JP2007112805A - Composition for promoting propagation of lactobacillus casei subsp. casei - Google Patents
Composition for promoting propagation of lactobacillus casei subsp. casei Download PDFInfo
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本発明は、種々の生物学的活性を有する乳酸菌の1種であるラクトバシラス・カゼイ 亜種 カゼイ(Lactobacillus casei subsp.casei)(以下、「L.casei subsp.casei」と略記する。)を選択的に増殖させるための、デキストランを含む菌増殖促進用組成物、及び、さらにL.casei subsp.caseiを含む該菌増殖促進用組成物に関する。より具体的には、本発明は該菌増殖促進用組成物を含む生物学的活性を高めるための医薬組成物、並びに該菌増殖促進用組成物を含む生物学的活性を高めるための健康用食品及び飼料に関する。 In the present invention, Lactobacillus casei subsp. Casei (hereinafter abbreviated as “L. casei subsp. Casei”), which is one of lactic acid bacteria having various biological activities, is selectively used. And a composition for promoting bacterial growth containing dextran, and L. casei subsp. The present invention relates to a composition for promoting bacterial growth comprising casei. More specifically, the present invention relates to a pharmaceutical composition for enhancing biological activity containing the composition for promoting bacterial growth, and for health use for enhancing biological activity containing the composition for promoting bacterial growth. It relates to food and feed.
近年プロバイオティックスと呼ばれ、生きた乳酸菌等の有用微生物からなる「微生物製剤」を整腸効果や感染症の防止、免疫賦活等を目的として摂取することが注目されている。しかしながら、その有用微生物がヒトや動物等の腸内で生育・定着できるかが疑問視されている。有用微生物が腸内で生育・定着できなければ、期待されるその有用微生物の持つ生物学的活性が持続的に得られないことになる。それを補うための対策としては、その微生物製剤を随時補給する事が提唱・実行されているのが実情である。一方、プロバイオティックスに含まれる有用微生物が資化できるオリゴ糖等をプレバイオティックスと呼び、またそれをプロバイオティックスと一緒にしたものをシンバイオティックスと呼び、それらもまた、上記目的のために、ヒトや動物等に投与されている。しかしながら、そのプレバイオティックスがかなり多くの目的とする菌以外の腸内細菌に資化される場合には、腸内でそのプレバイオティックスの奪い合いになり目的の有用微生物を選択的に生育・定着させることは難しい。 In recent years, it is called probiotics, and it has attracted attention to take “microbe preparations” composed of useful microorganisms such as live lactic acid bacteria for the purpose of intestinal regulation, prevention of infectious diseases, immune activation, and the like. However, it has been questioned whether the useful microorganisms can grow and settle in the intestines of humans and animals. If useful microorganisms cannot grow and settle in the intestines, the expected biological activity of the useful microorganisms cannot be obtained continuously. As a countermeasure to compensate for this, the fact is that it is advocated and implemented to replenish the microorganism preparations as needed. On the other hand, oligosaccharides, etc. that can be assimilated by useful microorganisms contained in probiotics are called prebiotics, and those that are combined with probiotics are called symbiotics. It is administered to humans and animals. However, if the prebiotics are assimilated by a number of enteric bacteria other than the target fungus, the prebiotics compete with each other in the intestine to selectively grow and establish the desired useful microorganisms. It is difficult to let
本発明は、プロバイオティックスと呼ばれている有用微生物をヒトや動物等に摂取させた後に、これらの有用微生物を随時補給しなくても、腸内で選択的に発育増殖・定着させることで、また腸内に常在する有用微生物を腸内で選択的に発育増殖させることで、有用微生物由来の種々の生物学的活性を生体内で持続させることを課題とする。 The present invention allows humans and animals to ingest useful microorganisms called probiotics, and then selectively grows, proliferates and settles in the intestine without supplementing these useful microorganisms as needed. Another object of the present invention is to sustain various biological activities derived from useful microorganisms in vivo by selectively growing and proliferating useful microorganisms resident in the intestine.
本願発明者らは、数ある有用微生物の中で、デキストランを資化して発育増殖することが出来る菌種として、L.casei subsp.caseiを生体外(in vitro)試験において見出した。そのL.casei subsp.caseiは、アプルーブドリスト(Approved Lists)(1980)において分類・命名されているものである。この菌はヒトや動物等の腸内にも常在するグラム陽性の通性嫌気性桿菌であり、他の菌と共に腸内細菌叢を形成している。 Among the many useful microorganisms, the present inventors have identified L. cerevisiae as a bacterial species that can assimilate and grow dextran. casei subsp. casei was found in an in vitro test. The L. casei subsp. Casei is classified and named in the Approved Lists (1980). This bacterium is a Gram-positive facultative anaerobe that is also resident in the intestines of humans and animals and forms an intestinal flora together with other bacteria.
