JP2020191793A - Experiment non-human animal and use thereof - Google Patents
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Abstract
Description
本発明は、異種動物の腸内細菌叢に含まれる細菌群を腸内細菌として有する実験用非ヒト動物及びその使用に関する。 The present invention relates to an experimental non-human animal having a bacterial group contained in the intestinal flora of a heterologous animal as an intestinal bacterium and its use.
ヒトや動物の腸管内には多種多様な腸内細菌が生息している。一般的に、健全なヒトの成人の腸管内には、１０００種類以上、総数４０兆個以上にもおよぶ腸内細菌が生息していると言われており、これはおよそ３７兆個と言われるヒトの細胞数を上回っている。このような膨大な数の腸内細菌が、互いに相互作用を及ぼし合いながら一定のバランスが保たれた複雑な生態系を宿主の腸管内で形成しており、腸内細菌叢（腸内フローラ）と呼ばれる。また、腸管は、食餌や外界からの病原微生物等の異物に常にさらされる場所であり、消化吸収器官であると同時に、多くの免疫細胞が集中する免疫器官でもある。腸内細菌叢を構成する細菌群の多くは難培養性で解析困難であったが、近年、次世代シーケンサーの登場等により、複合微生物やその代謝物等の評価を行うオミクス解析技術が発展し、腸内細菌叢についての解明が進みつつある。その中で、腸内細菌叢は、宿主の代謝・生理機能や免疫機能とも大きく関連していることが明らかになってきた。 A wide variety of intestinal bacteria inhabit the intestinal tract of humans and animals. In general, it is said that the intestinal tract of a healthy human adult is inhabited by more than 1000 types and a total of more than 40 trillion intestinal bacteria, which is said to be about 37 trillion. It exceeds the number of human cells. Such a huge number of intestinal bacteria form a complex ecosystem in the host's intestinal tract that interacts with each other and maintains a certain balance, and the intestinal flora (intestinal flora). Is called. In addition, the intestinal tract is a place that is constantly exposed to foreign substances such as food and pathogenic microorganisms from the outside world, and is a digestive and absorptive organ as well as an immune organ in which many immune cells are concentrated. Many of the bacterial groups that make up the intestinal flora are difficult to culture and difficult to analyze, but in recent years, with the advent of next-generation sequencers, omics analysis technology for evaluating complex microorganisms and their biotransformers has developed. , The elucidation of the intestinal flora is progressing. Among them, it has become clear that the intestinal flora is closely related to the metabolic / physiological function and immune function of the host.
宿主が健康な場合、腸内細菌叢のバランスはおおよそ一定で、恒常性が保たれている。しかし、遺伝的あるいは過度の外的環境要因によりその恒常性が破綻し、腸内細菌叢のバランスが乱れると、炎症性腸疾患や大腸がんといった腸の疾患のみならず、自己免疫疾患や代謝疾患といった全身性の疾患にもつながりうる（例えば、非特許文献１参照）。このように、腸内細菌叢は、宿主と相互作用し、宿主の健康に大きく関与していることから、近年、薬剤やプロバイオティクス等によって腸内細菌叢を制御して宿主の腸内環境を整えるための技術に関心が寄せられている。これらの薬剤やプロバイオティクス等の安全性や効能等の評価を行う場合、対象とする動物を用いて投与実験を行うことが最も好ましい。しかし、対象とする動物が、産業動物や愛玩動物のような動物実験に用いるのに困難性を有する動物である場合は、その代替として、マウス等の実験用動物を用いて評価を行っているのが現状である。 When the host is healthy, the balance of the gut flora is approximately constant and homeostatic. However, when the homeostasis is disrupted by genetic or excessive external environmental factors and the balance of the intestinal flora is disturbed, not only intestinal diseases such as inflammatory bowel disease and colon cancer, but also autoimmune diseases and metabolism It can also lead to systemic diseases such as diseases (see, for example, Non-Patent Document 1). In this way, the intestinal flora interacts with the host and is greatly involved in the health of the host. Therefore, in recent years, the intestinal flora is controlled by drugs, probiotics, etc. to control the intestinal environment of the host. There is an interest in the technology for preparing the host. When evaluating the safety and efficacy of these drugs and probiotics, it is most preferable to carry out administration experiments using the target animals. However, if the target animal is an animal that is difficult to use in animal experiments such as industrial animals and pet animals, an experimental animal such as a mouse is used as an alternative for evaluation. is the current situation.
また、プロバイオティクスは、乳酸菌のような常温領域で活動する中温菌が用いられることが主であるため、中温菌が動物の生体に与える影響については盛んに研究が行われているが、５０℃以上の高い温度領域で活動する好熱菌が動物の生体について与える影響についてはあまり研究されていなかった。一方、本発明者らは、甲殻類や魚類等の海産物資源を７５℃前後の高温下で発酵させて得られる高温発酵物中に含まれる好熱性複合微生物群が、動植物に与える影響について長年研究を行ってきた。その一連の研究の中で、その好熱性複合微生物群から、新種の好熱菌バチルス・ヒサシイ（Bacillus hisashii）N-11T株（受託番号：NITE BP-863）の単離に成功しており、この好熱菌バチルス・ヒサシイ（Bacillus hisashii）N-11T株をマウスや豚に経口投与することにより、当該マウスや豚の腸内細菌叢のポピュレーションが変化し、粘膜免疫の賦活化や内臓脂肪の蓄積軽減といった代謝機能や免疫機能に好影響をもたらすことを明らかにしている（特許文献１及び非特許文献２参照）。 In addition, since probiotics mainly use thermophiles that are active in the normal temperature range, such as lactic acid bacteria, the effects of thermophiles on the living body of animals have been actively studied. Little research has been done on the effects of thermophiles operating in the high temperature range above ° C on the living body of animals. On the other hand, the present inventors have long studied the effects of thermophilic complex microorganisms contained in high-temperature fermented products obtained by fermenting marine resources such as crustaceans and fish at a high temperature of around 75 ° C. on animals and plants. I went to. In the series of studies, a new species of thermophile Bacillus hisashii N-11 T strain (accession number: NITE BP-863) was successfully isolated from the thermophilic complex microbial community. By oral administration of this thermophile Bacillus hisashii N-11 T strain to mice and pigs, the population of the intestinal flora of the mice and pigs is changed, and mucosal immunity is activated. It has been clarified that it has a positive effect on metabolic function and immune function such as reduction of visceral fat accumulation (see Patent Document 1 and Non-Patent Document 2).
上記のように、プロバイオティクスや薬剤等の評価を行う場合に、対象とする動物の代替として、マウス等の実験用動物が用いられることがある。しかし、従来の実験用動物は、その実験用動物本来の腸内細菌叢を有するもの、または無菌のものが用いられ、対象とする動物の腸内細菌叢に関する観点が無かった。上記のように、腸内細菌叢は、宿主の代謝機能、生理機能、病態等に大きく関連しているため、対象とする動物の腸内細菌叢の特徴を有しない従来の実験用動物では、対象とする動物の腸内細菌叢に依存した影響を評価できずに、実験用動物で得られた結果が、対象とする動物では確認できない可能性があった。 As described above, when evaluating probiotics, drugs, etc., experimental animals such as mice may be used as a substitute for the target animal. However, as the conventional laboratory animals, those having the original intestinal flora of the laboratory animals or those which are sterile are used, and there is no viewpoint regarding the intestinal flora of the target animal. As described above, since the intestinal flora is greatly related to the metabolic function, physiological function, pathological condition, etc. of the host, in conventional experimental animals that do not have the characteristics of the intestinal flora of the target animal, It was not possible to assess the intestinal flora-dependent effects of the animals of interest, and the results obtained in the experimental animals could not be confirmed in the animals of interest.
