JP2015517807A - Newly isolated Bacillus licheniformis and probiotics using the same - Google Patents
Newly isolated Bacillus licheniformis and probiotics using the same Download PDFInfo
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- JP2015517807A JP2015517807A JP2015504500A JP2015504500A JP2015517807A JP 2015517807 A JP2015517807 A JP 2015517807A JP 2015504500 A JP2015504500 A JP 2015504500A JP 2015504500 A JP2015504500 A JP 2015504500A JP 2015517807 A JP2015517807 A JP 2015517807A
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- bacillus licheniformis
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- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/22—Nature of the water, waste water, sewage or sludge to be treated from the processing of animals, e.g. poultry, fish, or parts thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/10—Bacillus licheniformis
Abstract
本発明は、消化酵素を生産し、アンモニアと亜硝酸の酸化能力を有する新たなバチルス・リケニフォルミスＣＪＭＰＢ２８３（Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ ＣＪＭＰＢ２８３；寄託番号ＫＣＣＭ１１２７０Ｐ）及びその用途に関するものである。【選択図】図５The present invention relates to a new Bacillus licheniformis CJMPB283 (Bacillus licheniformis CJMPB283; deposit number KCCM11270P) that produces digestive enzymes and has the ability to oxidize ammonia and nitrite, and uses thereof. [Selection] Figure 5
Description
本発明は、新たなプロバイオティクス及びその用途に関するものである。 The present invention relates to a new probiotic and its use.
養殖業は多くの国で主要な経済的手段であり、養殖魚類の疾病発生は経済的発展に大きく影響を及ぶ（非特許文献１）。 The aquaculture industry is a major economic means in many countries, and the disease outbreak of cultured fish greatly affects economic development (Non-patent Document 1).
一般的に、養殖魚類に発病する疾病と水質改善のために抗生剤、抗菌剤、化工薬品を使用するが、これらの過度な使用により耐性菌の増加と体内残留をはじめ、周辺の水質汚染のどの問題が発生し、その使用を制限している現状である。また、水産食品の抗生剤使用有無に関する消費者の関心が高まることに従い、抗生剤の使用を根源的に遮断するため、有機酸、生菌剤、非特異免疫増強剤、天然物質などの代替物質が開発が盛んに行われている（魚類疾病の診断と治療対策、海洋水産部、２００１）。 In general, antibiotics, antibacterial agents, and chemicals are used to improve disease quality and water quality in cultured fish, but excessive use of these causes increases in resistant bacteria and residuals in the body. Which problem has occurred and is currently limiting its use. In addition, as the consumer's interest in the use of antibiotics in seafood is growing, alternative substances such as organic acids, viable bacteria, non-specific immunity enhancing agents, natural substances, etc. are used to fundamentally block the use of antibiotics. Are being actively developed (diagnosis and treatment of fish diseases, Marine Fisheries Department, 2001).
養殖においてのプロバイオティクスは、飼料に添加したり、水に付随的に投与している。最近、海老などの養魚において、プロバイオティクスを与えることで病原性への免疫力増強と水質改善による疾病改善効果に関する研究が盛んに行われている（非特許文献２）。 Probiotics in aquaculture are added to feed or administered incidentally to water. Recently, in fish farms such as shrimp, research on the effect of disease improvement by enhancing immunity to pathogenicity and improving water quality by providing probiotics has been actively conducted (Non-patent Document 2).
プロバイオティクスは、抗生物質を意味する抗生剤（antibiotics）と反対の語源的意味を有するものであり、腸内微生物の均衡に役立つ微生物製剤、又は微生物成分と定義され、ラクトバチルス菌とビフィズス菌などの乳酸菌と称される菌種が代表的である。 Probiotics have the etymological meaning opposite to antibiotics, which means antibiotics, and are defined as microbial preparations or microbial components that help balance intestinal microorganisms. Lactobacillus and Bifidobacteria The bacterial species called lactic acid bacteria are representative.
プロバイオティクス として使用されているバチルスはグラム陽性の桿菌であり、内生胞子を形成し、プロバイオティクスとして用いられる菌の中で独特な形態を有する。バチルスは、内生胞子を形成しないラクトバチルスより耐熱性が優秀である。また、胃壁の低いｐＨにも生存するため、投与したほとんどの菌が小腸まで達することができる（非特許文献３）。 Bacillus used as a probiotic is a Gram-positive gonococcus, forms endospores, and has a unique form among the fungi used as probiotics. Bacillus has better heat resistance than Lactobacillus which does not form endospores. Moreover, since it survives even at a low pH of the stomach wall, most of the administered bacteria can reach the small intestine (Non-patent Document 3).
水質改善の目的で用いられている物質と微生物に関する先行技術を調べると、特許文献１には亜硝酸塩−酸化バクテリアを利用し水生環境で亜硝酸塩の蓄積を予防・緩和する方法が開示されている。前記亜硝酸塩−酸化バクテリアはニトロコッカス（Nitrococcus）とニトロスピラ（Nitrospira）である。また、特許文献２には、従属栄養アンモニア酸化バクテリアを利用して廃水から炭素と窒素汚染物質を除去する方法を開示し、前記文献で開示された栄養アンモニア酸化バクテリアは、バチルス・シュードファーマスＮＨ−２（Bacillus pseudofimus NH-2）、アルスロバクター・グロビフォルミスＷＲ−２（Arthrobacter globiformis ER-2）を意味する。上記の文献で開示されたニトロコッカスとニトロスピラはＧＲＡＳ菌株ではないためプロバイオティクスとして使用できず、古細菌に属するため生産が非常に難しいので、産業上の利用が不可能である。また、ＧＲＡＳ菌株と知られているバチルス・シュードファーマスＷＲ−２はプロバイオティクスとして登録されてない菌株である。 Examining prior art related to substances and microorganisms used for the purpose of water quality improvement, Patent Document 1 discloses a method for preventing / mitigating nitrite accumulation in an aquatic environment using nitrite-oxidizing bacteria. . The nitrite-oxidizing bacteria are nitrococcus and nitrospira. Patent Document 2 discloses a method for removing carbon and nitrogen pollutants from wastewater using heterotrophic ammonia-oxidizing bacteria, and the nutrient-ammonia oxidizing bacteria disclosed in the above-mentioned literature is Bacillus pseudopharmaceus NH-. 2 (Bacillus pseudofimus NH-2), Arthrobacter globiformis ER-2. Nitrococcus and nitrospira disclosed in the above literature cannot be used as probiotics because they are not GRAS strains, and because they belong to archaea, they are very difficult to produce, and therefore cannot be used industrially. In addition, Bacillus pseudopharmas WR-2, known as a GRAS strain, is a strain that is not registered as probiotics.
本発明者らは、韓国の伝統的発酵食品であるテンジャンから、消化酵素活性を有してアンモニアと亜硝酸を酸化させる菌株を分離し、当該菌株の形態学的、生化学的、遺伝的特性を確認し、本発明を完成した。 The present inventors isolated a strain that has digestive enzyme activity and oxidizes ammonia and nitrous acid from Tenjang, a traditional Korean fermented food, and the morphological, biochemical and genetic characteristics of the strain. The present invention was completed.
本発明の目的は、消化酵素の生産、アンモニアと亜硝酸の酸化能力を有する、新たなバチルス・リケニフォルミスＣＪＭＰＢ２８３を提供することである。 An object of the present invention is to provide a new Bacillus licheniformis CJMPB283 having digestive enzyme production and the ability to oxidize ammonia and nitrite.
本発明の他の目的は、前記バチルス・リケニフォルミスＣＪＭＰＢ２８３の培養液、その濃縮液及びその乾燥物からなる群より選択される培養物を提供することである。 Another object of the present invention is to provide a culture selected from the group consisting of the culture solution of Bacillus licheniformis CJMPB283, a concentrated solution thereof, and a dried product thereof.
本発明の他の目的は、前記バチルス・リケニフォルミスＣＪＭＰＢ２８３、又は培養物を含むプロバイオティクス製剤を提供することである。
本発明の他の目的は、前記プロバイオティクス製剤を含む飼料添加剤を提供することである。
Another object of the present invention is to provide a probiotic preparation comprising the Bacillus licheniformis CJMPB283 or culture.
Another object of the present invention is to provide a feed additive comprising the probiotic preparation.
本発明の他の目的は、バチルス・リケニフォルミスＣＪＭＰＢ２８３、その培養液、その濃縮液、又はその乾燥物を含む水産養殖用の水質改善剤を提供することである。 Another object of the present invention is to provide a water quality improving agent for aquaculture comprising Bacillus licheniformis CJMPB283, a culture solution, a concentrate thereof, or a dried product thereof.
本発明のさらに他の目的は、養殖場の養殖前又は養殖段階において、前記水質改善剤を撒布し前記養殖場の水質改善方法を提供することである。 Still another object of the present invention is to provide a method for improving the water quality of the farm by spreading the water quality improving agent before or during the cultivation of the farm.
本発明のある実施様態において、消化酵素を生産し、アンモニアと亜硝酸の酸化能力を有する新たなバチルス・リケニフォルミスＣＪＭＰＢ２８３が提供される。 In one embodiment of the present invention, a new Bacillus licheniformis CJMPB283 is provided that produces digestive enzymes and has the ability to oxidize ammonia and nitrite.
