帯別に培養液をＢＨＩ固体培地に塗抹して総菌数を測定し、９５℃で１０分間熱処理した培養液をＢＨＩアガー（Ａｇａｒ）培地に塗抹して、内生胞子の数を測定した。 BHI liquid medium was inoculated with 0.1% of bacteria and cultured at 37 ° C. and 200 rpm for 24 hours and 48 hours. The culture solution was smeared on a BHI solid medium at each time period to measure the total number of bacteria, and the culture solution heat-treated at 95 ° C. for 10 minutes was smeared on a BHI agar medium to measure the number of endospores. .
下表５は、前記内生胞子数の測定結果を示す。
従って、本発明のバチルス・サブチルスＣＪＭＰＢ９５７ ２４時間以上培養したとき、高い内生胞子形成能を有し、プロバイオティクスとして利用したら動物の消化器官内で高い生存率の維持が可能であると判断される。 Accordingly, when the Bacillus subtilis CJMPB957 of the present invention is cultured for 24 hours or more, it is judged that it has a high endospore-forming ability and can maintain a high survival rate in the digestive tract of animals when used as a probiotic. The
実施例７
ＣＪＭＰＢ９５７菌株の耐酸性と耐胆汁性
プロバイオティクス菌株は、口腔から摂取され最終目的部位である腸まで生きたまま到達するためには強酸性の胃液と胆汁液に対する抵抗性が必要である。よって、本発明のバチルス・サブチルスＣＪＭＰＢ９５７がプロバイオティクス菌株としての応用できるかを確認するため、耐酸性及び耐胆汁性を確認した。
Example 7
The acid-resistant and bile-resistant probiotic strain of CJMPB957 strain requires resistance to strongly acidic gastric juice and bile fluid in order to reach the intestine which is the final target site when ingested from the oral cavity. Therefore, in order to confirm whether the Bacillus subtilis CJMPB957 of the present invention can be applied as a probiotic strain, acid resistance and bile resistance were confirmed.
（１）耐酸性及び人工胃液に対する抵抗性
人工胃液は、ＨＣｌを用いてｐＨ２．５に調整した０．０５Ｍのリン酸ナトリウム（ｓｏｄｉｕｍ ｐｈｏｓｐｈａｔｅ）溶液にペプシン（Ｐｅｐｓｉｎ；Ｓｉｇｍａ、ＵＳＡ）１％（ｗ／ｖ）を添加して製造した。
ＣＪＭＰＢ９５７菌株をＢＨＩ液体培地に、３７℃、２００ｒｐｍで２４時間培養した後、１３，０００ｒｐｍで５分間遠心分離し上澄液は捨てて菌体を回収し、人工胃液を上澄液の同量に添加し、３７℃で３時間培養した。培養後、ＢＨＩ培地に塗抹して菌数を測定し、耐酸性、及び人工胃液に対する抵抗性を確認した。
(1) Acid resistance and resistance to artificial gastric juice Artificial gastric juice was prepared by adding 1% (w) of pepsin (Sigma, USA) to 0.05 M sodium phosphate solution adjusted to pH 2.5 using HCl. / V) was added to produce.
CJMPB957 strain was cultured in BHI liquid medium at 37 ° C and 200 rpm for 24 hours, then centrifuged at 13,000 rpm for 5 minutes, the supernatant was discarded and the cells were recovered, and the artificial gastric juice was made up to the same amount of the supernatant. The mixture was added and cultured at 37 ° C. for 3 hours. After culturing, the cells were smeared on a BHI medium and the number of bacteria was measured to confirm acid resistance and resistance to artificial gastric juice.