一方、上記のように、本発明者らは好熱菌バチルス・ヒサシイ（Bacillus hisashii）N-11T株をマウスや豚に投与することで、当該マウスや豚の腸内細菌叢を改善して代謝機能に好影響を与えることを見出した。しかし、異種動物の腸内細菌叢を移植された動物に対してバチルス・ヒサシイを投与した場合の知見は無かった。 On the other hand, as described above, the present inventors improved the intestinal flora of the mice and pigs by administering the thermophile Bacillus hisashii N-11 T strain to the mice and pigs. It was found to have a positive effect on metabolic function. However, there was no finding when Bacillus hisashii was administered to animals transplanted with the intestinal flora of heterologous animals.
本発明は上記事情に鑑みてなされたものであって、異種動物の腸内微生物叢の特徴を備えた腸内微生物叢を有しながら、少なくとも代謝機能が健全に保たれており、当該異種動物の腸内微生物叢の影響を評価可能なモデル動物として用いることができる実験用非ヒト動物を提供することを目的とする。 The present invention has been made in view of the above circumstances, and the heterologous animal has at least a healthy metabolic function while having an intestinal microflora having the characteristics of the intestinal microflora of a heterologous animal. It is an object of the present invention to provide an experimental non-human animal that can be used as a model animal capable of evaluating the influence of the intestinal microflora.
上記課題を解決するために、本発明に係る実験用非ヒト動物は、無菌非ヒト動物に異種動物の腸内細菌叢に含まれる細菌群とバチルス・ヒサシイ（Bacillus hisashii）とが投与されてなることを特徴とする。 In order to solve the above problems, the experimental non-human animal according to the present invention comprises a sterile non-human animal administered with a bacterial group contained in the intestinal flora of a heterologous animal and Bacillus hisashii. It is characterized by that.
好ましくは、前記細菌群が、乳酸菌を含むことを特徴とする。 Preferably, the bacterial group is characterized by containing lactic acid bacteria.
好ましくは、前記乳酸菌が、ラクトバチルス属（Lactobacillus）、ビフィドバクテリウム属（Bifidobacterium）、ストレプトコッカス属（Streptococcus）及びエンテロコッカス属（Enterococcus）のうち１種類以上の細菌を含むことを特徴とする。 Preferably, the lactic acid bacterium is characterized by containing one or more of the genus Lactobacillus, the genus Bifidobacterium, the genus Streptococcus and the genus Enterococcus.
好ましくは、さらに乳酸資化菌を含むことを特徴とする。 Preferably, it further contains a lactic acid assimilating bacterium.
好ましくは、前記乳酸資化菌が、メガスファエラ属（Megasphaera）及びセレノモナス属（Selenomonas）のうち１種類以上の細菌を含むことを特徴とする。 Preferably, the lactic acid assimilating bacterium is characterized by containing one or more of the genus Megasphaera and the genus Selenomonas.
好ましくは、前記細菌群が、クロストリジウム属クラスターＸＩ（Clostridium cluster XI）、バクテロイデス属（Bacteroides）、プレボテラ属（Prevotella）、アロバキュラム属（Allobaculum）、ディアリスター属（Dialister）、トゥリシバクター属（Turicibacter）、コプロコッカス属（Coprococcus）、ミツオケラ属（Mitsuokella）及びメタノブレビバクター属（Methanobrevibacter）のうち１種類以上の細菌を含むことを特徴とする。 Preferably, the bacterial group is Clostridium cluster XI, Bacteroides, Prevotella, Allobaculum, Dialister, Turicibacter. , Coprococcus, Mitsuokella, and Methanobrevibacter, which are characterized by containing one or more types of bacteria.
好ましくは、前記非ヒト動物が、マウス、ラット及びモルモットのいずれかから選択されることを特徴とする。 Preferably, the non-human animal is selected from any of mice, rats and guinea pigs.
好ましくは、前記異種動物が、産業動物または愛玩動物から選択されることを特徴とする。 Preferably, the heterologous animal is selected from industrial animals or pet animals.
好ましくは、前記産業動物または愛玩動物が、豚、鶏、牛及び犬のいずれかであることを特徴とする。 Preferably, the industrial animal or pet animal is any of pigs, chickens, cows and dogs.
無菌非ヒト動物に異種動物の腸内細菌叢に含まれる細菌群とバチルス・ヒサシイ（Bacillus hisashii）とが投与されてなる本発明に係る実験用非ヒト動物が、前記異種動物の腸内環境の挙動及び生理的影響を評価するモデル動物として使用されることを特徴とする。 The experimental non-human animal according to the present invention, which comprises a sterile non-human animal administered with a bacterial group contained in the intestinal flora of a heterologous animal and Bacillus hisashii, is a non-human animal of the heterogeneous animal in the intestinal environment. It is characterized by being used as a model animal for evaluating behavior and physiological effects.
無菌非ヒト動物に異種動物の腸内細菌叢に含まれる細菌群とバチルス・ヒサシイ（Bacillus hisashii）とが投与されてなる本発明に係る実験用非ヒト動物が、前記異種動物に被検物質の投与実験を行う前段階として前記被験物質のスクリーニングを行うモデル動物として使用されることを特徴とする。 The experimental non-human animal according to the present invention, which comprises administering a bacterial group contained in the intestinal flora of a heterologous animal and Bacillus hisashii to a sterile non-human animal, is a test substance of the heterologous animal. It is characterized in that it is used as a model animal for screening the test substance as a pre-stage for performing an administration experiment.