具体的に、本発明のバチルス・リケニフォルミスＣＪＭＰＢ２８３は、韓国の伝統食品であるテンジャンから分離して得られたものである。本発明の菌株の形態学的特徴は、グラム陽性桿菌であり（図６）、配列番号１の１６ｓ ｒＤＮＡ塩基配列を有する。前記塩基配列の分析結果、バチルス・リケニフォルミス（Bacillus licheniformis）と９７％の相同性を有していた。よって、本発明者らは、新たに分離したバチルス・リケニフォルミスＣＪＭＰＢ２８３を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ＫＣＣＭ１１２７０Ｐとして寄託した。 Specifically, Bacillus licheniformis CJMPB283 of the present invention is obtained by separating from Tenjang, a traditional Korean food. The morphological feature of the strain of the present invention is Gram-positive gonococci (FIG. 6), which has the 16s rDNA base sequence of SEQ ID NO: 1. As a result of analyzing the base sequence, it had 97% homology with Bacillus licheniformis. Accordingly, the inventors of the present invention newly separated Bacillus licheniformis CJMPB283 on Korean Culture Center of Microorganisms (Korea Culture Center of Microorganisms, Seoul, Dongdaemun-gu, 361-221) on March 22, 2012. Deposited under the deposit number KCCM11270P.
具体的に、塩漬（メジュを食塩液に漬けること）して４０日目の韓国の伝統食品であるメジュから菌株を採取し、ＢＨＩ（Brain heart infusion）固体培地から分離した。分離菌株は、アミラーゼ、セルラーゼ、プロテアーゼなどの有用な消化酵素活性が優秀であり、アンモニアと亜硝酸を酸化させる特徴を有した。また、通性嫌気と嫌気的条件での生長が可能であり、１０％塩化ナトリウムに対する耐塩性があるため、様々な水質環境で生長が可能である。また、ヒラメの稚魚を対照として水質浄化能力を評価した結果、ヒラメの飼育水槽内のアンモニア制御効率が優秀であり、低い斃死率を示した。 Specifically, the strain was pickled (soaked in a salt solution), the strain was collected from the Korean traditional food on the 40th day, and separated from a BHI (Brain heart infusion) solid medium. The isolated strain was excellent in useful digestive enzyme activities such as amylase, cellulase and protease, and had the characteristics of oxidizing ammonia and nitrous acid. In addition, it can grow under facultative anaerobic and anaerobic conditions, and has salt resistance to 10% sodium chloride, so it can grow in various water quality environments. Moreover, as a result of evaluating the water purification ability using flounder larvae as a control, the ammonia control efficiency in the flounder breeding aquarium was excellent, and the mortality rate was low.
また他の実施様態において、本発明の新たに分離した菌株の培養液、その濃縮液、及びその乾燥物からなる群より選択される培養物が提供される。 In another embodiment, a culture selected from the group consisting of a culture solution of the newly isolated strain of the present invention, a concentrated solution thereof, and a dried product thereof is provided.
本発明の新たに分離した菌株は、通常的なバチルス菌株の培養方法により培養することができる。培地は、天然培地、又は合成培地を用いることができる。培地の炭素原は、例えば、グルコース、スクロース、デキストリン、グリセロール、デンプンなどがあり、窒素原としてペプトン、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があるが、これらの成分に限らない。培地に含まれる無機塩は、マグネシウム、マンガン、カルシウム、鉄、カルシウムなどがあるが、これらの成分に限らない。前記炭素原、窒素原、及び無機塩の成分以外に、アミノ酸、ビタミン、核酸、及びそれに関わる化合物が培地に添加され得る。本発明の新たに分離した菌株は、２０〜４０℃の温度条件範囲で１２時間〜４日間培養することができる。 The newly isolated strain of the present invention can be cultured by a usual method for culturing Bacillus strains. As the medium, a natural medium or a synthetic medium can be used. The carbon source of the medium includes, for example, glucose, sucrose, dextrin, glycerol, starch, etc., and peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate, and other organics as nitrogen source -There are inorganic nitrogen-containing compounds, but not limited to these components. Inorganic salts contained in the medium include magnesium, manganese, calcium, iron, calcium, and the like, but are not limited to these components. In addition to the carbon source, nitrogen source, and inorganic salt components, amino acids, vitamins, nucleic acids, and related compounds may be added to the medium. The newly isolated strain of the present invention can be cultured in a temperature condition range of 20 to 40 ° C. for 12 hours to 4 days.
具体的に、新たに分離した菌株の培養液は菌体を含む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。前記培養液の組成には通常のバチルス培養に必要な成分だけでなく、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。 Specifically, the culture solution of the newly isolated strain can be a culture stock solution containing bacterial cells, a solution obtained by removing the culture supernatant from the culture stock solution, and / or a concentrated solution thereof. . The composition of the culture solution may include not only components necessary for normal Bacillus culture but also components that act to increase the growth of Bacillus, and the resulting composition has ordinary knowledge in the art. Can be easily selected by a person.
また、菌株の状態は、液状状態、又は乾燥状態であり得る。乾燥方法は、通風乾燥、自然乾燥、噴霧乾燥、凍結乾燥ができるが、これらに限らない。 Moreover, the state of a strain may be a liquid state or a dry state. The drying method can be, but is not limited to, ventilation drying, natural drying, spray drying, and freeze drying.
また他の実施様態において、本発明の新たに分離した菌株、又は培養物を有効成分として含むプロバイオティクス製剤が提供される。 In another embodiment, a probiotic preparation comprising the newly isolated strain or culture of the present invention as an active ingredient is provided.
プロバイオティクスは、腸内の消化管の壁に定着し、有害菌の定着と病原菌の増殖を抑制する。また、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で飼料要求率を改善する。 Probiotics colonize the digestive tract wall in the intestine and suppress the colonization of harmful bacteria and the growth of pathogenic bacteria. The beneficial digestive enzymes produced by probiotics also improve feed requirements by facilitating the absorption and utilization of nutrients.
本発明のプロバイオティクス製剤は、５×１０４ないし５×１０１０ＣＦＵ／ｍｌのバチルス・リケニフォルミスＣＪＭＰＢ２８３を含み、好ましくは１×１０６ないし１×１０９ＣＦＵ／ｍｌのバチルス・リケニフォルミスＣＪＭＰＢ２８３を含む。 The probiotic formulation of the present invention comprises 5 × 10 4 to 5 × 10 10 CFU / ml Bacillus licheniformis CJMPB283, preferably 1 × 10 6 to 1 × 10 9 CFU / ml Bacillus licheniformis CJMPB283. .
本発明の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、食品及び飼料添加剤として提供できる。 The probiotic preparation of the present invention can further contain a pharmaceutically acceptable carrier, and can be formulated with the carrier and provided as a food and feed additive.
本発明において用語“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、又は希釈剤をいう。 In the present invention, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity or properties of the administered compound.
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許容できる薬学的担体として、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、グリセロール、及びこれらの成分の一つ以上を混合して用いることができ、必要に応じて抗酸化剤、緩衝液、静菌剤などの他の通常の添加剤を添加することができる。また、希釈剤、分散剤、界面活性剤、結合剤、及び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。また、プロバイオティクス製剤の品質低下を防止するため添加する決着剤、乳化剤、保存剤、効用増大のため飼料に添加するアミノ酸剤、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、オリゴ糖などがある。その他、飼料混合剤などをさらに含むこともできるが、これらに限定されない。 Pharmaceutical carriers acceptable for probiotic formulations formulated as liquid solutions are sterile and biocompatible with saline, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol , And one or more of these components can be mixed and used, and other conventional additives such as antioxidants, buffers, bacteriostatics, and the like can be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added in addition to injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. It can be formulated. In addition, it is a finalizer, emulsifier, preservative, added to prevent deterioration of the quality of probiotic preparations, amino acids, vitamins, enzymes, flavoring agents, non-protein nitrogen compounds, There are acid salt, buffer, extractant, oligosaccharide and the like. In addition, although it can further contain a feed mixture etc., it is not limited to these.
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剤形は、例えば、錠剤、トローチ、ローゼンジ、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、又はエリキシル剤に製剤化することができる。錠剤、及びカプセルなどの剤形として製剤化するために、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剤を含むことができ、カプセル剤形の場合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。 Oral dosage forms containing the probiotic formulation of the present invention as an active ingredient are, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispensing powders or granules, emulsions, hard or soft capsules, syrups, or It can be formulated into an elixir. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin, excipients such as dicalcium phosphate, corn starch, or sweet potato for formulation as dosage forms such as tablets and capsules Disintegrants such as starch, lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax can be included. In the case of capsule dosage forms, liquid carriers such as fatty oils can be used in addition to the substances described above. Further can be included.
さらに他の実施様態において、本発明は前記プロバイオティクス製剤を有効成分として含む水産養殖用の飼料添加剤に関するものである。 In still another embodiment, the present invention relates to a feed additive for aquaculture containing the probiotic preparation as an active ingredient.
通常的に、バチルス種は、内生胞子を形成し熱に非常に安定的特徴を有する。したがって、新たに分離した前記バチルス・リケニフォルミスＣＪＭＰＢ２８３は、飼料添加剤形態に別に製造して飼料に混合するか、飼料製造のときに直接添加して製造することができる。本発明の飼料内のバチルス・リケニフォルミスＣＪＭＰＢ２８３は、液状、又は乾燥状態であることができ、好ましくは乾燥された粉末形態である。乾燥方法は、通風乾燥、自然乾燥、噴霧乾燥、凍結乾燥ができるが、これらに限定されない。本発明のバチルス・リケニフォルミスＣＪＭＰＢ２８３は粉末形態で、飼料重量の０．０５ないし１０重量％、好ましくは０．１ないし１重量％の成分比で混合できる。また、前記水産養殖用の飼料添加剤は、本発明のバチルス・リケニフォルミスＣＪＭＰＢ２８３の他に飼料の保存性を高められる通常の添加剤をさらに含むことができる。 Usually, Bacillus species form endospores and have very stable characteristics to heat. Therefore, the newly separated Bacillus licheniformis CJMPB283 can be produced separately in the form of a feed additive and mixed with the feed, or can be directly added during the production of the feed. The Bacillus licheniformis CJMPB283 in the feed of the present invention can be in a liquid or dry state, preferably in a dried powder form. The drying method can be, but is not limited to, air drying, natural drying, spray drying, and freeze drying. The Bacillus licheniformis CJMPB283 of the present invention can be mixed in a powder form at a component ratio of 0.05 to 10% by weight, preferably 0.1 to 1% by weight of the feed weight. Moreover, the feed additive for aquaculture can further include a normal additive capable of enhancing the storage stability of the feed in addition to the Bacillus licheniformis CJMPB283 of the present invention.