そこで、まず、この菌自体に生物学的活性、1例として皮下免疫の際の免疫賦活作用が存在するかどうかを確認したところ、体液性免疫及び細胞性免疫を増強・維持することが分かった。次に、デキストランを経口摂取させることで、L.casei subsp.caseiをヒトや動物等の腸内で選択的に発育増殖・定着させ、その菌の有する生物学的活性を持続させることができるかどうかを確認するために、マウスを用いて初回のみにL.casei subsp.caseiを投与し、その後、デキストランをマウスに経口で継続的に摂取させたところ、本菌はデキストランを資化して腸内で発育増殖・定着し、その菌の有する生物学的活性、例えば、体液性免疫及び細胞性免疫を増強・維持することを生体内(in vivo)試験において見出すに至った。 Therefore, first, it was confirmed whether or not the bacterium itself has biological activity, for example, an immunostimulatory action in the case of subcutaneous immunity. As a result, it was found that humoral immunity and cellular immunity are enhanced and maintained. . Next, by ingesting dextran, L. casei subsp. In order to confirm whether casei can selectively grow, proliferate and settle in the intestines of humans, animals, etc., and maintain the biological activity of the fungus, L. casei is used only for the first time using a mouse. casei subsp. Casei was administered, and then dextran was continuously ingested orally by mice. As a result, this bacterium utilized dextran to grow, grow and settle in the intestine, and its biological activity, for example, body fluid It has been found in in vivo tests to enhance and maintain sexual immunity and cellular immunity.
他方、デキストラン自身にも種々の生物学的活性を有することが知られているが、デキストラン単独投与に比べ、デキストランをL.casei subsp.caseiと併行投与した場合は、高い効果が維持され、相乗効果が大きい事が確認された。 On the other hand, dextran itself is known to have various biological activities. casei subsp. When co-administered with casei, it was confirmed that the high effect was maintained and the synergistic effect was large.
本発明は、以上のようにして達成された。本発明の要旨は、以下のとおりである。
(1) ラクトバシラス・カゼイ 亜種 カゼイを選択的に増殖させるための、デキストランを含む菌増殖促進用組成物。
(2) さらに、ラクトバシラス・カゼイ 亜種 カゼイを含む(1)に記載の菌増殖促進用組成物。
(3) デキストランの分子量が2,000〜4,000万である(1)に記載の菌増殖促進用組成物。
(4) (1)〜(3)のいずれかに記載の菌増殖促進用組成物を含む医薬組成物。
(5) (1)〜(3)のいずれかに記載の菌増殖促進用組成物を含む健康用食品。
(6) (1)〜(3)のいずれかに記載の菌増殖促進用組成物を含む飼料。
The present invention has been achieved as described above. The gist of the present invention is as follows.
(1) Lactobacillus casei Subspecies A composition for promoting bacterial growth comprising dextran for selectively growing casei.
(2) The composition for promoting bacterial growth according to (1), further comprising Lactobacillus casei subspecies casei.
(3) The composition for promoting bacterial growth according to (1), wherein the molecular weight of dextran is 2,000 to 40 million.
(4) A pharmaceutical composition comprising the composition for promoting bacterial growth according to any one of (1) to (3).
(5) A health food containing the composition for promoting bacterial growth according to any one of (1) to (3).
(6) A feed comprising the composition for promoting bacterial growth according to any one of (1) to (3).
本発明によれば、L.casei subsp.caseiを随時摂取しなくても、L.casei subsp.caseiをヒトや動物等の腸内で選択的に発育増殖・定着させることで、また腸内に常在するL.casei subsp.caseiを腸内で選択的に発育増殖させることで、L.casei subsp.casei由来の種々の生物学的活性を生体内で持続させることができる。 According to the present invention, L. casei subsp. Even without taking casei from time to time, casei subsp. By selectively growing, proliferating and colonizing casei in the intestines of humans and animals, L. casei casei subsp. by selectively growing and growing casei in the intestine, casei subsp. Various biological activities derived from casei can be sustained in vivo.