本発明に係る実験用非ヒト動物は、無菌非ヒト動物に異種動物の腸内細菌叢に含まれる細菌群とバチルス・ヒサシイ（Bacillus hisashii）とが投与されてなることを特徴としており、バチルス・ヒサシイの投与の効果により、本発明に係る実験用非ヒト動物は、少なくとも代謝機能が健全に保たれた状態で、異種動物の腸内細菌叢に含まれる細菌群を、腸内で腸内細菌叢として維持することができる。無菌非ヒト動物に投与される異種動物の腸内細菌叢に含まれる細菌群として、異種動物の腸内細菌叢それ自体、異種動物の腸内細菌叢の一部の特徴的な優占菌群、またはそれら優占菌群を構成する各細菌の標準菌株により構成された細菌群を用いることができる。また、投与される細菌群には異種動物の生理的変化と呼応する特徴的な菌群を含めても良い。それによって本発明に係る実験用非ヒト動物の腸内細菌叢は、当該異種動物の腸内細菌叢の特徴が模倣されたものとなる。従って、本発明に係る実験用非ヒト動物は、異種動物の腸内環境の挙動や生理的影響を評価するためのモデル動物として用いることができる。動物の腸内細菌叢の構成は、動物種、地域、食餌、生育段階等によって異なるため、それらの異なる腸内細菌叢の特徴を模倣した本発明に係る実験用非ヒト動物を用いることで、腸内細菌叢の特性を考慮したオーダーメード型の薬剤、栄養剤、サプリメント、生菌材等の開発や運用が可能となる。また、非ヒト動物に、マウス、ラットまたはモルモットのような容易に入手及び管理可能な動物を用い、異種動物に、豚、牛、鶏等の産業動物や、犬等の愛玩動物のような高価で管理が煩雑な動物や動物愛護の観点から動物実験が困難な動物を用いることにより、安価かつ簡便に当該異種動物の腸内細菌叢の影響を加味した生体実験を行うことができる。従って、産業動物や愛玩動物に対して、目的とする薬剤、栄養剤、サプリメント、生菌材等の投与試験を最初から実施するのではなく、その前段階として当該実験用非ヒト動物を用いてスクリーニングを実施して、比較的簡便かつ安価に生体への影響を評価することができる。それによって、産業動物や愛玩動物に対して投与する成功確率の高い薬剤、栄養剤、サプリメント、生菌材等の選抜が可能となり、コスト、試験の難易度、評価の難しさという点を軽減することができる。 The experimental non-human animal according to the present invention is characterized in that a sterile non-human animal is administered with a bacterial group contained in the intestinal flora of a heterologous animal and Bacillus hisashii. Due to the effect of administration of Hisashii, the experimental non-human animal according to the present invention has a group of bacteria contained in the intestinal flora of a heterologous animal, at least in a state where the metabolic function is maintained in a healthy state. It can be maintained as a flora. As a bacterial group contained in the intestinal flora of a heterologous animal administered to a sterile non-human animal, the intestinal flora of the heterogeneous animal itself itself is a characteristic dominant bacterial group of a part of the intestinal flora of the heterogeneous animal. , Or a bacterial group composed of standard strains of each bacterium constituting those dominant bacterial groups can be used. In addition, the group of bacteria to be administered may include a characteristic group of bacteria that responds to physiological changes in different animals. As a result, the intestinal flora of the experimental non-human animal according to the present invention mimics the characteristics of the intestinal flora of the heterologous animal. Therefore, the experimental non-human animal according to the present invention can be used as a model animal for evaluating the behavior and physiological effects of the intestinal environment of different animals. Since the composition of the intestinal flora of an animal differs depending on the animal species, region, diet, growth stage, etc., by using the experimental non-human animal according to the present invention that mimics the characteristics of these different intestinal flora. It will be possible to develop and operate custom-made drugs, nutritional supplements, supplements, viable bacterial materials, etc. in consideration of the characteristics of the intestinal flora. In addition, for non-human animals, easily available and manageable animals such as mice, rats or guinea pigs are used, and for heterogeneous animals, industrial animals such as pigs, cows and chickens, and expensive animals such as pet animals such as dogs are used. By using animals that are complicated to manage and animals that are difficult to carry out animal experiments from the viewpoint of animal protection, it is possible to carry out biological experiments that take into account the effects of the intestinal flora of the heterologous animals inexpensively and easily. Therefore, instead of conducting an administration test of the target drug, nutritional supplement, supplement, viable bacterial material, etc. on industrial animals and pet animals from the beginning, the non-human animal for the experiment is used as a preliminary step. Screening can be performed to evaluate the effects on living organisms relatively easily and inexpensively. This makes it possible to select drugs, nutritional supplements, supplements, live bacterial materials, etc. that have a high probability of success for administration to industrial animals and pet animals, reducing costs, test difficulty, and evaluation difficulty. be able to.
本発明の一実施形態について、以下、図面を参照しつつ説明する。以下は、あくまで本発明の実施形態を例示的に示すものであるため、本発明の範囲は下記実施形態のみに限定されるものではなく、本発明の思想を逸脱しない範囲で適宜変更可能である。 An embodiment of the present invention will be described below with reference to the drawings. Since the following is merely an example of an embodiment of the present invention, the scope of the present invention is not limited to the following embodiments, and can be appropriately changed without departing from the idea of the present invention. ..
本実施形態に係る実験用非ヒト動物は、無菌非ヒト動物に、該非ヒト動物と種の異なる異種動物の腸内細菌叢に含まれる細菌群と、バチルス・ヒサシイ（Bacillus hisashii）とを投与することによって作出することができる。バチルス・ヒサシイの投与によって、無菌非ヒト動物に投与された異種動物の腸内細菌叢に含まれる細菌群が、当該非ヒト動物の腸内で腸内細菌叢として維持される。そのため、図１に示すように、本実施形態に係る実験用非ヒト動物は、該非ヒト動物と種の異なる異種動物の腸内細菌叢の特徴を模倣することができるので、当該異種動物の腸内環境の挙動や生理的影響を評価するための実験用のモデル動物として用いることができる。 The experimental non-human animal according to the present embodiment administers a bacterial group contained in the intestinal flora of a heterologous animal of a different species from the non-human animal and Bacillus hisashii to the sterile non-human animal. It can be created by. By administration of Bacillus hisashii, the bacterial group contained in the intestinal flora of a heterologous animal administered to a sterile non-human animal is maintained as an intestinal flora in the intestine of the non-human animal. Therefore, as shown in FIG. 1, the experimental non-human animal according to the present embodiment can mimic the characteristics of the intestinal flora of a heterologous animal of a different species from the non-human animal, and thus the intestine of the heterogeneous animal. It can be used as a model animal for experiments to evaluate the behavior and physiological effects of the internal environment.
本実施形態に係る非ヒト動物として、マウス、ラット及びモルモットのいずれかが好適に用いられる。これら以外の非ヒト動物を用いてもよいが、マウス、ラット及びモルモットは実験用動物として広く用いられており、無菌状態のものも安価で容易に入手あるいは作出することができ、飼育や管理も容易である。無菌非ヒト動物は、例えば、妊娠末期の該非ヒト動物の母獣の帝王切開あるいは子宮切断によって無菌状態で胎児を取り出し、無菌のアイソレータ内で無菌の飼料や飲水等によって飼育する、といったような一般的な無菌操作によって作出可能である。 As the non-human animal according to the present embodiment, any one of mouse, rat and guinea pig is preferably used. Non-human animals other than these may be used, but mice, rats and guinea pigs are widely used as experimental animals, and sterile ones can be easily obtained or produced at low cost, and can be bred and managed. It's easy. Aseptic non-human animals are generally such that, for example, a foetation is taken out in a sterile state by cesarean section or uterine cutting of the mother animal of the non-human animal at the end of pregnancy and is bred in a sterile isolator with sterile feed or drinking water. It can be produced by aseptic operation.
本実施形態に係る異種動物は、本実施形態で用いる非ヒト動物とは種の異なる動物であり、産業動物や愛玩動物のような高価で管理が煩雑であったり動物愛護の観点等から動物実験に直接用いることが困難な動物が好適である。具体的には、豚、鶏、牛、犬等が挙げられ、これらのうちの１つの動物の腸内細菌叢に含まれる細菌群が無菌非ヒト動物に投与される。 The heterologous animal according to the present embodiment is an animal of a different species from the non-human animal used in the present embodiment, and is expensive and complicated to manage such as industrial animals and pet animals, and animal experiments from the viewpoint of animal protection and the like. Animals that are difficult to use directly in are suitable. Specific examples thereof include pigs, chickens, cows, dogs and the like, and a bacterial group contained in the intestinal flora of one of these animals is administered to a sterile non-human animal.
無菌非ヒト動物に投与される異種動物の腸内細菌叢に含まれる細菌群としては、異種動物の腸内細菌叢それ自体、異種動物の腸内細菌叢に特徴的な優占菌群、及び、それら優占菌群の各細菌の標準菌株によって構成した細菌群のうちのいずれかを用いることが好適である。また、無菌非ヒト動物に投与される細菌群は、異種動物の生理的変化と呼応する特徴的な菌群を含んでいても良い。これによって、本実施形態に係る実験用非ヒト動物が有する腸内細菌叢は、当該異種動物の腸内細菌叢の特徴が模倣されたものとなる。 Bacterial groups contained in the intestinal flora of heterologous animals administered to sterile non-human animals include the intestinal flora of heterogeneous animals itself, the dominant bacterial group characteristic of the intestinal flora of heterologous animals, and , It is preferable to use any one of the bacterial groups composed of the standard strains of each bacterium of the dominant bacterial group. In addition, the bacterial group administered to sterile non-human animals may include a characteristic bacterial group that responds to physiological changes in heterologous animals. As a result, the intestinal flora of the experimental non-human animal according to the present embodiment mimics the characteristics of the intestinal flora of the heterologous animal.