本発明の飼料には、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、魚粉などがあり、これらに限定されない。 In the feed of the present invention, cereals, root fruits, food processing by-products, algae, fibers, fats and oils, starches, fukube, cereal by-products, etc. as animals, such as proteins, inorganics , Oils and fats, minerals, oils and fats, single cell proteins, zooplanktons, fish meal, and the like, but are not limited thereto.
本発明における水産養殖用の飼料添加剤は、浸漬、噴霧、又は混合して、前記水産養殖用飼料に添加して利用することができる。本発明の飼料添加剤が添加された飼料が使用できる魚種は、魚類、甲殻類などがあり、より具体的に、ヒラメ、鯛、海老、ティラピア、サケ、ウナギ、マスなどの養殖魚類が含まれるが、これらに限定されない。 The feed additive for aquaculture in the present invention can be dipped, sprayed, or mixed and added to the aquaculture feed. Fish species that can be used with the feed to which the feed additive of the present invention is added include fish and crustaceans, and more specifically, cultured fish such as flounder, salmon, shrimp, tilapia, salmon, eel and trout. However, it is not limited to these.
本発明の飼料添加剤を含む水産養殖用飼料の配合方法は、前記飼料添加剤を動物飼料に乾燥重量を基準として０．０５ないし０．５重量％混合する。 In the method for blending an aquaculture feed containing the feed additive of the present invention, the feed additive is mixed with animal feed at 0.05 to 0.5% by weight based on the dry weight.
また他の実施様態において、前記バチルス・リケニフォルミスＣＪＭＰＢ２８３を有効成分として含む水産養殖用の水質改善剤；及び養殖場で養殖前、又は養殖段階で前記水質改善剤を撒布し、前記養殖場の水質を改善する方法に関するものである。 In another embodiment, the water quality improver for aquaculture containing the Bacillus licheniformis CJMPB283 as an active ingredient; and the water quality improver is distributed before or at the aquaculture stage, and the water quality of the aquaculture area is increased. It's about how to improve.
本発明の新たに分離したバチルス・リケニフォルミスＣＪＭＰＢ２８３は、水産養殖環境内に存在するアンモニアと亜硝酸の含量を減少させるこちに利用できる。 The newly isolated Bacillus licheniformis CJMPB283 of the present invention can be used to reduce the contents of ammonia and nitrous acid present in the aquaculture environment.
本発明の水質改善剤が利用できる養殖場の魚種は、魚類、甲殻類などがあり、より具体的に、ヒラメ、鯛、海老、ティラピア、サケ、ウナギ、マスなどの養殖魚類が含まれるが、これらに限定されない。 Fish species in the farm where the water quality-improving agent of the present invention can be used include fish and crustaceans, and more specifically, cultured fish such as flounder, salmon, shrimp, tilapia, salmon, eel and trout. However, it is not limited to these.
本発明の水質改善剤は新たに分離したバチルス・リケニフォルミスＣＪＭＰＢ２８３を水質改善剤の形態として別に製造したり、前記菌株、及び／又は、プロバイオティクス製剤を直接撒布したりすることもできる。本発明の水質改善剤内のバチルス・リケニフォルミスＣＪＭＰＢ２８３は、液状、又は乾燥状態であることができ、好ましくは乾燥された粉末形態である。乾燥方法は、通風乾燥、自然乾燥、噴霧乾燥、凍結乾燥ができるが、これらに限定されない。本発明のバチルス・リケニフォルミスＣＪＭＰＢ２８３は粉末形態で、飼料重量の０．０５ないし１０重量％、好ましくは０．１ないし１重量％の成分比で混合できる。 The water quality-improving agent of the present invention can be produced by separately producing Bacillus licheniformis CJMPB283 as a form of the water quality-improving agent, or directly distributing the strain and / or probiotic preparation. The Bacillus licheniformis CJMPB283 in the water quality improver of the present invention can be in a liquid or dry state, and is preferably in a dried powder form. The drying method can be, but is not limited to, air drying, natural drying, spray drying, and freeze drying. The Bacillus licheniformis CJMPB283 of the present invention can be mixed in a powder form at a component ratio of 0.05 to 10% by weight, preferably 0.1 to 1% by weight of the feed weight.
水質改善剤において許容される担体は、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、グリセロール、及びこれらの成分から一つの成分以上を混合して使用でき、必要に応じて、抗酸化剤、緩衝液、静菌剤など通常の添加剤を添加することができる。また、希釈剤、分散剤、界面活性剤、結合剤、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。 Carriers that are acceptable in water quality improvers are those that are sterile and biocompatible, including saline, sterile water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and one component from these components The above can be mixed and used, and usual additives, such as an antioxidant, a buffer solution, and a bacteriostatic agent, can be added as needed. In addition, diluents, dispersants, surfactants, binders, lubricants can be added to prepare injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Can be
本発明において、新たに分離したバチルス・リケニフォルミスＣＪＭＰＢ２８３は、アミラーゼ、セルラーゼ、プロテアーゼなどの消化酵素の活性が優秀で、アンモニア、及び亜硝酸に対する酸化能が優秀であり、養魚と海老の養殖段階で水質を改善できる効用を有する。 In the present invention, newly isolated Bacillus licheniformis CJMPB 283 has excellent activity of digestive enzymes such as amylase, cellulase, protease, etc., and has excellent oxidation ability to ammonia and nitrite, and the water quality at the stage of aquaculture of fish and shrimp It has the utility that can be improved.
したがって、新たに分離したバチルス・リケニフォルミスＣＪＭＰＢ２８３は、プロバイオティクスとして利用可能であるだけでなく、水質改善剤として利用できる。 Therefore, the newly isolated Bacillus licheniformis CJMPB283 can be used not only as a probiotic but also as a water quality improver.
下記において、実施例により本発明をより詳細に説明する。しかし、これらの実施例は本発明を例示的に説明するもので、本発明の範囲がこれらの実施例に限られることではない。 In the following, the invention is explained in more detail by means of examples. However, these examples are illustrative of the present invention, and the scope of the present invention is not limited to these examples.
実施例１：アンモニアと亜硝酸の酸化菌株を分離
（１）試料の用意と菌株の分離
韓国の伝統食品であるテンジャン、メジュ、醤油などから名品醤類由来菌株を用意した。用意した試料を３％塩化ナトリウムが添加されたＢＨＩ個体培地（Ｄｉｆｃｏ、ＵＳＡ）に塗抹した後、３７℃の条件で２４時間培養した。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て菌株を分離した。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て培養する方法により分離し、純粋培養した菌を２０％グリセロールが添加された培地に入れ、−７０℃以下で保存した。
Example 1: Separation of oxidized strains of ammonia and nitrous acid (1) Preparation of samples and isolation of strains A famous soy sauce-derived strain was prepared from Korean traditional foods such as Tenjang, Medju and soy sauce. The prepared sample was smeared on a BHI solid medium (Difco, USA) supplemented with 3% sodium chloride, and then cultured at 37 ° C. for 24 hours. Strains isolated from each sample were grouped by colony observation to isolate the strains. The selected colonies were transferred to a new medium three times and separated by culturing, and the purely cultured bacteria were placed in a medium supplemented with 20% glycerol and stored at −70 ° C. or lower.
（２）アンモニアと亜硝酸の酸化の評価
水質汚染の主要原因であるアンモニアと亜硝酸を酸化させる微生物を１次選抜するため、アンモニアと亜硝酸に対する酸化評価を行った。
(2) Evaluation of oxidation of ammonia and nitrous acid In order to primarily select microorganisms that oxidize ammonia and nitrous acid, which are the main causes of water pollution, oxidation evaluation of ammonia and nitrous acid was performed.