本発明の組成物は、L.casei subsp.caseiを選択的に増殖させるための、デキストランを含む菌増殖促進用組成物、及び、さらにL.casei subsp.caseiを含む該菌増殖促進用組成物である。本発明の組成物に用いられるデキストランは、当業者によく知られた通常の方法により合成、調整等を行うことが可能である。また、本発明に用いられる L.casei subsp.caseiの培養、菌体の調製・乾燥方法、遺伝子組換え等については、当業者によく知られた通常の方法によって行うことができる。 The composition of the present invention comprises L. casei subsp. A composition for promoting bacterial growth containing dextran for selectively growing casei, and L. casei subsp. The composition for promoting bacterial growth comprising casei. Dextran used in the composition of the present invention can be synthesized, adjusted, and the like by ordinary methods well known to those skilled in the art. In addition, L. used in the present invention. casei subsp. Casei culture, cell preparation / drying method, gene recombination and the like can be carried out by conventional methods well known to those skilled in the art.
(本発明に用いられるデキストランの説明)
デキストランはα1,6結合を主体としたグルコースの多糖体である。本発明に用いられるデキストランとしては、例えば、市場で入手したものを用いてもよく、または、リューウコノストク(Leuconostoc)属、ストレプトコッカス(streptococcus)属からなる群より選ばれる細菌、例えば、リューウコノストク・メゼンテロイデス(Leuconostoc mesenteroides)等を、通常の培養条件にて、例えば、ショ糖含有栄養培地で培養して得られたものを用いてもよい。
(Description of dextran used in the present invention)
Dextran is a polysaccharide of glucose mainly composed of α1,6 bonds. As the dextran used in the present invention, for example, commercially available ones may be used, or bacteria selected from the group consisting of the genus Leuconostoc and the genus Streptococcus, for example, You may use what was obtained by culture | cultivating Konostok mesenteroides (Leuconostoc mesenteroides) etc. on a normal culture condition, for example with a sucrose containing nutrient medium.
(本発明に用いられるデキストランの分子量)
本発明に用いられるデキストランの分子量としては、分子量が2,000〜4,000万、好ましくは4,000〜500万、更に好ましくは4,000〜200万である。なお、デキストランの分子量が2,000未満であると、L.casei subsp.casei以外の細菌が増殖することがあるため好ましくない。本発明に用いられるデキストランとしては、例えば、上記細菌の培養により得られたデキストランをそのまま、または、更にこれを部分的加水分解処理して適当な分子量のデキストランにしたものを用いることが可能である。
(Molecular weight of dextran used in the present invention)
The molecular weight of dextran used in the present invention is 2,000 to 40 million, preferably 4,000 to 5,000,000, more preferably 4,000 to 2,000,000. When the molecular weight of dextran is less than 2,000, L.P. casei subsp. Since bacteria other than casei may grow, it is not preferable. As the dextran used in the present invention, it is possible to use, for example, dextran obtained by culturing the above bacteria as it is or by further subjecting it to partial hydrolysis to obtain a dextran having an appropriate molecular weight. .
(本発明に用いられるL.casei subsp.casei)
本発明においてL.casei subsp.caseiは、生菌または発育増殖可能な乾燥菌体等の形態で使用することができる。本発明に用いられるL.casei subsp.caseiは、野生型の他、本発明の菌増殖用組成物に含まれるデキストランに対する資化性を損なわない範囲におけるL.casei subsp.caseiの遺伝子組換え体を用いることもできる。また、L.casei subsp.casei由来のデキストラン資化性に関与する遺伝子を他の微生物に導入した遺伝子組換え体を用いることもできる。好ましくは、遺伝子組換え体は、L.casei subsp.caseiに遺伝子組換えによって新たな生物学的活性を発現させた新規微生物、またはL.casei subsp.casei由来のデキストラン資化性に関与する遺伝子を他の微生物に導入した別の新規微生物である。
(L. casei subsp. Casei used in the present invention)
In the present invention, L. casei subsp. casei can be used in the form of live cells or dry cells that can grow and proliferate. L. used in the present invention. casei subsp. casei is an L. coli in a range that does not impair the assimilation property to dextran contained in the composition for bacterial growth of the present invention in addition to the wild type. casei subsp. Casei recombinants can also be used. L. casei subsp. It is also possible to use a recombinant gene in which a gene related to casei-derived dextran utilization is introduced into another microorganism. Preferably, the genetic recombinant is L. casei subsp. a novel microorganism in which a new biological activity is expressed by genetic recombination in casei, or L. casei subsp. This is another novel microorganism in which a gene involved in casei-derived dextran utilization is introduced into another microorganism.