図２に示すように、動物の腸内細菌叢の構成は、動物種によって異なる。そのため、目的の動物種に応じて、本実施形態に係る実験用非ヒト動物で模倣する腸内細菌叢も変化させる。図２からも分かるように、産業動物や愛玩動物の腸内細菌叢には、通常、ラクトバチルス属（Lactobacillus）、ビフィドバクテリウム属（Bifidobacterium）、ストレプトコッカス属（Streptococcus）、エンテロコッカス属（Enterococcus）といった乳酸菌が１種類以上、優占菌群として含まれていることが多い。従って、無菌非ヒト動物に投与される細菌群として、乳酸菌を有効に用いることができる。 As shown in FIG. 2, the composition of the intestinal flora of an animal varies depending on the animal species. Therefore, the intestinal flora mimicked by the experimental non-human animal according to the present embodiment is also changed according to the target animal species. As can be seen from FIG. 2, the intestinal flora of industrial animals and pet animals usually includes the genus Lactobacillus, the genus Bifidobacterium, the genus Streptococcus, and the genus Enterococcus. In many cases, one or more types of lactic acid bacteria such as are included as a dominant bacterial group. Therefore, lactic acid bacteria can be effectively used as a bacterial group to be administered to sterile non-human animals.
異種動物が豚の場合、腸内細菌叢で特徴的な乳酸菌としては、ラクトバチルス・アミロボラス（Lactobacillus amylovorus）、ラクトバチルス・アシドフィラス（Lactobacillus acidophilus）、ラクトバチルス・キタサトニス（Lactobacillus kitasatonis）、ラクトバチルス・ルミニス（Lactobacillus ruminis）、ラクトバチルス・ポンティス（Lactobacillus pontis）、ストレプトコッカス・アラクトリティカス（Streptococcus alactolyticus）及びストレプトコッカス・ヒオインテスティナリス（Streptococcus hyointestinalis）が挙げられ、これらの少なくとも１つを活用することが出来る。 When the heterogeneous animal is a pig, the characteristic lactic lactic bacteria in the intestinal flora include Lactobacillus amylovorus, Lactobacillus acidophilus, Lactobacillus kitasatonis, and Lactobacillus luminis. (Lactobacillus ruminis), Lactobacillus pontis, Streptococcus alactolyticus and Streptococcus hyointestinalis, at least one of which can be utilized. ..
異種動物が鶏の場合、腸内細菌叢に特徴的な乳酸菌としては、ラクトバチルス・ジョンソニー（Lactobacillus johnsonii）、ラクトバチルス・バギナリス（Lactobacillus vaginalis）、ラクトバチルス・クリスパタス（Lactobacillus crispatus）、ラクトバチルス・サリバリアス（Lactobacillus salivarius）、ラクトバチルス・ガリナラム（Lactobacillus gallinarum）、ラクトバチルス・アビアリエス（Lactobacillus aviaries）、ラクトバチルス・ロイテリ（Lactobacillus reuteri）、エンテロコッカス・フェカリス（Enterococcus faecalis）及びエンテロコッカス・カセリフラバス（Enterococcus casseliflavus）が挙げられ、これらの少なくとも１つを活用することが出来る。 When the heterogeneous animal is a chicken, Lactobacillus johnsonii, Lactobacillus vaginalis, Lactobacillus crispatus, Lactobacillus crispatus, Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus crispatus, Lactobacillus johnsonii, Lactobacillus crispatus, Lactobacillus salivarius, Lactobacillus gallinarum, Lactobacillus aviaries, Lactobacillus reuteri, Lactobacillus reuteri, Enterococcus enteroccus fa And at least one of these can be utilized.
異種動物が牛の場合、腸内細菌叢に特徴的な乳酸菌としては、ビフィドバクテリウム・シュードロンガム（Bifidobaciterium pseudolongum）が挙げられ、これを活用することが出来る。 When the heterologous animal is cattle, examples of lactic acid bacteria characteristic of the intestinal flora include Bifidobaciterium pseudolongum, which can be utilized.
異種動物が犬の場合、腸内細菌叢に特徴的な乳酸菌としては、ラクトバチルス・ジョンソニー（Lactobacillus johnsonii）、ラクトバチルス・ロイテリ（Lactobacillus reuteri）及びストレプトコッカス・ルテティエンシス（Streptococcus lutetiensis）が挙げられ、これらの少なくとも１つを活用することが出来る。 When the heterologous animal is a dog, Lactobacillus johnsonii, Lactobacillus reuteri, and Streptococcus lutetiensis are examples of lactic acid bacteria characteristic of the intestinal flora. , At least one of these can be utilized.
以上のように動物種によって異なる特徴的な乳酸菌を、無菌非ヒト動物に投与される細菌群に含むようにして良い。また、乳酸菌は炭水化物を消化して乳酸を産生する細菌であり、乳酸菌が腸内に生息している場合、乳酸菌によって産生された乳酸を消化して、酪酸、プロピオン酸、酢酸といった短鎖脂肪酸を産生する乳酸資化菌も生息していることが多い。乳酸資化菌によって産生される短鎖脂肪酸は、宿主の動物のエネルギー源として利用されたり、腸内を弱酸性に保って有害な細菌の増殖を抑制して腸内環境を健全に保ったりというような宿主に有益な効果をもたらすことが知られている。従って、無菌非ヒト動物に投与される細菌群に、異種動物の動物種によって特徴的な乳酸菌と併せて乳酸資化菌を含むようにして良い。ここで用いられる乳酸資化菌としては、メガスファエラ属（Megasphaera）及びセレノモナス属（Selenomonas）のうち１種類以上の乳酸資化菌を含むことが好ましい。さらに好ましくは、メガスファエラ・エルスデニー（Megasphaera elsedinii）及びセレノモナス・ボビス（Selenomonas bovis）のうち１種類以上の乳酸資化菌を含むようにして良い。 As described above, characteristic lactic acid bacteria that differ depending on the animal species may be included in the bacterial group administered to sterile non-human animals. Lactic acid bacteria are bacteria that digest carbohydrates to produce lactic acid, and when lactic acid bacteria live in the intestine, they digest lactic acid produced by lactic acid bacteria to produce short-chain fatty acids such as butyric acid, propionic acid, and acetic acid. In many cases, the lactic acid bacteria that produce it also inhabit. Short-chain fatty acids produced by lactic acid-utilizing bacteria are used as an energy source for host animals, and keep the intestines weakly acidic to suppress the growth of harmful bacteria and keep the intestinal environment healthy. It is known to have beneficial effects on such hosts. Therefore, the bacterial group administered to sterile non-human animals may include lactic acid-utilizing bacteria in addition to lactic acid bacteria characteristic of different animal species. The lactic acid-utilizing bacterium used here preferably contains one or more types of lactic acid-utilizing bacteria from the genus Megasphaera and the genus Selenomonas. More preferably, it may contain one or more lactic acid-utilizing bacteria of Megasphaera elsedinii and Selenomonas bovis.