アンモニア酸化の評価をするため、アンモニアが添加されたイオン培地（（ＮＨ４）２ＳＯ４４．９５ｇ／Ｌ、 Ｋ２ＨＰＯ４ ８．８２ｇ／Ｌ、ＭｇＳＯ４（１Ｍ溶液）１．１ｍｌ／Ｌ、ＣａＣｌ２（１Ｍ溶液） ０．３ｍｌ／Ｌ、ＦｅＳＯ４（３０ｍＭ溶液） ０．５ｍｌ／Ｌ、ＣｕＳＯ４（５０ｍＭ溶液） ０．０４ｍｌ／Ｌ、ＮａＨ２ＰＯ４０．７ｇ／Ｌ、５％（Ｗ／Ｖ） Ｎａ２ＣＯ３ ａｎｈｙｄｒｏｕｓ １２ｍｌ／Ｌ）と、亜硝酸が添加されたイオン培地（ＣａＣｌ２０．０１ｇ／Ｌ、ＭｇＳＯ４・７Ｈ２Ｏ ０．１ｇ／Ｌ、ＥＤＴＡ １．４ｍｇ／Ｌ、ＦｅＳＯ４・７Ｈ２Ｏ ５ｍｇ／Ｌ、Ｈ２ＳＯ４ ０．５ μｌ／Ｌ、Ｎａ２ＭｏＯ４・２Ｈ２Ｏ ０．０５ｍｇ／Ｌ、ＭｎＣｌ２・４Ｈ２Ｏ ０．１ｍｇ／Ｌ、ＣｏＣｌ２・６Ｈ２Ｏ ０．００１ｍｇ／Ｌ、ＺｎＳＯ４・７Ｈ２Ｏ ０．０５ｍｇ／Ｌ、ＣｕＳＯ４・５Ｈ２Ｏ ０．０１ｍｇ／Ｌ、ＮａＮＯ２０．２１ｇ／Ｌ、Ｋ２ＨＰＯ４ ０．３ｍｇ／Ｌ）を製造した。前記製造された二つの培地５ｍｌに選抜菌株を０．０１％接種した後、３０℃で１４日間培養した。培養液を遠心分離して培養上澄液を１ｍｌずつ各々採取した後、亜硝酸が存在するとき色反応を表す指示薬（sulanilic acid、N、N-Dimethyl-1-naphylamine）を１００μｌずつ添加した。指示薬が添加された培養上澄液を、２５℃で１０分間反応した後に色変化有無を観察した。亜硝酸の存在や生成があった場合は培養液の色が濃い紫色に変わり、亜硝酸が存在しないか消耗された場合は培養液の色変化が観察されなかった。 In order to evaluate ammonia oxidation, ion medium to which ammonia was added ((NH 4 ) 2 SO 4 4.95 g / L, K 2 HPO 4 8.82 g / L, MgSO 4 (1M solution) 1.1 ml / L , CaCl 2 (1M solution) 0.3 ml / L, FeSO 4 (30 mM solution) 0.5 ml / L, CuSO 4 (50 mM solution) 0.04 ml / L, NaH 2 PO 4 0.7 g / L, 5% W / V) Na 2 CO 3 anhydrous 12 ml / L) and ionic medium supplemented with nitrous acid (CaCl 2 0.01 g / L, MgSO 4 .7H 2 O 0.1 g / L, EDTA 1.4 mg / L) , FeSO 4 · 7H 2 O 5mg / L, H 2 SO 4 0.5 μl / L, Na 2 MoO 4 · 2H 2 O 0.05mg / L, MnCl 2 · 4H 2 O 0.1mg / L CoCl 2 · 6H 2 O 0.001mg / L, ZnSO 4 · 7H 2 O 0.05mg / L, CuSO4 · 5H 2 O 0.01mg / L, NaNO 2 0.21g / L, K 2 HPO 4 0.3mg / L) was produced. After inoculating 0.01% of the selected strain into 5 ml of the two prepared media, the medium was cultured at 30 ° C. for 14 days. After centrifuging the culture solution and collecting 1 ml each of the culture supernatant, 100 μl each of an indicator (sulanilic acid, N, N-dimethyl-1-naphylamine) showing a color reaction when nitrous acid was present was added. After the culture supernatant to which the indicator was added was reacted at 25 ° C. for 10 minutes, the presence or absence of a color change was observed. The color of the culture broth turned dark purple when nitrous acid was present or formed, and no color change was observed when nitrous acid was absent or was consumed.
約１０００種くらいの分離菌株を評価した結果、表１に示したとおり、アンモニアと亜硝酸の両方を酸化させる菌株７種、アンモニア分解の優秀な菌株２種、亜硝酸分解菌株１種を１次選抜した。 As a result of evaluating about 1,000 kinds of isolated strains, as shown in Table 1, seven strains that oxidize both ammonia and nitrous acid, two strains that excel in ammonia degradation, and one nitrite-degrading strain are primary. Selected.
実施例２：アンモニアと亜硝酸酸化のin vitro評価
（１）１次選抜菌株のアンモニア定量評価
１次選抜菌株のアンモニア酸化による低減効果を評価するため、前記選抜菌株１０種に対し、アンモニアの添加培地でのアンモニア消耗量を定量した。
Example 2 In Vitro Evaluation of Ammonia and Nitrite Oxidation (1) Ammonia Quantitative Evaluation of Primary Selected Bacteria In order to evaluate the reduction effect of primary selected strains by ammonia oxidation, addition of ammonia to the 10 selected strains The amount of ammonia consumption in the medium was quantified.
１次選抜された１０種の菌株をＢＨＩ液体培地に０．１％ずつ接種した後、３７℃、２００ｒｐｍで１５時間培養し、菌株を活性化させた。アンモニア含有培地（（ＮＨ４）２ＳＯ４０．５ｇ／Ｌ、ＮａＨ２ＰＯ４ １３．５ｇ／Ｌ、Ｋ２ＨＰＯ４ ０．７ｇ／Ｌ、ＭｇＳＯ４・７Ｈ２Ｏ ０．１ｇ／Ｌ、ＣａＣｌ２・２Ｈ２Ｏ ０．１８ｇ／Ｌ、ＮａＨＣＯ３ ０．５ｇ／Ｌ、ＦｅＣｌ３・６Ｈ２Ｏ ０．０１４ｇ／Ｌ、ｇｌｕｃｏｓｅ ０．５ｇ／Ｌ）に、活性化された菌を１％接種し、３７℃、２００ｒｐｍで培養した。培養過程中、６時間、１２時間、２４時間、３６時間の培養液を採取して遠心分離した後、培養上澄液だけを回収した。回収された培養上澄液においてのアンモニア残余量を定量した。 Ten strains selected first were inoculated 0.1% each in a BHI liquid medium, and then cultured at 37 ° C. and 200 rpm for 15 hours to activate the strains. Ammonia-containing medium ((NH 4 ) 2 SO 4 0.5 g / L, NaH 2 PO 4 13.5 g / L, K 2 HPO 4 0.7 g / L, MgSO 4 .7H 2 O 0.1 g / L, CaCl 2 · 2H 2 O 0.18 g / L, NaHCO 3 0.5 g / L, FeCl 3 · 6H 2 O 0.014 g / L, glucose 0.5 g / L) The cells were cultured at 37 ° C. and 200 rpm. During the culture process, 6 hours, 12 hours, 24 hours, and 36 hours of culture broth were collected and centrifuged, and then only the culture supernatant was collected. The ammonia residual amount in the collected culture supernatant was quantified.
その結果、図１のとおり、アンモニア含有培地で選抜菌株１０種すべて培養上澄液内のアンモニア含量が減少したことを確認でき、特に２８３番、２９６番、３０３番、３９６番の菌株は培養初期である６時間目からアンモニアの含量が急激に減っていくことを確認した。アンモニアの酸化により亜硝酸が生成されたかを確認するため、亜硝酸が存在するとき色反応を表す指示薬を添加して色反応を観察した結果、アンモニアを含む初期培地からは色変化観察されなく、培養が終了したとき採取した各サンプルでは、培養上澄液の色が全て濃い紫色に変わった。これによって、１次選抜菌株は全てアンモニアを亜硝酸に酸化させることをわかった。 As a result, as shown in FIG. 1, it was confirmed that the ammonia content in the culture supernatant was reduced in all 10 selected strains in the ammonia-containing medium. In particular, the strains 283, 296, 303, and 396 were in the initial stage of culture. It was confirmed that the ammonia content rapidly decreased from the 6th hour. In order to confirm whether nitrous acid was produced by oxidation of ammonia, as a result of observing the color reaction by adding an indicator indicating the color reaction when nitrous acid was present, no color change was observed from the initial medium containing ammonia. In each sample collected when the culture was completed, the color of the culture supernatant changed to dark purple. As a result, it was found that all the primary selection strains oxidize ammonia to nitrous acid.
（２）１次選抜菌株における亜硝酸の定量評価
１次選抜菌株の亜硝酸酸化による低減効果を評価するため、前記選抜菌株１０種に対して、亜硝酸が添加が培地から定量して亜硝酸の消耗量を定量した。
(2) Quantitative evaluation of nitrous acid in primary selected strains In order to evaluate the reduction effect of nitrite oxidation on primary selected strains, nitrite was added to the 10 selected strains and nitrite was quantified from the medium. The amount of consumption was quantified.
１次選抜された１０種の菌株をＢＨＩ液体培地に０．１％ずつ接種した後、３７℃、２００ｒｐｍで１５時間培養して菌株を活性化させた。亜硝酸含有培地（ＮａＮＯ２０．５ｇ／Ｌ、 ＮａＨ２ＰＯ４ １３．５ｇ／Ｌ、 Ｋ２ＨＰＯ４ ０．７ｇ／Ｌ、 ＭｇＳＯ４・７Ｈ２Ｏ ０．１ｇ／Ｌ、 ＣａＣｌ２・２Ｈ２Ｏ ０．１８ｇ／Ｌ、 ＮａＨＣＯ３ ０．５ｇ／Ｌ、 ＦｅＣｌ３・６Ｈ２Ｏ ０．０１４ｇ／Ｌ、 ｇｌｕｃｏｓｅ ０．５ｇ／Ｌ）に活性化された菌を１％接種し、３７℃、２００ｒｐｍで培養した。培養過程中、６時間、９時間、１２時間、２４時間、３０時間の培養液を採取して遠心分離した後、培養上澄液だけを回収した。回収された培養上澄液の亜硝酸の残余量を定量した。 Ten strains selected primarily were inoculated 0.1% each in a BHI liquid medium, and then cultured at 37 ° C. and 200 rpm for 15 hours to activate the strains. Nitrite-containing medium (NaNO 2 0.5 g / L, NaH 2 PO 4 13.5 g / L, K 2 HPO 4 0.7 g / L, MgSO 4 .7H 2 O 0.1 g / L, CaCl 2 .2H 2 O 0.18g / L, NaHCO 3 0.5g / L, FeCl 3 · 6H 2 O 0.014g / L, the activated bacteria glucose 0.5 g / L) was inoculated 1%, 37 ℃, 200rpm In culture. During the culturing process, 6 hours, 9 hours, 12 hours, 24 hours, and 30 hours of culture broth were collected and centrifuged, and then only the culture supernatant was collected. The remaining amount of nitrous acid in the collected culture supernatant was quantified.