本発明の組成物は、L.casei subsp.caseiを選択的に増殖させるための、デキストランを含む菌増殖促進用組成物、及び、さらにL.casei subsp.caseiを含む該菌増殖促進用組成物である。本発明の組成物は、デキストラン、または、さらにL.casei subsp.caseiを含むこと以外は、当業者によく知られた通常の方法を採用し、調製、投与等をすることが可能である。 The composition of the present invention comprises L. casei subsp. A composition for promoting bacterial growth containing dextran for selectively growing casei, and L. casei subsp. The composition for promoting bacterial growth comprising casei. The composition of the present invention may be dextran, or even L.I. casei subsp. Except for containing casei, it is possible to prepare, administer, etc. by adopting ordinary methods well known to those skilled in the art.
(本発明の組成物の投与形態)
本発明の組成物は、デキストラン単体、またはデキストランとL.casei subsp.caseiの混合物であってもよいが、好ましくは、これらを製剤学上よく知られた製剤学的に許容し得る希釈剤乃至担体等と組合わせて、デキストランを含む固状、半固状または液状の組成物、または、デキストランとL.casei subsp.caseiを共に加工した固状、半固状または液状の組成物として提供することができる。なお、本発明の組成物とは、本発明のデキストランとL.casei subsp.caseiを組み合わせた形態の他、本発明のデキストランとL.casei subsp.caseiを個別に加工したものを使用時に組み合わせる形態を含む。本発明の組成物における希釈剤乃至担体等の含有量は目的や用法に応じて適宜変化させられ、特に制限されるものではないが、一般的に組成物重量に基づいて約0.1〜約99.9重量%、好ましくは約1〜約99重量%、更に好ましくは約5〜約95重量%の含有量を例示することができる。
(Dosage form of the composition of the present invention)
The composition of the present invention includes dextran alone or dextran and L. casei subsp. The casei may be a mixture, but it is preferable that these are combined with a pharmaceutically acceptable diluent or carrier well known in pharmaceutics to form a solid, semi-solid or liquid containing dextran. Or dextran and L. casei subsp. Casei can be provided as a solid, semi-solid or liquid composition processed together. The composition of the present invention includes dextran of the present invention and L.P. casei subsp. In addition to the combined form of casei, dextran of the present invention and L. casei subsp. Including the case of combining casei individually processed at the time of use. The content of the diluent or carrier in the composition of the present invention can be appropriately changed according to the purpose and usage and is not particularly limited, but is generally about 0.1 to about 0.1 based on the weight of the composition. A content of 99.9% by weight, preferably about 1 to about 99% by weight, more preferably about 5 to about 95% by weight can be exemplified.
尚、液状でデキストランとL.casei subsp.caseiを併存させると、菌の資化によりデキストラン含量が次第に減少していくので、長期間の併存は避ける方が好ましい。そこで、個別に加工・包装し、摂取時に混合するか、個別に摂取することが望ましい。 In addition, dextran and L. casei subsp. When casei coexists, the dextran content gradually decreases due to the assimilation of bacteria, so it is preferable to avoid coexistence for a long period of time. Therefore, it is desirable to process and package them individually and mix them at the time of ingestion or ingest them individually.
本発明の医薬組成物は、本発明の組成物単体であってもよいが、好ましくは、これらを薬学上よく知られた薬学的に許容し得る希釈剤乃至担体等と組合わせて、軟カプセル剤、硬カプセル剤、顆粒剤、丸剤、散剤、錠剤、シロップ剤、トローチ剤、エリキシル剤等の如き剤型に調製されたものであってもよい。このような剤型への調製手段は当業者によく知られている。このような剤型への調製において、各剤型に適した薬学上よく知られた薬学的に許容し得る希釈剤乃至担体等を更に含有させることができる。 The pharmaceutical composition of the present invention may be the composition of the present invention alone, but preferably a soft capsule by combining these with a pharmaceutically acceptable diluent or carrier that is well known in pharmacy. It may be prepared in a dosage form such as an agent, hard capsule, granule, pill, powder, tablet, syrup, troche, elixir. Means for preparing such dosage forms are well known to those skilled in the art. In preparation of such a dosage form, a pharmacologically well-known pharmaceutically acceptable diluent or carrier suitable for each dosage form can be further contained.
本発明の医薬組成物を、ヒトや動物等の腸内においてL.casei subsp.caseiを選択的に発育増殖・定着させ、または、腸内に常在するL.casei subsp.caseiを腸内で選択的に発育増殖させ、L.casei subsp.casei由来の種々の生物学的活性を生体内で持続させるための医薬として用いることができる。 The pharmaceutical composition of the present invention can be used in the intestines of humans and animals. casei subsp. casei selectively grows, proliferates and settles, or is resident in the intestine. casei subsp. casei is selectively grown and proliferated in the intestine. casei subsp. It can be used as a medicament for sustaining various biological activities derived from casei in vivo.