また、異種動物の生理的変化と呼応する特徴的な菌群として、コハク酸からプロピオン酸を産生するディアリスター・サクシナティフィラス（Dialister succinatiphilus）、バクテロイデス・コプロコラ（Bacteroides coprocola）、クロストリジウム属クラスターＸＩ（Clostridium cluster XI）にカテゴライズされるクロストリジウム・マヨンベイ（Clostridium mayombei）、クロストリジウム・バテリ（Clostridium baterri）、クロストリジウム・リツセブレンス（Clostridium lituseburense）、エスエムビー（SMB）、クロストリジウム・ベイジェリンキー（Clostridium beijerinckii）、犬に特徴的なアロバキュラム・ステロコリカニス（Allobaculum stercoricanis）、環境微生物としてありふれているトゥリシバクター・サングイニス（Turicibacter sanguinis）、インシュリン抵抗性を誘導するプレボテラ・コプリ（Prevotella copri）、並びにその近縁種であるプレボテラ・ロエシェイイ（Prevotella loescheii）、プレボテラ・オウロラム（Prevotella oulorum）、脂質代謝に関与するコプロコッカス・カタス（Coprococcus catus）、ミツオケラ・ジャラルディニイ（Mitsuokella jalaludinii）、メタンガスの産生と脂質代謝に関与するメタノブレビバクター・スミスィ（Methanobrevibacter smithii）等が挙げられる。これらの菌群は、異種動物の腸内細菌叢に当該異種動物の動物種に応じて固有に含まれ、当該異種動物の固有の腸内細菌叢を起点にして、当該異種動物に特徴的な成長、脂肪蓄積、免疫系の賦活化、温暖化ガスの発生抑制等の上記のような生理的変化に寄与することが示唆されている。従って、腸内細菌叢を模倣しようとする異種動物に応じて、無菌非ヒト動物に投与される細菌群にこれらのうち１種類以上の細菌を含むようにして良い。それによって、作出される本実施形態に係る実験用非ヒト動物における異種動物の腸内細菌叢の模倣度を向上させることができる。また、これらの菌群が、本実施形態に係る実験用非ヒト動物の腸内細菌叢に定着し難い場合には、当該実験用非ヒト動物がスクリーニング等の実験に用いられるまで、当該実験用非ヒト動物にこれらの菌群を連続的に投与することによって、腸内細菌叢にこれらの菌群を含む実験用非ヒト動物を作出するようにしてもよい。 In addition, as a characteristic bacterial group that responds to physiological changes in heterologous animals, Dialister succinatiphilus, Bacteroides coprocola, and Clostridium cluster XI, which produce propionic acid from succinic acid, (Clostridium cluster XI) categorized as Clostridium mayombei, Clostridium baterri, Clostridium lituseburense, SMB, Clostridium beijerinckii Allobaculum stercoricanis, which is characteristic of Clostridium, Turicibacter sanguinis, which is a common environmental microorganism, Prevotella copri, which induces insulin resistance, and its related species, Prevotella copri. -Roescheii (Prevotella loescheii), Prevotella oulorum, Coprococcus catus involved in lipid metabolism, Mitsuokella jalaludinii, Metanobredinii involved in methane gas production and lipid metabolism Bacter Smithy (Methanobrevibacter smithii) and the like can be mentioned. These bacterial groups are uniquely contained in the intestinal flora of the heterologous animal according to the animal species of the heterologous animal, and are characteristic of the heterologous animal starting from the unique intestinal flora of the heterogeneous animal. It has been suggested that it contributes to the above-mentioned physiological changes such as growth, fat accumulation, activation of the immune system, and suppression of generation of warming gas. Therefore, the group of bacteria administered to sterile non-human animals may include one or more of these bacteria, depending on the heterologous animal attempting to mimic the gut flora. Thereby, the degree of imitation of the intestinal flora of a heterologous animal in the experimental non-human animal according to the present embodiment produced can be improved. In addition, when these bacterial groups are difficult to settle in the intestinal flora of the experimental non-human animal according to the present embodiment, the experimental non-human animal is used for the experiment until it is used for an experiment such as screening. By continuously administering these bacterial groups to non-human animals, experimental non-human animals containing these bacterial groups in the intestinal flora may be produced.
本実施形態に係る無菌非ヒト動物に投与される細菌群として、腸内細菌叢を模倣しようとする目的の異種動物の動物種に応じて、以上に示したような各動物種の腸内細菌叢に特徴的な優占菌群や当該動物種の生理的変化と呼応するような特徴的な菌群を適宜含ませるようにすることで、図３に示すように、各動物種の腸内細菌叢に特徴的な優占菌群を腸内細菌叢として有する本実施形態に係る実験用非ヒト動物をそれぞれ作出することができる。また、図４に示すように、動物種が同じでも、生育段階によって腸内細菌叢の構成が異なる可能性がある。そのため、生育段階別の腸内細菌叢に特徴的な優占菌群を含む細菌群を本実施形態に係る無菌非ヒト動物に投与することで、生育段階別の腸内細菌叢に特徴的な優占菌群を腸内細菌叢として有する本実施形態に係る実験用非ヒト動物をそれぞれ作出することができる。従って、動物種別及び生育段階別の腸内細菌叢の特徴を模倣した本実施形態に係る実験用非ヒト動物によって、より効果の高いオーダーメード型の薬剤、栄養剤、サプリメント、生菌材等の開発や運用を安価かつ簡便に行うことが可能となる。 As the bacterial group administered to the sterile non-human animal according to the present embodiment, the intestinal bacteria of each animal species as shown above depend on the animal species of the heterologous animal for which the intestinal flora is to be imitated. As shown in FIG. 3, the gut flora of each animal species is appropriately included by appropriately including the dominant bacterial group characteristic of the flora and the characteristic bacterial group corresponding to the physiological changes of the animal species. It is possible to produce experimental non-human animals according to the present embodiment, each of which has a predominant bacterial flora characteristic of the bacterial flora as an intestinal flora. Further, as shown in FIG. 4, even if the animal species are the same, the composition of the intestinal flora may differ depending on the growth stage. Therefore, by administering a bacterial group including a dominant bacterial group characteristic of the intestinal flora according to the growth stage to the sterile non-human animal according to the present embodiment, the intestinal bacterial flora characteristic of each growth stage is characteristic. Experimental non-human animals according to the present embodiment having a dominant bacterial group as an intestinal flora can be produced. Therefore, by using the experimental non-human animals according to the present embodiment that mimic the characteristics of the intestinal flora according to the animal type and growth stage, more effective custom-made drugs, nutritional supplements, supplements, viable bacterial materials, etc. It will be possible to carry out development and operation inexpensively and easily.
本実施形態に係る異種動物の腸内細菌叢に含まれる細菌群は、異種動物の糞便から採取可能である。それに限らず、異種動物の腸管から採取してもよい。また、糞便そのものを用いてもよいし、糞便から一部の細菌群を取り出して用いてもよい。また、腸内細菌叢を構成する各細菌と同等の標準菌株を用いてもよい。これら細菌群は、無菌非ヒト動物に経口的に投与されてよい。 The bacterial group contained in the intestinal flora of the heterologous animal according to the present embodiment can be collected from the feces of the heterologous animal. Not limited to this, it may be collected from the intestinal tract of a different animal. Further, the stool itself may be used, or a part of the bacterial group may be extracted from the stool and used. In addition, a standard strain equivalent to each bacterium constituting the intestinal flora may be used. These bacterial groups may be administered orally to sterile non-human animals.
バチルス・ヒサシイは、２０１０年１月１５日付（受託日）でバチルス・ヒサシイN-11T株（Bacillus hisashii N-11T）が国際寄託当局である独立行政法人製品評価技術基盤機構特許微生物寄託センター(NPMD)（日本国千葉県木更津市かずさ鎌足２−５−８ １２２号室）に受託番号NITE BP-863として国際寄託されており、当該当局から入手可能である。バチルス・ヒサシイは、例えば飲水に添加する等によって無菌非ヒト動物に経口的に投与されてよく、以後も実験用非ヒト動物が実験等に使用されるまで継続的に投与されることが好ましい。 Bacillus hisashii N-11 T strain (Bacillus hisashii N-11 T ) is an international depositary authority on January 15, 2010 (consignment date). National Institute of Technology and Evaluation Patent Microorganisms Depositary Center It has been internationally deposited under (NPMD) (Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE BP-863, and is available from the relevant authorities. Bacillus hisashii may be orally administered to sterile non-human animals, for example by adding it to drinking water, and is preferably continuously administered thereafter until the experimental non-human animals are used in experiments and the like.