その結果、図２に示したとおり、亜硝酸含有培地で選抜菌株１０種は全て培養上澄液内の亜硝酸含量が減少されたことを確認でき、特に１０２番、１０９番、２５１番、２５３番、２６８番の菌株は培養初期の６時間目から亜硝酸の含量が急激に減っていくことを確認した。亜硝酸の酸化有無をするため、亜硝酸が存在するとき色反応を表す指示薬を添加して色反応を観察した結果、亜硝酸を含有する初期培地では濃い紫色が観察され、培養が終了して採取した各サンプルでは培養上澄液の変色がなかった。即ち、次選抜菌株が亜硝酸を酸化させることにより、亜硝酸が酸化されて存在しないことを分かった。 As a result, as shown in FIG. 2, it was confirmed that all the 10 selected strains in the nitrite-containing medium had a reduced nitrite content in the culture supernatant, and in particular, Nos. 102, 109, 251, and 253. No. 268 strain confirmed that the content of nitrous acid decreased sharply from the 6th hour of the initial culture. In order to check whether or not nitrous acid is oxidized, an indicator indicating the color reaction was added when nitrous acid was present, and as a result of observing the color reaction, a dark purple color was observed in the initial medium containing nitrous acid, and the culture was terminated. In each sample collected, there was no discoloration of the culture supernatant. That is, it was found that nitrite was oxidized and did not exist when the next selected strain oxidized nitrous acid.
（３）生長性の評価
水質環境は、好気、通性嫌気、嫌気的条件が共存し、特に嫌気的条件でアンモニアが硝酸に酸化するアナモックス（Anammox、嫌気的条件でアンモニアが硝酸に酸化する過程）が行われるため選抜菌株が多様な条件で生長することは重要な因子の一つである。そして、１次選抜菌株１０種に対し、通性嫌気と嫌気条件においての生長性を評価した。
(3) Evaluation of growth The water quality environment is anaerobic, facultative anaerobic, and anaerobic conditions. Anammox, where ammonia is oxidized to nitric acid under anaerobic conditions (Anammox, ammonia is oxidized to nitric acid under anaerobic conditions) It is one of the important factors that the selected strain grows under various conditions. And the growth property in facultative anaerobic and anaerobic conditions was evaluated with respect to 10 types of primary selection strains.
１次選抜菌株１０種をＢＨＩ液体培地に０．１％ずつ接種した後、３７℃の通性嫌気、嫌気的条件で２４時間培養を行った。選抜菌株の大部分は通性嫌気、嫌気的条件で全て生長することを確認し、特に２５３番と３６８番の菌株が嫌気的条件における生長性が優秀であり、２８３番の菌株は通性嫌気的条件における生長性が最も優秀であった（図３）。そして、通性嫌気と嫌気的条件においての生長性が優秀であった２５３番、２６８番、２８３番の菌株を２次選抜した。 After inoculating 0.1% each of 10 primary selected strains in BHI liquid medium, the cells were cultured for 24 hours under 37 ° C. facultative and anaerobic conditions. Most of the selected strains were confirmed to grow under facultative anaerobic and anaerobic conditions. Especially, strains 253 and 368 were excellent in anaerobic conditions, and strain 283 was facultative anaerobic. The growth was the best under the conditions (Fig. 3). Then, strains Nos. 253, 268 and 283, which were excellent in facultative anaerobic and anaerobic conditions, were secondarily selected.
（４）耐塩性の評価
淡水条件と海水条件における菌株の活性を評価するため、塩化ナトリウムが添加された培地で２次選抜された菌株の生長性評価を行った。
(4) Evaluation of salt tolerance In order to evaluate the activity of the strain in freshwater and seawater conditions, the growth of the secondarily selected strain was evaluated in a medium to which sodium chloride was added.
塩化ナトリウムが０％、３％、５％、１０％添加されたＢＨＩ液体培地に、選抜された候補菌株３種を０．１％ずつ接種し、３７℃、２００ｒｐｍで培養した。培養過程中、８時間、１０時間、２２時間目の各サンプルを採取し、吸光度を測定した。 BHI liquid medium supplemented with 0%, 3%, 5%, or 10% sodium chloride was inoculated with 0.1% of the three selected candidate strains and cultured at 37 ° C. and 200 rpm. During the culturing process, samples at 8 hours, 10 hours, and 22 hours were collected and the absorbance was measured.
その結果、図４に示したとおり、菌株３種全て３％塩化ナトリウム添加培地で菌が増殖することを確認し、２８３番の菌株は５％の塩化ナトリウム添加培地で他の菌株より生長性が優秀であった。したがって、２次選抜された３種の菌株、即ち、２５３、２６８、２８３は、淡水条件だけでなく、海水条件でもその適用が可能であることを確認した。 As a result, as shown in FIG. 4, all three strains were confirmed to grow on 3% sodium chloride-added medium, and strain 283 was more viable than other strains on 5% sodium chloride-added medium. It was excellent. Therefore, it was confirmed that the three selected strains, ie, 253, 268, and 283, can be applied not only in freshwater conditions but also in seawater conditions.
実施例３：in vivo評価と最終菌株選抜
２次選抜された菌株３種に対する水質浄化能力をヒラメ稚魚をに評価した。ヒラメ稚魚に市販飼料を満腹で供給した後、実験水槽（６４Ｌ）でランダムに配置した。実験魚を鉢した後、全ての実験飼育水槽の飼育水を綺麗に換水し、０時間、２４時間、４８時間、７２時間、９２時間、１２０時間目に各々５０ｍｌずつ飼育水をサンプリングした。２次選抜菌株の投与の２４時間の間隔で約３ｍｌに１ｘ１０５ＣＦＵ菌数になるよう希釈した後、水槽に５日間投与した。対照区の場合は同量の海水を投与した。サンプルを採取してアンモニアの濃度を分析し、分析して残った海水サンプルは−２０℃で保管した後、亜硝酸塩と硝酸塩の分析に用いた。水質分析は、アンモニア、亜硝酸塩、硝酸塩の３つの項目を分析した。これらの分析は水質分析器（RQflex 10、Merck、ドイツ）を利用し、キットの試薬と各々反応した後分析した。
Example 3: In vivo evaluation and final strain selection The water purifying ability of three secondary strains was evaluated for flounder larvae. After supplying commercial feed to flounder larvae with a full stomach, they were randomly placed in an experimental water tank (64 L). After potting the experimental fish, the breeding water in all experimental breeding tanks was neatly changed, and 50 ml of the breeding water was sampled at 0, 24, 48, 72, 92, and 120 hours. After diluting to about 3 ml at a 24-hour interval after administration of the secondary selection strain to 1 × 10 5 CFU bacteria count, it was administered to a water tank for 5 days. In the case of the control group, the same amount of seawater was administered. A sample was collected and analyzed for ammonia concentration, and the seawater sample remaining after analysis was stored at −20 ° C. and then used for analysis of nitrite and nitrate. The water quality analysis analyzed three items, ammonia, nitrite, and nitrate. These analyzes were carried out using a water quality analyzer (RQflex 10, Merck, Germany) and reacting with each reagent of the kit.
実験結果、７２時間目からアンモニアの除去効果が表れ（図５）、１２０時間目はもっと大きな差を見せた。１４０時間後には全体水槽でアンモニア毒性による斃死が起こり始めた。２次選抜された菌株３種中、２５３番と２８３番の菌株が対照区に比べて有意的に高いアンモニア除去効果を示し、斃死率も対照区より低い斃死率を示し、飼育水（ヒラメ（魚）を飼育する際用いる水）内のアンモニアと亜硝酸の酸化菌株の添加はヒラメの生存率を高めることがわかった。したがって、ヒラメ飼育において、アンモニア除去効果が優秀で、通性嫌気と嫌気条件における生長性が優秀であった２８３番菌株を最終選抜した。 As a result of the experiment, an effect of removing ammonia appeared from the 72nd hour (FIG. 5), and a larger difference was shown at the 120th hour. After 140 hours, drowning due to ammonia toxicity began to occur in the entire tank. Of the three strains selected for the second time, the strains Nos. 253 and 283 showed a significantly higher ammonia removal effect than the control group, and the mortality rate was lower than that of the control group. It was found that the addition of an oxidizing strain of ammonia and nitrous acid in water) used for rearing fish increased the survival rate of Japanese flounder. Therefore, in the flounder breeding, the 283th strain having excellent ammonia removal effect and facultative growth under facultative anaerobic and anaerobic conditions was finally selected.
実施例４：選抜菌株の同定と生理・生化学的特性の調査
（１）形態学的、生化学的特性の調査
最終選抜された菌株の種定のため、１次的に形態学的、生化学的調査を行った。形態的特徴において、グラム染色結果グラム陽性であり、電子顕微鏡撮影の結果桿菌であることを確認した（図６）。生化学的特性を分析するためＡＰＩ５０ ＣＨＢ system（Biomerieux、フランス）で菌株の糖発酵パターンを分析した（表２）。
Example 4: Identification of selected strains and investigation of physiological and biochemical characteristics (1) Investigation of morphological and biochemical characteristics For the final selection of strains, primary morphological and biochemical characteristics A chemical investigation was conducted. In morphological characteristics, the Gram staining result was Gram positive, and as a result of electron microscopic photography, it was confirmed that it was Neisseria gonorrhoeae (FIG. 6). To analyze the biochemical properties, the sugar fermentation pattern of the strain was analyzed with the API50 CHB system (Biomerieux, France) (Table 2).