本発明の医薬組成物の調製において、本発明の組成物の添加量は、下記本発明の組成物
の投与量及び投与周期に関する記載を参照して適宜決定することができる。
In the preparation of the pharmaceutical composition of the present invention, the addition amount of the composition of the present invention can be appropriately determined with reference to the following description regarding the dosage and administration cycle of the composition of the present invention.
本発明の健康用食品は、本発明の組成物と、食品や飲料等の組成物を含むものである。なお、本発明の健康用食品とは、本発明の組成物と食品や飲料等の組成物を組み合わせた形態の他、本発明の組成物と食品や飲料等の組成物を個別に加工したものを摂取時に組み合わせる形態を含む。このような食品または飲料組成物としては、例えば、キャンディー、チョコレート、ビスケット等の菓子類、パンや麺類、粉末及び液体等の飲料、ヨーグルト等の乳製品等を挙げることができる。特にヨーグルトの場合、このL.casei subsp.caseiを使ってヨーグルトを製造することができ、摂取時にデキストランを混合するか、個別に摂取することが望ましい。 The health food of the present invention comprises the composition of the present invention and a composition such as food or beverage. In addition, the health food of the present invention is a product obtained by individually processing the composition of the present invention and a composition such as a food or beverage, in addition to a combination of the composition of the present invention and a composition such as food or beverage. Including forms that are combined at the time of ingestion. Examples of such a food or beverage composition include confectionery such as candy, chocolate and biscuits, bread and noodles, beverages such as powder and liquid, and dairy products such as yogurt. Particularly in the case of yogurt, this L.P. casei subsp. Casei can be used to produce yogurt, preferably mixed with dextran at the time of ingestion or ingested separately.
本発明の飼料は、本発明の組成物と、家畜・家禽や魚類、ペット等の配合飼料を含むものである。なお、本発明の飼料とは、本発明の組成物と飼料組成物を組み合わせた形態の他、本発明の組成物と飼料組成物を個別に加工したものを摂取時に組み合わせる形態を含む。 The feed of the present invention includes the composition of the present invention and a mixed feed of livestock, poultry, fish, pets and the like. In addition, the feed of this invention includes the form which combined what processed the composition and feed composition of this invention separately in addition to the form which combined the composition and feed composition of this invention at the time of ingestion.
本発明の健康用食品または飼料をヒトや動物等の腸内において L.casei subsp.caseiを選択的に発育増殖・定着させ、または、腸内に常在するL.casei subsp.caseiを腸内で選択的に発育増殖させ、L.casei subsp.casei由来の種々の生物学的活性を生体内で持続させるために用いることができる。 In the intestines of humans and animals, etc. casei subsp. casei selectively grows, proliferates and settles, or is resident in the intestine. casei subsp. casei is selectively grown and proliferated in the intestine. casei subsp. It can be used to sustain various biological activities derived from casei in vivo.
本発明の健康用食品及び飼料の調製において、本発明の組成物の添加量は、下記本発明の組成物の投与量及び投与周期に関する記載を参照して適宜決定することができる。 In the preparation of the health food and feed of the present invention, the amount of the composition of the present invention can be appropriately determined with reference to the following description regarding the dose and administration cycle of the composition of the present invention.
(本発明の組成物の投与量及び投与周期)
本発明の組成物の投与量としては、組成物中に含まれるデキストランの投与量として、0.01mg〜2g/kg体重・日、好ましくは0.1mg〜1g/kg体重・日、更に好ましくは1mg〜100mg/kg体重・日であり、少なくとも本発明のデキストランを含む組成物を通常毎日投与することが好ましい。なお、L.casei subsp.caseiは本来ヒトや動物等の腸内常在菌であるが、L.casei subsp.caseiの生物学的活性の確実な効果を誘導するためには、生菌または発育増殖可能な乾燥菌体を少なくとも1回、好ましくは適当な間隔で投与することが好ましい。また、より確実な効果を誘導するために、毎日投与することもできる。
(Dosage and administration cycle of the composition of the present invention)
The dose of the composition of the present invention is 0.01 mg to 2 g / kg body weight / day, preferably 0.1 mg to 1 g / kg body weight / day, more preferably, as the dose of dextran contained in the composition. It is preferably 1 mg to 100 mg / kg body weight / day, and a composition containing at least dextran of the present invention is usually administered daily. In addition, L. casei subsp. Casei is originally an intestinal bacteria such as humans and animals. casei subsp. In order to induce a reliable effect of the biological activity of casei, it is preferable to administer live bacteria or dry cells capable of growth and growth at least once, preferably at an appropriate interval. It can also be administered daily to induce a more reliable effect.