（２）人工胆汁に対する抵抗性
人工胆汁は０．０５Ｍリン酸ナトリウム溶液に１％（ｗ／ｖ）のパンクレアチン（Ｐａｎｃｒｅａｔｉｎ；Ｓｉｇｍａ、ＵＳＡ）を添加し滅菌した後、滅菌１０％（ｗ／ｖ）ｏｘａｇａｌｌ（Ｄｉｆｃｏ Ｃｏ．）溶液を培地の１％（ｖ／ｖ）になるように添加し、ｐＨを６．８に調整して製造した。
(2) Resistance to artificial bile Artificial bile is sterilized by adding 1% (w / v) pancreatin (Sigma, USA) to 0.05 M sodium phosphate solution, and then sterilized 10% (w / v) ) Oxagall (Difco Co.) solution was added to 1% (v / v) of the medium, and the pH was adjusted to 6.8.
前記（１）の人工胃液で３時間培養したＣＪＭＰＢ９５７菌株を、１３，０００ｒｐｍで１０分間遠心分離し上澄液は捨てて菌体を回収し前記人工胆汁を上澄液と同量に添加し、３７℃で２４時間培養した。培養後、ＢＨＩ培地に塗抹して菌数を測定し耐酸性、及び人工胆汁に対する抵抗性を確認した。 CJMPB957 strain cultured for 3 hours in the artificial gastric juice of (1) above, centrifuged at 13,000 rpm for 10 minutes, the supernatant was discarded, the cells were collected, and the artificial bile was added in the same amount as the supernatant, The cells were cultured at 37 ° C. for 24 hours. After culturing, it was smeared on a BHI medium and the number of bacteria was measured to confirm acid resistance and resistance to artificial bile.
前記耐酸性、及び人工胃液と人工胆汁に対する抵抗性測定の結果は、図６に示した。
図６に示したとおり、ＣＪＭＰＢ９５７をｐＨ２．５の人工胃液で２時間処理したとき、１００％の生存率を表し、人工胃液処理後に人工胆汁を２４時間処理したときにも同様に１００％が生存率が維持された。
The acid resistance and the results of measuring resistance to artificial gastric juice and artificial bile are shown in FIG.
As shown in FIG. 6, when CJMPB957 was treated with artificial gastric fluid at pH 2.5 for 2 hours, it showed 100% survival rate, and 100% survived when artificial bile was treated for 24 hours after artificial gastric fluid treatment. The rate was maintained.
したがって、本発明のバチルス・サブチルスＣＪＭＰＢ９５７は人工胃液と人工胆汁の連続処理後においても、高い生存率を表し、プロバイオティクス菌株として有用に利用できることが確認された。 Therefore, it was confirmed that Bacillus subtilis CJMPB957 of the present invention exhibits a high survival rate even after continuous treatment of artificial gastric juice and artificial bile, and can be usefully used as a probiotic strain.
実施例８
ＣＪＭＰＢ９５７菌株の安定性
ＣＪＭＰＢ９５７の安定性調べるため溶血性（β−ｈｅｍｏｌｙｓｉｓ）を調査した。β−溶血は、有害菌の中でリン脂質酵素を生産し赤血球により供給されるリン脂質を加水分解して赤血球を溶血させる作用である。
Example 8
Stability of CJMPB957 strain In order to investigate the stability of CJMPB957, hemolysis (β-hemolysis) was investigated. β-hemolysis is an action of producing phospholipid enzymes in harmful bacteria and hydrolyzing phospholipids supplied by erythrocytes to lyse erythrocytes.
バチルス・サブチルスＣＪＭＰＢ９５７の溶血性を調べるため５％の羊血液（ｓｈｅｅｐ ｂｌｏｏｄ、ギサンバイオテック、韓国）が含まれているＴＳＡ（Ｄｉｆｃｏ、ＵＳＡ）を製造した。製造された前記血液寒天培地に画線接種（ｓｔｒｅａｋｉｎｇ）した後、３７℃で２４時間培養し溶血有無を確認した結果、図７に示したとおり、溶血性を表さないことを確認した。 To examine the hemolytic properties of Bacillus subtilis CJMPB957, TSA (Difco, USA) containing 5% sheep blood (Gisan Biotech, Korea) was manufactured. After streaking the manufactured blood agar medium, the cells were cultured at 37 ° C. for 24 hours to confirm the presence or absence of hemolysis, and as a result, as shown in FIG.