（１）菌株の同定
正確な菌株同定をするため、ＤＮＡ塩基配列による分子系統分類学的方法を行った。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’ＧＧYＴＡＣＣＴＴＧＴＴＡＣＧＡＣＴＴ 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。分離菌株の分析された１６ｓ ｒＤＮＡの塩基配列は配列番号１に示す。前記分析結果、バチルス・リケニフォルミスと９６％の相同性を有する微生物として同定された。よって、選抜された前記菌株をバチルス・リケニフォルミスＣＪＭＰＢ２８３に命名し、前述の方法により同定された本発明の新たな微生物は、２０１２年３月２２日、韓国微生物保存センターに、"Bacillus licheniformis ＣＪＭＰＢ２８３"として寄託された（寄託番号ＫＣＣＭ１１２７０Ｐ）。
(1) Identification of strains In order to accurately identify strains, molecular phylogenetic methods based on DNA base sequences were performed. For the nucleotide sequence analysis, a 16s rDNA gene was amplified using PCR premix (Bionia, Korea) and universal primer 27F (5′AGAGTTTGATCMTGGCTCAG3 ′: SEQ ID NO: 2) and 1492R (5′GGYTACCTTGTACCGACTT 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 the isolated strain is shown in SEQ ID NO: 1. As a result of the analysis, the microorganism was identified as 96% homologous to Bacillus licheniformis. Therefore, the selected strain was named Bacillus licheniformis CJMPB283, and a new microorganism of the present invention identified by the above-mentioned method was designated as “Bacillus licheniformis CJMPB283” on March 22, 2012 at the Korea Microbiology Conservation Center. Deposited (deposit number KCCM11270P).
実施例５：バチルス・リケニフォルミスＣＪＭＰＢ２８３菌株のプロバイオティクス特性を分析
（１）分離菌株の消化酵素活性
１）助酵素液の採取
前記分離した菌株をＢＨＩ液体培地で、２４時間培養した後、培養液を酵素活性の分析のための助酵素液にして、下記の通り各酵素別に各々の基質が入っている培地を用いて基質分解の程度を判定した。
Example 5: Analysis of probiotic characteristics of Bacillus licheniformis CJMPB283 strain (1) Digestive enzyme activity of isolated strain 1) Collection of coenzyme solution The isolated strain was cultured in a BHI liquid medium for 24 hours, and then the culture solution Was used as a coenzyme solution for analysis of enzyme activity, and the degree of substrate degradation was determined using a medium containing each substrate for each enzyme as described below.
２）プロテアーゼ（Protease）活性
脱脂粉乳（skim milk、Sigma、USA）を２％添加したＹＭ（Difco、USA）培地を製造した。上記で採取した助酵素液２μｌを基質培地に分株し、３７℃で１５時間反応した後、透明環（clear zone）形成の程度により酵素活性能力を測定した。
2) Protease activity A YM (Difco, USA) medium supplemented with 2% skim milk (Sigma, USA) was produced. 2 μl of the coenzyme solution collected above 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 clear zone formation.
３）セルラーゼ（Cellulase）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２μｌ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（clear zone）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. 2 μl of the coenzyme solution collected above was divided into a substrate medium, reacted at 37 ° C. for 15 hours, stained with 0.2% aqueous Congo red solution for 30 minutes, and decolorized with 1M NaCl aqueous solution. . The activity of the enzyme was measured based on the degree of formation of a clear zone formed by decomposition of the substrate around the coenzyme solution.
４）アミラーゼ（Amylase）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３μｌずつ基質培地に分株したし、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（clear zone）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. 3 μl of the coenzyme solution collected above was divided into a substrate medium and reacted at 37 ° C. for 15 hours. After staining with an aqueous solution in which I 2 and KI were added at 0.1% and 2%, respectively, the activity of the enzyme was measured by the degree of formation of a clear zone formed by decomposition of the substrate around the coenzyme solution. .
最終選抜された前記菌株バチルス・リケニフォルミスＣＪＭＰＢ２８３は、表３に示したとおり、プロテアーゼ、アミラーゼ、セルラーゼに対する消化酵素活性を有した。特に、プロテアーゼに対する活性が優秀であった。 The final selected Bacillus licheniformis CJMPB283 had digestive enzyme activities for protease, amylase and cellulase as shown in Table 3. In particular, the activity against protease was excellent.
（２）内生胞子の形成能
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。よって、バチルス・リケニフォルミスＣＪＭＰＢ２８３を長時間培養し、内生胞子の形成能を確認した。
(2) Ability to form endospores Bacillus forms endospores for survival when subjected to stress such as the depletion of one or more of the essential nutrients. 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. Therefore, Bacillus licheniformis CJMPB283 was cultured for a long time to confirm the ability to form endospores.
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 for each time period, and the total number of bacteria was measured. The culture solution heat-treated at 95 ° C. for 10 minutes was smeared on a BHI Agar medium, and the number of endospores was measured. .
上記表４に示した通り、バチルス・リケニフォルミスＣＪＭＰＢ２８３を２４時間培養したとき約２４．５％が内生胞子を形成し、４８時間培養したときには１００％が内生胞子を形成した。本発明のバチルス・リケニフォルミスＣＪＭＰＢ２８３は、４８時間以上培養するとき、高い内生胞子形成能を有し、プロバイオティクスとして使用したら、高い生存率の特性を維持することができる。 As shown in Table 4 above, about 24.5% formed endospores when Bacillus licheniformis CJMPB283 was cultured for 24 hours, and 100% formed endospores when cultured for 48 hours. The Bacillus licheniformis CJMPB283 of the present invention has a high endospore-forming ability when cultured for 48 hours or longer, and can maintain high survival characteristics when used as a probiotic.
本発明新たに分離したバチルス・リケニフォルミスＣＪＭＰＢ２８３は、アミラーゼ、セルラーゼ、プロテアーゼなどの消化酵素生産が優秀で、アンモニアと亜硝酸に対する酸化効果が優秀であり、養魚と海老の養殖段階で、プロバイオティクスと水質改善の効果がある。したがって、新たに分離したバチルス・リケニフォルミスＣＪＭＰＢ２８３は、プロバイオティクスとして利用できるだけでなく、水質改善剤としても利用できる。
The newly isolated Bacillus licheniformis CJMPB283 of the present invention has excellent production of digestive enzymes such as amylase, cellulase, protease, etc., and has an excellent oxidizing effect on ammonia and nitrous acid. Effective in improving water quality. Therefore, the newly isolated Bacillus licheniformis CJMPB283 can be used not only as a probiotic but also as a water quality improver.
養殖業は多くの国で主要な経済的手段であり、養殖魚類の疾病発生は経済的発展に大きく影響を及ぼす（非特許文献１）。
Aquaculture is a major economic means in many countries, the disease occurrence aquaculture fish greatly affects the及boss economic development (Non-Patent Document 1).
一般的に、養殖魚類に発病する疾病と水質改善のために抗生剤、抗菌剤、化工薬品を使用するが、これらの過度な使用により耐性菌の増加と体内残留をはじめ、周辺の水質汚染のどの問題が発生し、その使用を制限している現状である。また、水産食品の抗生剤使用有無に関する消費者の関心が高まることに従い、抗生剤の使用を根源的に遮断するため、有機酸、生菌剤、非特異免疫増強剤、天然物質などの代替物質の開発が盛んに行われている（魚類疾病の診断と治療対策、海洋水産部、２００１）。
In general, antibiotics, antibacterial agents, and chemicals are used to improve disease quality and water quality in cultured fish, but excessive use of these causes increases in resistant bacteria and residuals in the body. Which problem has occurred and is currently limiting its use. In addition, as the consumer's interest in the use of antibiotics in seafood is growing, alternative substances such as organic acids, viable bacteria, non-specific immunity enhancing agents, natural substances, etc. are used to fundamentally block the use of antibiotics. development of has been actively conducted (diagnosis and treatment measures of fish disease, Maritime Affairs and Fisheries, 2001).
水質改善の目的で用いられている物質と微生物に関する先行技術を調べると、特許文献１には亜硝酸塩−酸化バクテリアを利用し水生環境で亜硝酸塩の蓄積を予防・緩和する方法が開示されている。前記亜硝酸塩−酸化バクテリアはニトロコッカス（Nitrococcus）とニトロスピラ（Nitrospira）である。また、特許文献２には、従属栄養アンモニア酸化バクテリアを利用して廃水から炭素と窒素汚染物質を除去する方法を開示し、前記文献で開示された栄養アンモニア酸化バクテリアは、バチルス・シュードファーマスＮＨ−２（Bacillus pseudofimus NH-2）、アルスロバクター・グロビフォルミスＷＲ−２（Arthrobacter globiformis ER-2）を意味する。上記の文献で開示されたニトロコッカスとニトロスピラはＧＲＡＳ菌株ではないためプロバイオティクスとして使用できず、古細菌に属するため生産が非常に難しいので、産業上の利用が不可能である。また、ＧＲＡＳ菌株と知られているバチルス・シュードファーマスＮＨ−２はプロバイオティクスとして登録されてない菌株である。
Examining prior art related to substances and microorganisms used for the purpose of water quality improvement, Patent Document 1 discloses a method for preventing / mitigating nitrite accumulation in an aquatic environment using nitrite-oxidizing bacteria. . The nitrite-oxidizing bacteria are nitrococcus and nitrospira. Patent Document 2 discloses a method for removing carbon and nitrogen pollutants from wastewater using heterotrophic ammonia-oxidizing bacteria, and the nutrient-ammonia oxidizing bacteria disclosed in the above-mentioned literature is Bacillus pseudopharmaceus NH-. 2 (Bacillus pseudofimus NH-2), Arthrobacter globiformis ER-2. Nitrococcus and nitrospira disclosed in the above literature cannot be used as probiotics because they are not GRAS strains, and because they belong to archaea, they are very difficult to produce, and therefore cannot be used industrially. In addition, Bacillus pseudopharmas NH- 2, which is known as a GRAS strain, is a strain that is not registered as a probiotic.