(本発明の組成物の適用対象)
本発明の組成物は、ヒト含む哺乳類、鳥類、爬虫類,魚類、甲殻類、昆虫等あらゆる生物を対象とすることができる。
(Application target of the composition of the present invention)
The composition of the present invention can be used for all living organisms such as mammals including humans, birds, reptiles, fishes, crustaceans and insects.
(本発明の組成物により付与及び/または向上されることが期待できる生物学的活性)
免疫賦活作用の他、一般的に言われている乳酸菌の有する生物学的活性、例えば、抗腫瘍、血圧降下、抗酸化、抗潰瘍、抗HIV、脂質代謝改善、悪性腫瘍再発抑制、抗炎症、自己免疫疾患予防・抑制、血清コレステロール低下、血糖低下、IgE抗体産生抑制、抗アレルギー、インターロイキン12産生促進、ミネラル吸収促進、ミネラル強化、アルコール吸収代謝調整、尿路感染症予防治療、有害物質産生抑制、整腸、便秘防止、美容、高アンモニア血症や肝性脳症等の治療・防止、感染予防、糞便悪臭抑制、家畜・家禽腸内pH低下、生育向上、肥育促進、及び産卵助長等の作用が期待できる。
(Biological activity that can be expected to be imparted and / or improved by the composition of the present invention)
In addition to the immunostimulatory action, the biological activity of lactic acid bacteria generally said, such as anti-tumor, blood pressure lowering, antioxidant, anti-ulcer, anti-HIV, lipid metabolism improvement, malignant tumor recurrence suppression, anti-inflammatory, Autoimmune disease prevention / suppression, serum cholesterol lowering, blood glucose lowering, IgE antibody production suppression, antiallergy, interleukin 12 production promotion, mineral absorption promotion, mineral enhancement, alcohol absorption metabolism adjustment, urinary tract infection prevention treatment, harmful substance production Suppression, bowel control, prevention of constipation, beauty, treatment / prevention of hyperammonemia and hepatic encephalopathy, infection prevention, fecal malodor control, lowering intestinal pH in domestic animals and poultry, improving growth, promoting fattening, and promoting spawning Expected to work.
以下、本発明を実施例によって、更に詳細に説明するが、本発明はこれらに限定される
ものではない。
<実施例1> ショ糖を主原料としたデキストランの製造方法
ショ糖を主原料とした培地(ショ糖含有栄養培地)に、乳酸菌の一種であるLeuconostoc mesenteroidesを接種し、25℃、24時間、嫌気性下で培養した後、培養上澄にメタノールを加え、得られた沈殿物を蒸留水に溶解し、不溶物を除いた後に、再度メタノールを加え、得られた沈殿物を蒸留水に再溶解し、噴霧乾燥をしてデキストランを得た。更にこれを部分的加水分解処理してメタノール分画、噴霧乾燥を経て各種分子量のデキストランを得た。
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.
<Example 1> Method for producing dextran using sucrose as a main raw material A medium containing sucrose as a main raw material (sucrose-containing nutrient medium) is inoculated with Leuconostoc mesenteroides, which is a kind of lactic acid bacteria, at 25 ° C for 24 hours. After culturing under anaerobic condition, methanol is added to the culture supernatant, the resulting precipitate is dissolved in distilled water, insoluble matter is removed, methanol is added again, and the resulting precipitate is reconstituted in distilled water. Dissolved and spray dried to obtain dextran. Furthermore, this was partially hydrolyzed and subjected to methanol fractionation and spray drying to obtain dextran having various molecular weights.