本発明のさらなる実施様態において、前記バチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株、前記菌株の培養物、又は前記プロバイオティクス製剤を含む飼料添加剤が提供される。
本発明のさらなる実施様態において、前記バチルス・サブチルスＣＪＭＰＢ９５７（ＫＣＣＭ１１２７１Ｐ）菌株、前記菌株の培養物、前記プロバイオティクス製剤、又は前記飼料添加剤を含む飼料が提供される。
In a further embodiment of the present invention, there is provided a feed additive comprising the Bacillus subtilis CJMPB957 (KCCM11271P) strain, a culture of the strain, or the probiotic formulation.
In a further embodiment of the present invention, there is provided a feed comprising the Bacillus subtilis CJMPB957 (KCCM11271P) strain, a culture of the strain, the probiotic formulation, or the feed additive.
実施例１
バチルス・サブチルス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＣＪＭＰＢ９５７菌株の分離
（１）試料の用意と菌株の分離
伝統発酵食品であるメジュと各種醤類の試料を用意し、前記試料を段階的に希釈して３％塩化ナトリウムが添加されたＢＨＩ固体培地（Ｄｉｆｃｏ、ＵＳＡ）に塗抹した後、３７℃の条件で２４時間培養した。
Example 1
Isolation of Bacillus subtilis CJMPB957 strain (1) Preparation of sample and isolation of strain Prepared a sample of traditional fermented food, medyu and various soy sauces, and diluted the sample stepwise to 3% sodium chloride After smearing on a BHI solid medium (Difco, USA) to which was added, the cells were cultured at 37 ° C. for 24 hours.
上記表３において、’＋’は陽性、’−’は陰性を表し、対照区は基質のない試験群である。 In Table 3 above, “+” represents positive, “−” represents negative, and the control group is a test group having no substrate.
塩基配列分析はＰＣＲ ｐｒｅｍｉｘ（バイオニア、韓国）とユニバーサルプライマー２７Ｆ（５’ＡＧＡＧＴＴＴＧＡＴＣＭＴＧＧＣＴＣＡＧ３’：配列番号２）、及び１４９２Ｒ（５’ＴＡＣＧＧＹＴＡＣＣＴＴＧＴＴＡＣＧＡＣＴＴ３’： 配列番号３）を用いて、１６ｓ ｒＤＮＡの遺伝子を増幅した。遺伝子増幅の際、総反応液は２０μｌに合わせて、９４℃で１分、５６℃で１分、７２℃で１分を３０回繰り返し、増幅されたＤＮＡ塩基配列を分析した。ＣＪＭＰＢ９５７菌株の分析された１６ｓ ｒＤＮＡの塩基配列は配列番号１（図８）に示した。 The nucleotide sequence analysis was performed by amplifying a 16s rDNA gene using PCR premix (Bionia, Korea) and universal primer 27F (5 ′ AGAGTTGATCMTGGCTCAG 3 ′: SEQ ID NO: 2) and 1492R (5 ′ TACGYTACCTTTTACGACTTT 3 ′: SEQ ID NO: 3). did. At the time of gene amplification, the total reaction solution was adjusted to 20 μl, and the amplified DNA base sequence was analyzed by repeating 1 minute at 94 ° C., 1 minute at 56 ° C. and 1 minute at 72 ° C. 30 times. The analyzed base sequence of 16s rDNA of CJMPB957 strain is shown in SEQ ID NO: 1 (FIG. 8).
実施例４
ＣＪＭＰＢ９５７菌株の抗真菌活性
ＣＪＭＰＢ９５７の抗真菌活性を調べるため飼料において問題となっているアスペルギルス・フラブス（Ａｓｐｅｒｇｉｌｌｕｓ ｆｌａｖｕｓ）に対する抗真菌活性を評価した。
Example 4
Antifungal activity of CJMPB957 strain In order to investigate the antifungal activity of CJMPB957, the antifungal activity against Aspergillus flavus, which is a problem in feed, was evaluated.