本発明者らは、韓国の伝統的発酵食品であるテンジャンから、消化酵素活性を有してアンモニアと亜硝酸を酸化させる菌株を分離し、当該菌株の形態学的、生化学的、遺伝的特性を確認し、本発明を完成させた。
The present inventors isolated a strain that has digestive enzyme activity and oxidizes ammonia and nitrous acid from Tenjang, a traditional Korean fermented food, and the morphological, biochemical and genetic characteristics of the strain. Make sure, it has led to the completion of the present invention.
本発明の新たに分離した菌株は、通常的なバチルス菌株の培養方法により培養することができる。培地は、天然培地、又は合成培地を用いることができる。培地の炭素原は、例えば、グルコース、スクロース、デキストリン、グリセロール、デンプンなどがあり、窒素原としてペプトン、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があるが、これらの成分に限らない。培地に含まれる無機塩は、マグネシウム、マンガン、カルシウム、鉄、燐などがあるが、これらの成分に限らない。前記炭素原、窒素原、及び無機塩の成分以外に、アミノ酸、ビタミン、核酸、及びそれに関わる化合物が培地に添加され得る。本発明の新たに分離した菌株は、２０〜４０℃の温度条件範囲で１２時間〜４日間培養することができる。
The newly isolated strain of the present invention can be cultured by a usual method for culturing Bacillus strains. As the medium, a natural medium or a synthetic medium can be used. The carbon source of the medium includes, for example, glucose, sucrose, dextrin, glycerol, starch, etc., and peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate, and other organics as nitrogen source -There are inorganic nitrogen-containing compounds, but not limited to these components. Examples of inorganic salts contained in the medium include magnesium, manganese, calcium, iron, and phosphorus , but are not limited to these components. In addition to the carbon source, nitrogen source, and inorganic salt components, amino acids, vitamins, nucleic acids, and related compounds may be added to the medium. The newly isolated strain of the present invention can be cultured in a temperature condition range of 20 to 40 ° C. for 12 hours to 4 days.
具体的に、新たに分離した菌株の培養液は菌体を含む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。前記培養液の組成には通常のバチルス培養に必要な成分だけでなく、バチルスの生長に上昇的に作用する成分をさらに含むこともでき、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。
Specifically, the culture solution of the newly isolated strain can be a culture stock solution containing bacterial cells, a solution obtained by removing the culture supernatant from the culture stock solution, and / or a concentrated solution thereof. . The well components needed for normal Bacillus culture on the composition of the culture medium, can also be included in the al rising-acting component to growth of Bacillus, skilled in it by the art compositions It can be easily selected by those who have.
通常的に、バチルス種は、内生胞子を形成し熱に非常に安定的特徴を有する。したがって、新たに分離した前記バチルス・リケニフォルミスＣＪＭＰＢ２８３は、飼料添加剤形態に別に製造して飼料に混合するか、飼料製造のときに直接添加して製造することができる。本発明の飼料内のバチルス・リケニフォルミスＣＪＭＰＢ２８３は、液状、又は乾燥状態であることができ、好ましくは乾燥された粉末形態である。乾燥方法は、通風乾燥、自然乾燥、噴霧乾燥、凍結乾燥ができるが、これらに限定されない。本発明のバチルス・リケニフォルミスＣＪＭＰＢ２８３は粉末形態で、飼料重量の０．０５ないし１０重量％、好ましくは０．１ないし１重量％の成分比で混合できる。また、前記水産養殖用の飼料添加剤は、本発明の他に飼料の保存性を高められる通常の添加剤をさらに含むことができる。
Usually, Bacillus species form endospores and have very stable characteristics to heat. Therefore, the newly separated Bacillus licheniformis CJMPB283 can be produced separately in the form of a feed additive and mixed with the feed, or can be directly added during the production of the feed. The Bacillus licheniformis CJMPB283 in the feed of the present invention can be in a liquid or dry state, preferably in a dried powder form. The drying method can be, but is not limited to, air drying, natural drying, spray drying, and freeze drying. The Bacillus licheniformis CJMPB283 of the present invention can be mixed in a powder form at a component ratio of 0.05 to 10% by weight, preferably 0.1 to 1% by weight of the feed weight. The feed additive for the aquaculture may further include in addition to conventional additives for increasing the storage stability of the feed of the present invention.
本発明の飼料には、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、単細胞タンパク質、動物性プランクトン類、魚粉などがあり、これらに限定されない。
In the feed of the present invention, cereals, root fruits, food processing by-products, algae, fibers, fats and oils, starches, fukube, cereal by-products, etc. as animals, such as proteins, inorganics s, fats and oils, mineral acids, single cell protein, animal plankton, include fish meal, and the like.
本発明の飼料添加剤を含む水産養殖用飼料の配合組成物は、前記飼料添加剤を動物飼料に乾燥重量を基準として０．０５ないし０．５重量％混合する。
In the composition for aquaculture feed comprising the feed additive of the present invention, the feed additive is mixed with animal feed at 0.05 to 0.5% by weight based on the dry weight.
本発明の新たに分離したバチルス・リケニフォルミスＣＪＭＰＢ２８３は、水産養殖環境内に存在するアンモニアと亜硝酸の含量を減少させることに利用できる。
Bacillus licheniformis CJMPB283 freshly isolated of the present invention can be used in the this to reduce the content of ammonia and nitrite present in aquaculture environment.
実施例１：アンモニアと亜硝酸の酸化菌株を分離
（１）試料の用意と菌株の分離
韓国の伝統食品であるテンジャン、メジュ、醤油などから名品醤類由来菌株を用意した。用意した試料を３％塩化ナトリウムが添加されたＢＨＩ個体培地（Ｄｉｆｃｏ、ＵＳＡ）に塗抹した後、３７℃の条件で２４時間培養した。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て菌株を分離した。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て培養する方法により再分離し、純粋培養した菌を２０％グリセロールが添加された培地に入れ、−７０℃以下で保存した。
Example 1: Separation of oxidized strains of ammonia and nitrous acid (1) Preparation of samples and isolation of strains A famous soy sauce-derived strain was prepared from Korean traditional foods such as Tenjang, Medju and soy sauce. The prepared sample was smeared on a BHI solid medium (Difco, USA) supplemented with 3% sodium chloride, and then cultured at 37 ° C. for 24 hours. Strains isolated from each sample were grouped by colony observation to isolate the strains. The selected colonies were transferred to a new medium three times and re- separated by a method of culturing, and the purely cultured bacteria were placed in a medium supplemented with 20% glycerol and stored at −70 ° C. or lower.
その結果、図１のとおり、アンモニア含有培地で選抜菌株１０種すべて培養上澄液内のアンモニア含量が減少したことを確認でき、特に２８３番、２９６番、３０３番、３９６番の菌株は培養初期である６時間目からアンモニアの含量が急激に減っていくことを確認した。アンモニアの酸化により亜硝酸が生成されたかを確認するため、亜硝酸が存在するとき色反応を表す指示薬を添加して色反応を観察した結果、アンモニアを含む初期培地からは色変化観察されず、培養が終了したとき採取した各サンプルでは、培養上澄液の色が全て濃い紫色に変わった。これによって、１次選抜菌株は全てアンモニアを亜硝酸に酸化させることがわかった。
As a result, as shown in FIG. 1, it was confirmed that the ammonia content in the culture supernatant was reduced in all 10 selected strains in the ammonia-containing medium. In particular, the strains 283, 296, 303, and 396 were in the initial stage of culture. It was confirmed that the ammonia content rapidly decreased from the 6th hour. To confirm whether nitrite has been generated by the oxidation of ammonia is not color change observed from an initial medium containing results of observation of the color reaction by addition of an indicator representative of the color reaction, ammonia when nitrite is present, In each sample collected when the culture was completed, the color of the culture supernatant changed to dark purple. As a result, it was found that all the primary selected strains oxidize ammonia to nitrous acid.
（２）１次選抜菌株における亜硝酸の定量評価
１次選抜菌株の亜硝酸酸化による低減効果を評価するため、前記選抜菌株１０種に対して、亜硝酸が添加された培地から定量して亜硝酸の消耗量を定量した。
(2) Quantitative evaluation of nitrite in primary selected strains In order to evaluate the reduction effect of nitrite oxidation on primary selected strains, quantified from the medium added with nitrous acid for the 10 selected strains. The amount of nitric acid consumed was quantified.
その結果、図２に示したとおり、亜硝酸含有培地で選抜菌株１０種は全て培養上澄液内の亜硝酸含量が減少したことを確認でき、特に１０２番、１０９番、２５１番、２５３番、２６８番の菌株は培養初期の６時間目から亜硝酸の含量が急激に減っていくことを確認した。亜硝酸の酸化有無をするため、亜硝酸が存在するとき色反応を表す指示薬を添加して色反応を観察した結果、亜硝酸を含有する初期培地では濃い紫色が観察され、培養が終了して採取した各サンプルでは培養上澄液の変色がなかった。即ち、次選抜菌株が亜硝酸を酸化させることにより、亜硝酸が酸化されて存在しないことが分かった。
As a result, as shown in FIG. 2, all the selected strains 10 or nitrite-containing medium can be confirmed that the nitrite content of the culture supernatant liquid inside is reduced, in particular 102 number, 109 number, 251 number, 253 No. 268 strain confirmed that the content of nitrous acid decreased sharply from the 6th hour of the initial culture. In order to check whether or not nitrous acid is oxidized, an indicator indicating the color reaction was added when nitrous acid was present and the color reaction was observed. As a result, a dark purple color was observed in the initial medium containing nitrous acid, and the culture was terminated. In each sample collected, there was no discoloration of the culture supernatant. That is, it was found that nitrite was oxidized and was not present when the next selected strain oxidized nitrous acid.