<実施例2> 腸内細菌のデキストラン資化性試験
分子量(数平均)10,000のデキストラン(以下の実施例においても特に規定しない場合はこの分子量のデキストランを用いる)を0.5%添加したPYF培地(ペプトンイーストエキストラクトフィルデス液体培地)(Peptone yeast extract Fildes solution broth)に各種腸内関連細菌を接種し、デキストランの資化性を調べた。その結果、L.casei subsp.caseiの2株(JCM1134及びJCM8129)でデキストランの資化性を確認することができた。しかし、他のLactobacillus属やビフィドバクテリウム(Bifidobacterium)属等の乳酸菌、及び、その他の腸内関連細菌に関しては、デキストランの資化性は見られなかった。(表1参照)
<Example 2> Dextran utilization test of enterobacteria 0.5% of dextran having a molecular weight (number average) of 10,000 (use dextran having this molecular weight unless otherwise specified) is added. Various intestinal related bacteria were inoculated into PYF medium (Peptone yeast extract field fluid solution), and the utilization of dextran was examined. As a result, L. casei subsp. The utilization of dextran could be confirmed in two strains of casei (JCM1134 and JCM8129). However, lactic acid bacteria such as other Lactobacillus genus and Bifidobacterium genus and other intestinal related bacteria were not assimilated by dextran. (See Table 1)
更に、L.casei subsp.caseiの2株でデキストランの分子量の違いによる資化性を調べた。その結果、試験に供した全ての分子量のデキストランでデキストランの資化性を確認することができた。しかし、分子量が小さい方が資化されやすい傾向があった。(表2参照) In addition, L. casei subsp. The assimilation by the difference in the molecular weight of dextran was examined in two strains of casei. As a result, it was possible to confirm the utilization of dextran with dextran having all the molecular weights used in the test. However, the lower molecular weight tended to be assimilated. (See Table 2)
<実施例3> マウス糞便中のL.casei subsp.casei菌数に与えるデキストランの影響
8週齢のBALB/c雄マウス12匹を6匹ずつ2群に分けた。各群を(1)コントロール群と(2)デキストラン群とした。実験開始より試験期間を通して、(1)には通常食を、(2)にはデキストランを通常食1kgあたり75mg添加して与えた。試験開始より3日間にわたりどちらの群にもL.casei subsp.caseiをマウス1匹あたり106個経口接種した。28日目に解剖し大腸より採取した糞便をリン酸緩衝生理食塩水(PBS)で段階希釈してデキストラン添加LB培地に塗抹し37℃48時間培養して、コロニーをグラム染色で確認した上で、糞便中のL.casei subsp.casei菌数を求めた。その結果、デキストラン群のL.casei subsp.casei菌数はコントロール群のそれに比べて有意に多かった。(第1図参照)
<Example 3> L. in mouse feces. casei subsp. Effect of Dextran on Casei Bacterial Count Twelve 8-week-old BALB / c male mice were divided into two groups of 6 mice each. Each group was made into (1) control group and (2) dextran group. Throughout the test period from the start of the experiment, (1) was given a normal diet, and (2) was given dextran added at 75 mg / kg of the normal diet. In both groups, L. casei subsp. 10 6 casesi were orally inoculated per mouse. Feces dissected on day 28 and collected from the large intestine were serially diluted with phosphate buffered saline (PBS), smeared on dextran-added LB medium, cultured at 37 ° C. for 48 hours, and colonies were confirmed by Gram staining. , L. in feces. casei subsp. The number of casei bacteria was determined. As a result, dextran group L. casei subsp. The number of casei bacteria was significantly higher than that of the control group. (See Fig. 1)
<実施例4> 抗原経口投与における体液性免疫のデキストランによる増強
8週齢のBALB/c雄マウス30匹を6匹ずつ5群に分けた。各群は、(1)コントロール群、(2)BSA(牛血清アルブミン)群、(3)BSA+L.casei subsp.casei群、(4)BSA+デキストラン群、(5)BSA+L.casei
subsp.casei+デキストラン群とした。実験開始より試験期間を通して、(1)、(2)と(3)には通常食を、(4)と(5)にはデキストランを通常食1kgあたり75mg添加して与えた。試験開始より3日間にわたり(3)と(5)にはL.casei subsp.caseiをマウス1匹あたり106個経口接種した。3、4日目に(2)〜(5)にはBSAをPBSに10mg/mlの濃度で溶解し、マウス1匹あたり1mg(100μl)経口投与した。33、34日目に(2)〜(5)には3、4日目と同様にBSAをマウス1匹あたり1mg経口投与した。その5日後に眼下静脈より採血し、抗BSA抗体価をELISAにより調べた。その結果、(5)BSA+L.casei
subsp.casei+デキストラン群で特異抗体価が有意に高かった。(第2図参照)
<Example 4> Enhancement of humoral immunity by dextran in oral administration of antigen Thirty 8-week-old BALB / c male mice were divided into 5 groups of 6 mice each. Each group consists of (1) control group, (2) BSA (bovine serum albumin) group, (3) BSA + L. casei subsp. casei group, (4) BSA + dextran group, (5) BSA + L. casei