無菌非ヒト動物に異種動物の腸内細菌叢に含まれる細菌群が投与されることで、当該非ヒト動物は、腸内に当該細菌群を有する。本実施形態ではさらにバチルス・ヒサシイが投与され、バチルス・ヒサシイは、当該細菌群のうちの少なくとも１種の細菌のポピュレーションを変化させ、それによって当該非ヒト動物の代謝機能が健全に保たれた状態で、当該非ヒト動物の腸内に当該細菌群を腸内微生物叢として維持させる。図２に示すように、本実施形態で非ヒト動物として用いられるマウスが自然状態で生育した場合に本来有する腸内細菌叢は、異種動物として用いられる豚、鶏、牛等のものと異なる。このような本来では当該非ヒト動物の腸内細菌叢に定着しないまたはしにくい細菌を含む細菌群が無菌の当該非ヒト動物に投与された場合には、当該細菌群は、腸内で一定のバランスを保てずに腸内微生物叢として維持が困難であったり、当該非ヒト動物の代謝・生理機能等に悪影響を及ぼす可能性もある。 By administering a bacterial group contained in the intestinal flora of a heterologous animal to a sterile non-human animal, the non-human animal has the bacterial group in the intestine. In this embodiment, Bacillus hisashii is further administered, and the Bacillus hisashii alters the population of at least one bacterium in the bacterial group, thereby maintaining the healthy metabolic function of the non-human animal. In the state, the bacterial group is maintained as an intestinal microflora in the intestine of the non-human animal. As shown in FIG. 2, the intestinal flora originally possessed by the mouse used as a non-human animal in the present embodiment when grown in a natural state is different from that of pigs, chickens, cows and the like used as heterologous animals. When such a bacterial group containing bacteria that do not or are difficult to settle in the intestinal flora of the non-human animal is administered to the sterile non-human animal, the bacterial group is constant in the intestine. It may be difficult to maintain the intestinal microflora without maintaining the balance, or it may adversely affect the metabolic and physiological functions of the non-human animal.
例えば、豚の場合、豚の腸内細菌叢に特徴的な優占菌として、善玉菌であるラクトバチルス・アミロボラス（Lactobacillus amylovorus）及び日和見菌であるストレプトコッカス・アラクトリティカス（Streptococcus alactolyticus）の２つの乳酸菌が挙げられる。これら２つの細菌を含む細菌群を投与された本実施形態に係る実験用非ヒト動物の腸内細菌叢は、バチルス・ヒサシイの投与によって、善玉菌であるラクトバチルス・アミロボラスのポピュレーションが増加し、日和見菌であるストレプトコッカス・アラクトリティカスのポピュレーションが減少した状態で腸内に定着し、これらのポピュレーションの変化によって当該実験用非ヒト動物の代謝機能が健全に保たれる。また、ラクトバチルス・アミロボラス及びストレプトコッカス・アラクトリティカスは、畜産分野で一般的に使用される抗生物質であるテトラサイクリンに対して耐性を有するが、バチルス・ヒサシイは、これらのような抗生物質耐性菌についてもそのポピュレーションを制御しうる。このように、バチルス・ヒサシイは、本実施形態に係る非ヒト動物が本来腸内細菌叢として有しない異種動物の腸内細菌叢に含まれる細菌群が当該非ヒト動物に投与された場合でも、当該細菌群に含まれる少なくとも１種の細菌のポピュレーションを制御することで、当該非ヒト動物の代謝機能を健全に保ちながら、当該細菌群を腸内細菌叢として維持させることができる。 For example, in the case of pigs, the dominant bacteria characteristic of the intestinal flora of pigs are Lactobacillus amylovorus, which is a good bacterium, and Streptococcus alactolyticus, which is an opportunistic bacterium. There are two streptococci. In the intestinal flora of the experimental non-human animal according to the present embodiment to which a bacterial group containing these two bacteria was administered, the population of the good bacterium Lactobacillus amyloboras was increased by the administration of Bacillus hisashii. , The opportunistic bacterium Streptococcus actoriticus colonizes the intestine in a reduced state, and changes in these populations maintain the healthy metabolic function of the non-human animal for the experiment. In addition, Lactobacillus amylovoras and Streptococcus alactolicus are resistant to tetracycline, which is an antibiotic commonly used in the livestock field, while Bacillus hisashii is an antibiotic-resistant bacterium such as these. Can also control its population. As described above, Bacillus hisashii is produced even when a bacterial group contained in the intestinal flora of a heterologous animal that the non-human animal according to the present embodiment does not originally have as an intestinal flora is administered to the non-human animal. By controlling the population of at least one type of bacteria contained in the bacterial group, the bacterial group can be maintained as an intestinal flora while maintaining a healthy metabolic function of the non-human animal.
本実施形態に係る実験用非ヒト動物の腸内細菌叢は、必ずしも該当する動物種の腸内細菌叢のポピュレーションと一致しないことも想定されるが、該当する動物種由来の腸内細菌叢の存在下で飼育した実験用非ヒト動物の成長、生化学的・生理学的な詳細な解析が可能である。本実施形態に係る実験用非ヒト動物は、高額、かつ飼育管理技術において簡便ではない動物種を用いて、栄養剤、微生物製剤、新規抗生物質等の効能を評価する際の、その前段階のスクリーニング技術として有効に用いられる。 It is assumed that the gut flora of the experimental non-human animal according to the present embodiment does not necessarily match the population of the gut flora of the relevant animal species, but the gut flora derived from the relevant animal species. It is possible to analyze the growth, biochemical and physiological details of experimental non-human animals bred in the presence of. The experimental non-human animal according to the present embodiment is a pre-stage in evaluating the efficacy of nutritional supplements, microbial preparations, novel antibiotics, etc. using an animal species that is expensive and not convenient in breeding management technology. It is effectively used as a screening technique.
次に、試験例を示して、本発明を更に説明するが、本発明はこれらに限定されるものではない。 Next, the present invention will be further described with reference to test examples, but the present invention is not limited thereto.