（３）生長性の評価
水質環境は、好気、通性嫌気、嫌気的条件が共存し、特に嫌気的条件でアンモニアが硝酸に酸化するアナモックス（Anammox、嫌気的条件でアンモニアが硝酸に酸化する過程）が行われるため選抜菌株が多様な条件で生長することは重要なのである。そして、１次選抜菌株１０種に対し、通性嫌気と嫌気条件においての生長性を評価した。
(3) Evaluation of growth The water quality environment is anaerobic, facultative anaerobic, and anaerobic conditions. Anammox, where ammonia is oxidized to nitric acid under anaerobic conditions (Anammox, ammonia is oxidized to nitric acid under anaerobic conditions) it is important that selection strains for process) is performed to grow a variety of conditions. And the growth property in facultative anaerobic and anaerobic conditions was evaluated with respect to 10 types of primary selection strains.
１次選抜菌株１０種をＢＨＩ液体培地に０．１％ずつ接種した後、３７℃の通性嫌気、嫌気的条件で２４時間培養を行った。選抜菌株の大部分は通性嫌気、嫌気的条件で全て生長することを確認し、特に２５３番と２６８番の菌株が嫌気的条件における生長性が優秀であり、２８３番の菌株は通性嫌気的条件における生長性が最も優秀であった（図３）。そして、通性嫌気と嫌気的条件においての生長性が優秀であった２５３番、２６８番、２８３番の菌株を２次選抜した。
After inoculating 0.1% each of 10 primary selected strains in BHI liquid medium, the cells were cultured for 24 hours under 37 ° C. facultative and anaerobic conditions. Most of the selected strains were confirmed to grow under facultative anaerobic and anaerobic conditions. Especially, strains 253 and 268 were excellent in anaerobic conditions, and strain 283 was facultative anaerobic. The growth was the best under the conditions (Fig. 3). Then, strains Nos. 253, 268 and 283, which were excellent in facultative anaerobic and anaerobic conditions, were secondarily selected.
実施例３：in vivo評価と最終菌株選抜
２次選抜された菌株３種に対する水質浄化能力をヒラメ稚魚で評価した。ヒラメ稚魚に市販飼料を満腹で供給した後、実験水槽（６４Ｌ）でランダムに配置した。実験魚を鉢した後、全ての実験飼育水槽の飼育水を綺麗に換水し、０時間、２４時間、４８時間、７２時間、９２時間、１２０時間目に各々５０ｍｌずつ飼育水をサンプリングした。２次選抜菌株の投与の２４時間の間隔で約３ｍｌに１ｘ１０５ＣＦＵ菌数になるよう希釈した後、水槽に５日間投与した。対照区の場合は同量の海水を投与した。サンプルを採取してアンモニアの濃度を分析し、分析して残った海水サンプルは−２０℃で保管した後、亜硝酸塩と硝酸塩の分析に用いた。水質分析は、アンモニア、亜硝酸塩、硝酸塩の３つの項目を分析した。これらの分析は水質分析器（RQflex 10、Merck、ドイツ）を利用し、キットの試薬と各々反応した後分析した。
Example 3: In vivo evaluation and final strain selection The water purification ability of three secondary strains was evaluated with flounder larvae. After supplying commercial feed to flounder larvae with a full stomach, they were randomly placed in an experimental water tank (64 L). After potting the experimental fish, the breeding water in all experimental breeding tanks was neatly changed, and 50 ml of the breeding water was sampled at 0, 24, 48, 72, 92, and 120 hours. After diluting to about 3 ml at a 24-hour interval after administration of the secondary selection strain to 1 × 10 5 CFU bacteria count, it was administered to a water tank for 5 days. In the case of the control group, the same amount of seawater was administered. A sample was collected and analyzed for ammonia concentration, and the seawater sample remaining after analysis was stored at −20 ° C. and then used for analysis of nitrite and nitrate. The water quality analysis analyzed three items, ammonia, nitrite, and nitrate. These analyzes were carried out using a water quality analyzer (RQflex 10, Merck, Germany) and reacting with each reagent of the kit.
実施例４：選抜菌株の同定と形態学的・生化学的特性の調査
（１）形態学的、生化学的特性の調査
最終選抜された菌株の種定のため、１次的に形態学的、生化学的調査を行った。形態的特徴において、グラム染色結果グラム陽性であり、電子顕微鏡撮影の結果桿菌であることを確認した（図６）。生化学的特性を分析するためＡＰＩ５０ ＣＨＢ system（Biomerieux、フランス）で菌株の糖発酵パターンを分析した（表２）。
Example 4: Identification of selected strains and investigation of morphological and biochemical characteristics (1) Investigation of morphological and biochemical characteristics Primary morphological analysis for the selection of the final selected strains A biochemical investigation was conducted. In morphological characteristics, the Gram staining result was Gram positive, and as a result of electron microscopic photography, it was confirmed that it was Neisseria gonorrhoeae (FIG. 6). To analyze the biochemical properties, the sugar fermentation pattern of the strain was analyzed with the API50 CHB system (Biomerieux, France) (Table 2).
（２）菌株の同定
正確な菌株同定をするため、ＤＮＡ塩基配列による分子系統分類学的方法を行った。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’ＧＧYＴＡＣＣＴＴＧＴＴＡＣＧＡＣＴＴ 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。分離菌株の分析された１６ｓ ｒＤＮＡの塩基配列は配列番号１に示す。前記分析結果、バチルス・リケニフォルミスと９６％の相同性を有する微生物として同定された。よって、選抜された前記菌株をバチルス・リケニフォルミスＣＪＭＰＢ２８３に命名し、前述の方法により同定された本発明の新たな微生物は、２０１２年３月２２日、韓国微生物保存センターに、"Bacillus licheniformis ＣＪＭＰＢ２８３"として寄託された（寄託番号ＫＣＣＭ１１２７０Ｐ）。
( 2 ) Identification of strains In order to accurately identify strains, molecular phylogenetic methods based on DNA base sequences were performed. For the nucleotide sequence analysis, a 16s rDNA gene was amplified using PCR premix (Bionia, Korea) and universal primer 27F (5′AGAGTTTGATCMTGGCTCAG3 ′: SEQ ID NO: 2) and 1492R (5′GGYTACCTTTTACGAACTT 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 the isolated strain is shown in SEQ ID NO: 1. As a result of the analysis, the microorganism was identified as 96% homologous to Bacillus licheniformis. Therefore, the selected strain was named Bacillus licheniformis CJMPB283, and a new microorganism of the present invention identified by the above-mentioned method was designated as “Bacillus licheniformis CJMPB283” on March 22, 2012 at the Korea Microbiology Conservation Center. Deposited (deposit number KCCM11270P).
４）アミラーゼ（Amylase）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３μｌずつ基質培地に分株し、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（clear zone）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 auxiliary enzyme solution collected above was aliquoted to a substrate medium by 3 [mu] l, it was reacted for 15 hours at 37 ° C.. After staining with an aqueous solution in which I 2 and KI were added at 0.1% and 2%, respectively, the activity of the enzyme was measured by the degree of formation of a clear zone formed by decomposition of the substrate around the coenzyme solution. .
Claims (6)
The method for improving water quality of the farm, wherein the water quality improving agent according to claim 5 is distributed before or during the farming in the farm.
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| PCT/KR2013/002829 WO2013151364A1 (en) | 2012-04-05 | 2013-04-04 | Novel separated bacillus licheniformis and probiotics using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2020506872A (en) * | 2017-12-29 | 2020-03-05 | シージェイ チェイルジェダン コーポレイション | Feed composition for preventing or treating acute hepatopancreatic necrosis (AHPND) or white spot disease syndrome (WSS) comprising Bacillus subtilis strain as an active ingredient |
| JP2020127383A (en) * | 2019-02-08 | 2020-08-27 | 学校法人 東洋大学 | Culture method, culture apparatus, waste water treatment method, and waste water treatment apparatus |
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| CN104304142B (en) * | 2014-09-26 | 2017-03-29 | 浙江海洋学院 | A kind of environmental improvement method to Penaeus penicillatus (Alcock). nursery pond |
| WO2019132600A1 (en) * | 2017-12-29 | 2019-07-04 | 씨제이제일제당 (주) | Feed composition containing bacilius subtilus strain, bacilius pumilus strain, and bacilius lichenformis strain as active ingredients for preventing or treating acute hepatopancreatic necrosis disease or white spot syndrome |
| KR102242160B1 (en) | 2019-10-14 | 2021-04-20 | 단국대학교 천안캠퍼스 산학협력단 | Novel Bacillus subtilis MKHJ 1-1 and uses thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020506872A (en) * | 2017-12-29 | 2020-03-05 | シージェイ チェイルジェダン コーポレイション | Feed composition for preventing or treating acute hepatopancreatic necrosis (AHPND) or white spot disease syndrome (WSS) comprising Bacillus subtilis strain as an active ingredient |
| JP2020506873A (en) * | 2017-12-29 | 2020-03-05 | シージェイ チェイルジェダン コーポレイション | Feed composition for preventing or treating acute hepatopancreatic necrosis (AHPND) or white spot disease syndrome (WSS), comprising Bacillus subtilis strain, Bacillus pumilus strain and Bacillus licheniformis strain as active ingredients |
| US11432566B2 (en) | 2017-12-29 | 2022-09-06 | Cj Cheildejang Corporation | Feed composition for preventing or treating acute hepatopancreatic necrosis disease (AHPND) or white spot syndrome (WSS), comprising a Bacillus subtilis strain, a Bacillus pumilus strain, and a Bacillus licheniformis strain as active ingredients |
| JP2020127383A (en) * | 2019-02-08 | 2020-08-27 | 学校法人 東洋大学 | Culture method, culture apparatus, waste water treatment method, and waste water treatment apparatus |
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| MY174997A (en) | 2020-06-01 |
| JP6129294B2 (en) | 2017-05-17 |
| WO2013151364A1 (en) | 2013-10-10 |
| PH12014502245B1 (en) | 2014-12-15 |
| KR20130113108A (en) | 2013-10-15 |
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