ＢＨＩ液体培地に菌を０．１％接種し、３７℃、２００ｒｐｍで２４時間、４８時間培養した。各時間帯別に培養液をＢＨＩ寒天培地に塗抹して総菌数を測定し、９５℃で１０分間熱処理した培養液をＢＨＩアガー（Ａｇａｒ）培地に塗抹して、内生胞子の数を測定した。 BHI liquid medium was inoculated with 0.1% of bacteria and cultured at 37 ° C. and 200 rpm for 24 hours and 48 hours. The culture solution was smeared on a BHI agar medium for each time period, and the total number of bacteria was measured. The culture medium heat-treated at 95 ° C. for 10 minutes was smeared on a BHI agar medium, and the number of endospores was measured. .
実施例８
ＣＪＭＰＢ９５７菌株の安全性
ＣＪＭＰＢ９５７の安全性調べるため溶血性（β−ｈｅｍｏｌｙｓｉｓ）を調査した。β−溶血は、有害菌の中でリン脂質酵素を生産し赤血球により供給されるリン脂質を加水分解して赤血球を溶血させる作用である。
Example 8
CJMPB957 order to investigate safety sexual strains of safety CJMPB957 was investigated hemolytic (β-hemolysis). β-hemolysis is an action of producing phospholipid enzymes in harmful bacteria and hydrolyzing phospholipids supplied by erythrocytes to lyse erythrocytes.
バチルス・サブチルスＣＪＭＰＢ９５７の溶血性を調べるため５％の羊血液（ｓｈｅｅｐ ｂｌｏｏｄ、ギサンバイオテック、韓国）が含まれているＴＳＡ（Ｄｉｆｃｏ、ＵＳＡ）を製造した。製造された前記血液寒天培地に画線接種（ｓｔｒｅａｋｉｎｇ）した後、３７℃で２４時間培養し結果、図７に示したとおり、溶血性を表さないことを確認した。 To examine the hemolytic properties of Bacillus subtilis CJMPB957, TSA (Difco, USA) containing 5% sheep blood (Gisan Biotech, Korea) was manufactured. The prepared blood agar medium was streaked and then cultured at 37 ° C. for 24 hours. As a result, it was confirmed that it did not exhibit hemolysis as shown in FIG.
Claims (8)
家畜の細菌性又は真菌性の疾病を予防又は治療する、方法。 Including the step of administering to the livestock the Bacillus subtilis CJMPB957 strain according to paragraph 1, or a composition comprising the culture of the strain according to paragraph 2.
A method for preventing or treating a bacterial or fungal disease in livestock.
家畜の細菌性又は真菌性の疾病を予防又は治療する、第６項に記載の方法。 The composition is at least one selected from the group consisting of a probiotic formulation, a feed additive and a feed,
7. The method according to item 6, wherein a bacterial or fungal disease of livestock is prevented or treated.
家畜の細菌性又は真菌性の疾病を予防又は治療する、第６項に記載の方法。 The bacterial or fungal disease is at least one selected from the group consisting of diarrhea, colibosis, sepsis, salmonellosis, gangrenous enteritis, and aspergillosis. including,