（実施例１）
豚の腸内細菌叢を移植したマウスにおける飼育試験を行った。豚の腸内細菌叢としては豚糞を用いた。マウスはアイソレータ内で継代しているBALB/c系統を用いて無菌操作における飼育を行った。無菌マウスに106 cfu以上/mlに滅菌ＰＢＳ溶液において希釈調整した豚糞溶液をゾンデにて経口投与した後に無菌の通常食で飼育したマウス（対照区１）、無菌マウスに当該希釈調整豚糞溶液をゾンデにて経口投与した後に高脂肪食で飼育したマウス（対照区２）、及び無菌マウスに当該希釈調整豚糞溶液をゾンデにて経口投与した後にバチルス・ヒサシイBP-863株を終濃度として102 cfu以上/mlの濃度に調整して継続的に飲水給与しながら高脂肪食で飼育したマウス（試験区１）を作製した。なお、当該バチルス・ヒサシイBP-863株は、２−３日に一回の頻度で飲水ボトルの水を交換する際に投入した。また、対照区１、対照区２及び試験区１のマウスは、それぞれアイソレータ内の別々の飼育ケージで飼育を行った。これら各マウスの全身の体脂肪量を調べた結果を図５に示す。図５より、バチルス・ヒサシイBP-863株が投与されていない対照区１及び対照区２では、通常食より高脂肪の高脂肪食によって飼育された対照区２は、対照区１よりも体脂肪量が増加した。しかし、バチルス・ヒサシイBP-863株が継続給与されて対照区２と同様の高脂肪食によって飼育された試験区１は、有意差を持って対照区２よりも体脂肪量が少なく、通常食で飼育された対照区１と同等の体脂肪量となった。また、これら各マウスの腸内細菌叢における、豚の腸内細菌叢に特徴的な優占菌であるラクトバチルス・アミロボラス及びストレプトコッカス・アラクトリティカスの菌数を調べた結果を図６に示す。図６より、試験区１の腸内細菌叢では、対照区１及び対照区２と比較して、ラクトバチルス・アミロボラスの菌数が有意差を持って増加し、ストレプトコッカス・アラクトリティカスの菌数が有意差を持って減少している。このことから、バチルス・ヒサシイBP-863株によって、少なくともこれら２菌種のポピュレーションが制御されることが分かる。また、ラクトバチルス・アミロボラスは善玉菌で、ストレプトコッカス・アラクトリティカスは日和見菌であると考えられ、これらのポピュレーションの変化により、図５に示されるように、試験区１では代謝機能が向上して脂肪の蓄積軽減効果が引き起こされたものと考えられる。
(Example 1)
A breeding test was conducted on mice transplanted with the intestinal flora of pigs. Pig feces were used as the intestinal flora of pigs. Mice were bred in aseptic technique using the BALB / c strain passaged in the isolator. Diluted pig manure solution was orally administered to sterile mice in a sterile PBS solution to 10 6 cfu or more / ml with a sonde, and then fed to a sterile normal diet (control group 1). After oral administration of the solution in a sonde, mice fed a high-fat diet (Control Group 2) and sterile mice were orally administered the diluted-adjusted pig manure solution in a sonde, and then the final concentration of Bacillus hisashii BP-863 strain was added. As a result, mice (Test Group 1) bred on a high-fat diet were prepared while adjusting the concentration to 10 2 cfu or more / ml and continuously feeding with drinking water. The Bacillus Hisashii BP-863 strain was added when the water in the drinking bottle was changed once every 2-3 days. In addition, the mice in the control group 1, the control group 2, and the test group 1 were bred in separate breeding cages in the isolator. The results of examining the body fat mass of the whole body of each of these mice are shown in FIG. From FIG. 5, in the control group 1 and the control group 2 to which the Bacillus hisashii BP-863 strain was not administered, the control group 2 bred with a high-fat diet higher than the normal diet had more body fat than the control group 1. The amount has increased. However, the test group 1 in which the Bacillus hisashii BP-863 strain was continuously fed and bred on a high-fat diet similar to that in the control group 2 had a significantly smaller body fat mass than the control group 2 and had a normal diet. The body fat mass was equivalent to that of the control group 1 bred in. In addition, FIG. 6 shows the results of investigating the numbers of Lactobacillus amyloboras and Streptococcus aractiticus, which are the dominant bacteria characteristic of the intestinal flora of pigs, in the intestinal flora of each of these mice. .. From FIG. 6, in the intestinal flora of the test group 1, the number of Lactobacillus amyloboras increased significantly as compared with the control group 1 and the control group 2, and the bacteria of Streptococcus actoriticus increased significantly. The numbers are decreasing with a significant difference. From this, it can be seen that the Bacillus hisashii BP-863 strain controls the population of at least these two strains. In addition, Lactobacillus amylovoras is considered to be a good bacterium and Streptococcus alactolicus is considered to be an opportunistic bacterium, and changes in these populations improve metabolic function in Test Group 1 as shown in FIG. It is considered that the effect of reducing fat accumulation was caused.
（実施例２）
次に、豚の腸内細菌叢に特徴的な優占菌であるラクトバチルス・アミロボラス及びストレプトコッカス・アラクトリティカスの標準菌株の２菌株を移植したノトバイオートマウスにおける飼育試験を行った。当該２菌株の標準菌株として、理化学研究所バイオリソース研究センター（理研ＢＲＣ）から入手したラクトバチルス・アミロボラスJCM1126T株及びストレプトコッカス・アラクトリティカスJCM31116T株を用いた。マウスはアイソレータ内で継代しているBALB/c系統を用いて、実施例１と同様の飼育環境条件下において、アイソレータ内で無菌操作や飼育を行った。移植用標準菌２菌株は、それぞれ滅菌ＰＢＳ溶液によって少なくとも106 cfu/mlに調整された上で、200 μｌをそれぞれ無菌マウスに当該２菌株を投与した後に無菌の通常食で飼育したマウス（対照区３）、無菌マウスに当該２菌株を投与した後に無菌の高脂肪食で飼育したマウス（対照区４）、及び無菌マウスに当該２菌株を投与した後にバチルス・ヒサシイBP-863株を終濃度として106 cfu以上/mlに調整して継続的に飲水給与しながら無菌の高脂肪食で飼育したマウス（試験区２）を作製した。なお、対照区３、対照区４及び試験区２のマウスは、それぞれアイソレータ内の別々の飼育ケージで飼育を行った。これら各マウスの全身の体脂肪量を調べた結果を図７に示す。図７より、バチルス・ヒサシイBP-863株が継続給与されて対照区４と同様の高脂肪食によって飼育された試験区２は、有意差を持って対照区４よりも体脂肪量が少なく、通常食で飼育された対照区３と同等の体脂肪量となり、豚の腸内細菌叢に特徴的な優占菌の標準菌株を用いた場合においても、実施例１と同様に、脂肪蓄積が軽減した。また、図８に、当該２菌株が移植されたノトバイオートマウスにおいて、対照区、10 ppmのテトラサイクリン投与区、及び終濃度として106 cfu以上/mlに調整して継続的に飲水給与したBP-863給与区の腸内細菌叢における当該２菌株のポピュレーションについての結果を示す。なお、図８における対照区、テトラサイクリン投与区及びBP-863給与区のマウスは、それぞれアイソレータ内の別々の飼育ケージで飼育を行った。図８より、テトラサイクリン投与区では、当該２菌株のポピュレーションはどちらも減少しておらず、抗生物質であるテトラサイクリンに耐性があることが分かるが、BP-863給与区では、対照区と比べてラクトバチルス・アミロボラスのポピュレーションが増加し、ストレプトコッカス・アラクトリティカスのポピュレーションが減少している。従って、バチルス・ヒサシイBP-863株によって、当該２菌株のポピュレーションが制御されて、代謝機能が向上して脂肪蓄積が軽減していると考えられる。
(Example 2)
Next, a breeding test was conducted on notobiate mice transplanted with two strains of the standard strains of Lactobacillus amyloboras and Streptococcus aractolicus, which are the dominant bacteria characteristic of the intestinal flora of pigs. The as standard strain of 2 strains was used RIKEN Bioresource Research Center amylovorus Lactobacillus were from (RIKEN BRC) JCM1126 T strain and S. A lacto Utility Kas JCM31116 T strain. Mice were aseptically operated and bred in the isolator under the same breeding environment conditions as in Example 1 using the BALB / c strain subcultured in the isolator. Transplantation Standard bacteria 2 strains, after being adjusted to at least 10 6 cfu / ml by the respective sterile PBS solution, mouse (controls were housed 200 [mu] l with sterile normal diet after the administration of the two strains in sterile mice each Group 3), mice bred on a sterile high-fat diet after administration of the two strains to sterile mice (control group 4), and Bacillus hisashii BP-863 strain after administration of the two strains to sterile mice. Mice (test group 2) bred on a sterile high-fat diet while continuously drinking and feeding were prepared at 10 6 cfu or more / ml. The mice in the control group 3, the control group 4, and the test group 2 were bred in separate breeding cages in the isolator. The results of examining the body fat mass of the whole body of each of these mice are shown in FIG. From FIG. 7, the test group 2 in which the Bacillus hisashii BP-863 strain was continuously fed and bred on the same high-fat diet as the control group 4 had a significantly smaller body fat mass than the control group 4. The amount of body fat was the same as that of the control group 3 bred on a normal diet, and even when a standard strain of the dominant bacterium characteristic of the intestinal flora of pigs was used, fat accumulation was observed as in Example 1. Reduced. Further, in FIG. 8, in the notobioto mice transplanted with the two strains, a control group, a 10 ppm tetracycline-administered group, and a BP whose final concentration was adjusted to 10 6 cfu or more / ml and continuously fed with drinking water. -863 Shows the results for the population of the two strains in the gut flora of the feeding group. The mice in the control group, the tetracycline-administered group, and the BP-863 feeding group in FIG. 8 were bred in separate breeding cages in the isolator. From FIG. 8, it can be seen that the population of the two strains did not decrease in the tetracycline-administered group and that they were resistant to the antibiotic tetracycline, but in the BP-863 feeding group, compared with the control group. The population of Lactobacillus amylovoras has increased and the population of Streptococcus aractiticus has decreased. Therefore, it is considered that the Bacillus hisashii BP-863 strain controls the population of the two strains, improves the metabolic function, and reduces fat accumulation.
以上より、豚糞を移植したマウス及び豚糞中の優占菌を移植したマウスのどちらにおいても、バチルス・ヒサシイBP-863株の給与によって、脂肪蓄積が軽減されたモデルマウスが得られる。この効能の１つとしては、バチルス・ヒサシイBP-863株のゲノム上に、acetolactate synthase I/II/III large subunit (EC:2.2.1.6) (70% identity)、monooxygenase (EC:1.14.14.1) (66% identity)、monooxygenase yxeK (72% identity) 、D-lactate dehydrogenase (EC:1.1.2.4) (69% identity)、L-lactate dehydrogenase (EC:1.1.1.27) (73% identity)、Pyruvate dehydrogenase (EC:1.2.4.1) (72% identity)がある。acetolactate synthaseは、筋肉成長に関与するバリン、ロイシン、イソロイシンなどの分枝鎖アミノ酸の前駆体であるアセト乳酸の生合成に関与する酵素である。monooxygenaseは、酸素添加酵素であり、lactate 2-monooxygenaseは、Ｌ−乳酸を酢酸に資化する酵素である。また、lactate dehydrogenaseは乳酸からピルビン酸を、ピルビン酸から乳酸を産生する反応を促進する酵素である。さらに、pyruvate dehydrogenaseは、ピルビン酸から酢酸を資化する酵素である。酢酸は、脂肪蓄積の軽減や免疫系の賦活化などに広く関与することが明らかとなっている（非特許文献１）。これらの酵素反応のバランスは、腸内におけるニコチンアミドジヌクレオチドのＮＡＤ（酸化型）とＮＡＤＨ（還元型）の濃度バランスが影響する。バチルス・ヒサシイBP-863株は、これらの酵素反応と同様の働きをすることによって、特徴的な乳酸資化による酢酸の産生と、それによる脂肪蓄積の軽減効果と筋肉成長、並びに免疫系の賦活化をもたらすものと推察される。従って、脂肪蓄積が軽減されたモデルマウスによって、各種薬剤、栄養剤、サプリメント、酵素剤、生菌材等の試験が可能となる。また、このことから、豚糞中の優占菌種の移植によって、脂肪蓄積タイプ、脂肪非蓄積タイプの作成が可能であることが示された。なお、豚糞中の優占菌種の標準菌株は抗生物質耐性能が高いことから、抗生物質耐性の豚糞中の優占菌種の標準菌株の挙動を変化させる新規飼料添加物や新規薬剤のスクリーニングとして用いることができる。 From the above, in both the mouse transplanted with pig feces and the mouse transplanted with the dominant bacterium in pig feces, a model mouse in which fat accumulation is reduced can be obtained by feeding the Bacillus hisashii BP-863 strain. One of these effects is acetolactate synthase I / II / III large subunit (EC: 2.2.1.6) (70% identity), monooxygenase (EC: 1.14.14.1) on the genome of the Bacillus hisashii BP-863 strain. (66% identity), monooxygenase yxeK (72% identity), D-lactate dehydrogenase (EC: 1.1.2.4) (69% identity), L-lactate dehydrogenase (EC: 1.1.1.27) (73% identity), Pyruvate dehydrogenase There is (EC: 1.2.4.1) (72% identity). acetolactate synthase is an enzyme involved in the biosynthesis of acetolactate, which is a precursor of branched-chain amino acids such as valine, leucine, and isoleucine, which are involved in muscle growth. monooxygenase is an oxygenase, and lactate 2-monooxygenase is an enzyme that assimilates L-lactic acid to acetic acid. Lactate dehydrogenase is an enzyme that promotes the reaction of producing pyruvic acid from lactic acid and lactic acid from pyruvic acid. In addition, pyruvate dehydrogenase is an enzyme that assimilates acetic acid from pyruvate. It has been clarified that acetic acid is widely involved in the reduction of fat accumulation and the activation of the immune system (Non-Patent Document 1). The balance of these enzymatic reactions is affected by the concentration balance of NAD (oxidized form) and NADH (reduced form) of nicotinamide dinucleotide in the intestine. The Bacillus hisashii BP-863 strain acts in the same manner as these enzymatic reactions to produce acetic acid by characteristic lactic acid assimilation, thereby reducing fat accumulation and muscle growth, and activating the immune system. It is presumed that it will bring about the change. Therefore, the model mouse with reduced fat accumulation enables testing of various drugs, nutritional supplements, supplements, enzyme preparations, viable bacterial materials and the like. From this, it was shown that the fat accumulation type and the fat non-accumulation type can be produced by transplanting the dominant bacterial species in pig feces. Since the standard strain of the dominant strain in pig droppings has high antibiotic resistance, new feed additives and new drugs that change the behavior of the standard strain of the dominant strain in antibiotic-resistant pig droppings. It can be used as a screening for.
本発明に係る実験用非ヒト動物は、代謝機能が健全に保たれた状態で、異種動物の腸内細菌叢に含まれる細菌群を腸内に有するので、当該異種動物の腸内環境の挙動や生理的影響を評価するためのモデル動物として用いることができる。特に、異種動物として産業動物や愛玩動物等の動物実験に用いることが困難な動物を、無菌非ヒト動物としてマウス等の安価で管理の容易な動物を用いて作製された本発明に係る実験用非ヒト動物は、産業動物や愛玩動物に対する新規の飼料や薬剤等の効能を評価する際に、産業動物や愛玩動物に実際に投与実験を行う前段階のスクリーニングに好適に用いることができる。 Since the experimental non-human animal according to the present invention has a bacterial group in the intestine contained in the intestinal flora of the heterologous animal in a state where the metabolic function is maintained in a healthy state, the behavior of the intestinal environment of the heterologous animal. It can be used as a model animal for evaluating and physiological effects. In particular, for experiments according to the present invention, which are produced by using an animal such as an industrial animal or a pet animal which is difficult to use as a heterologous animal in an animal experiment, and an inexpensive and easily managed animal such as a mouse as a sterile non-human animal. Non-human animals can be suitably used for screening in the pre-stage of actually performing administration experiments on industrial animals and pet animals when evaluating the efficacy of new feeds and drugs on industrial animals and pet animals.
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