7. The method according to item 6, wherein a bacterial or fungal disease of livestock is prevented or treated.
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| PCT/KR2013/002827 WO2013151362A1 (en) | 2012-04-05 | 2013-04-04 | Novel bacillus subtilis |
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| JP2018082701A (en) * | 2016-11-25 | 2018-05-31 | ロッテ コンフェクショナリー カンパニー リミテッド | Bacillus subtilis lrcc1002 strain having enzyme production ability, bile resistance, and antimicrobial ability and thrombolytic ability to helicobacter pylori |
| JP2019520064A (en) * | 2016-05-31 | 2019-07-18 | エボニック デグサ ゲーエムベーハーＥｖｏｎｉｋ Ｄｅｇｕｓｓａ ＧｍｂＨ | Bacillus subtilis strain exhibiting probiotic activity |
| US11173184B2 (en) | 2016-05-31 | 2021-11-16 | Evonik Operations Gmbh | Bacillus subtilis strain with probiotic activity |
| JP7197204B2 (en) | 2017-11-10 | 2022-12-27 | エムティックスバイオ カンパニー リミテッド | Novel PEDIOCOCCUS PENTOSACEUS AB160011 Strain and Compositions Containing The Same |
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| EA028359B1 (en) * | 2012-04-13 | 2017-11-30 | Кр. Хансен А/С | Bacillus strain for adding to an animal feed, method for obtaining the same and use |
| KR101661745B1 (en) * | 2014-09-15 | 2016-10-04 | 녹십자수의약품(주) | Composition comprising novel bacillus subtilis GCB-13-001 having enzymatic profile for treating or preventing Clostridium sp. bacterial toxin |
| CN107691348A (en) * | 2017-09-19 | 2018-02-16 | 郝天伟 | A kind of cultural method of low fat reed catkins chicken |
| KR102005434B1 (en) | 2018-02-22 | 2019-07-30 | 강원대학교산학협력단 | Bacillus subtilis AF11 against Aspergillus flavus strain producing aflatoxin |
| KR102015823B1 (en) * | 2018-05-28 | 2019-10-23 | (주)에코비즈넷 | Bacillus subtilis EBN-YR47 having excellent inhibitory activity against mycotoxin-producing fungi |
| CN109924350A (en) * | 2019-04-04 | 2019-06-25 | 河南鑫牧生物科技有限公司 | A kind of feed addictive and preparation method thereof releasing mycotoxin |
| CN116024146B (en) * | 2023-03-28 | 2023-06-27 | 中国农业大学 | Bacillus subtilis with high antibacterial activity and fermentation condition optimization thereof |
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| KR100621657B1 (en) * | 2004-04-23 | 2006-09-19 | 주식회사 유니바이오테크 | New Bacillus subtilis UBT-M02 strain which has an acid and bile acid resistances, feed additive composition using it, and feed of animal having thereof |
| KR100850743B1 (en) * | 2006-12-07 | 2008-08-06 | (주)셀텍 | Bacillus subtilis and Probiotics Using the Same |
| KR20090066410A (en) * | 2007-12-20 | 2009-06-24 | (주) 비엔씨바이오팜 | Composition for forage additive comprising bacillus subtilis bc1212 |
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| KR101201184B1 (en) * | 2010-06-29 | 2012-11-13 | 몽고식품 주식회사 | A novel bacillus subtilis strain, and probiotics and low biogenic amine containing food by using the strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2019520064A (en) * | 2016-05-31 | 2019-07-18 | エボニック デグサ ゲーエムベーハーＥｖｏｎｉｋ Ｄｅｇｕｓｓａ ＧｍｂＨ | Bacillus subtilis strain exhibiting probiotic activity |
| US11173184B2 (en) | 2016-05-31 | 2021-11-16 | Evonik Operations Gmbh | Bacillus subtilis strain with probiotic activity |
| JP6991162B2 (en) | 2016-05-31 | 2022-02-04 | エボニック オペレーションズ ゲーエムベーハー | Bacillus subtilis strain showing probiotic activity |
| JP2018082701A (en) * | 2016-11-25 | 2018-05-31 | ロッテ コンフェクショナリー カンパニー リミテッド | Bacillus subtilis lrcc1002 strain having enzyme production ability, bile resistance, and antimicrobial ability and thrombolytic ability to helicobacter pylori |
| JP7197204B2 (en) | 2017-11-10 | 2022-12-27 | エムティックスバイオ カンパニー リミテッド | Novel PEDIOCOCCUS PENTOSACEUS AB160011 Strain and Compositions Containing The Same |
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