subsp. casei + dextran group. Throughout the test period from the start of the experiment, (1), (2) and (3) were given a normal diet, and (4) and (5) were given dextran added at 75 mg / kg of the normal diet. Over 3 days from the start of the test, L. casei subsp. 10 6 casesi were orally inoculated per mouse. On days 3 and 4, (2) to (5), BSA was dissolved in PBS at a concentration of 10 mg / ml, and 1 mg (100 μl) per mouse was orally administered. On days 33 and 34, (2) to (5) were orally administered with 1 mg of BSA per mouse in the same manner as on days 3 and 4. Five days later, blood was collected from the subocular vein, and the anti-BSA antibody titer was examined by ELISA. As a result, (5) BSA + L. casei
subsp. The specific antibody titer was significantly higher in the casei + dextran group. (See Fig. 2)
<実施例5> 抗原経口投与における細胞性免疫のデキストランによる増強
8週齢のBALB/c雄マウス30匹を6匹ずつ5群に分けた。各群は、(1)コントロール群、(2)BSA群、(3)BSA+L.casei subsp.casei群、(4)BSA+デキストラン群、(5)BSA+L.casei subsp.cas
ei+デキストラン群とした。実験開始より試験期間を通して、(1)、(2)と(3)には通常食を、(4)と(5)にはデキストランを通常食1kgあたり75mg添加して与えた。試験開始より3日間にわたり(3)と(5)にはL.casei subsp.caseiをマウス1匹あたり106個経口接種した。3、4日目に(2)〜(5)には、BSAをPBSに10mg/mlの濃度で溶解し、マウス1匹あたり1mg(100μl)経口投与した。33、34日目に(2)〜(5)には3、4日目と同様にBSAをマウス1匹あたり1mg経口投与した。その5日後、BSAをPBSに溶解し、マウス耳朶の皮内に投与し、24、48、72時間後に耳朶の厚みを測定した。その結果、(5)BSA+L.casei subsp.casei+デキストラン群の耳朶の厚みが有意に増加していた。(第3図参照)
<Example 5> Enhancement of cellular immunity by dextran in oral administration of antigen Thirty 8-week-old BALB / c male mice were divided into 5 groups of 6 mice each. Each group consists of (1) control group, (2) BSA group, (3) BSA + L. casei subsp. casei group, (4) BSA + dextran group, (5) BSA + L. casei subsp. cas
The ei + dextran group was used. Throughout the test period from the start of the experiment, (1), (2) and (3) were given a normal diet, and (4) and (5) were given dextran added at 75 mg / kg of the normal diet. Over 3 days from the start of the test, L. casei subsp. 10 6 casesi were orally inoculated per mouse. On days 3 and 4, (2) to (5), BSA was dissolved in PBS at a concentration of 10 mg / ml and orally administered at 1 mg (100 μl) per mouse. On days 33 and 34, (2) to (5) were orally administered with 1 mg of BSA per mouse in the same manner as on days 3 and 4. Five days later, BSA was dissolved in PBS and administered into the mouse earlobe skin, and the thickness of the earlobe was measured 24, 48, and 72 hours later. As a result, (5) BSA + L. casei subsp. The thickness of the earlobe of the casei + dextran group was significantly increased. (See Fig. 3)
<実施例6> 抗原皮下投与における体液性免疫のデキストランによる増強
8週齢のBALB/c雄マウス30匹を6匹ずつ5群に分けた。各群は、(1)コントロール群、(2)BSA群、(3)BSA+L.casei subsp.casei群、(4)BSA+デキストラン群、(5)BSA+L.casei subsp.casei+デキストラン群とした。実験開始より試験期間を通して、(1)、(2)と(3)には通常食を、(4)と(5)にはデキストランを通常食1kgあたり75mg添加して与えた。試験開始より3日間にわたり(3)と(5)にはL.casei subsp.caseiをマウス1匹あたり106個経口接種した。3日目にFIA(フロイント不完全アジュバント)(Freund's incomplete adjuvant)を用いて、それぞれ、(1)にはPBS(リン酸緩衝生理食塩水)+FIA、(2)〜(5)にはBSA5mg/ml+FIAをそれぞれ1:1で乳化させ、1匹あたり200μlを足蹠の皮下に投与した。35日目に3日目と同様にして追加免疫を行い、その5日後に眼下静脈より採血し、抗BSA抗体価をELISAにより調べた。その結果、(5)BSA+L.casei subsp.casei+デキストラン群で特異抗体価が有意に高かった。(第4図参照)
<Example 6> Enhancement of humoral immunity by dextran in subcutaneous administration of antigen Thirty 8-week-old BALB / c male mice were divided into 5 groups of 6 mice each. Each group consists of (1) control group, (2) BSA group, (3) BSA + L. casei subsp. casei group, (4) BSA + dextran group, (5) BSA + L. casei subsp. casei + dextran group. Throughout the test period from the start of the experiment, (1), (2) and (3) were given a normal diet, and (4) and (5) were given dextran added at 75 mg / kg of the normal diet. Over 3 days from the start of the test, L. casei subsp. 10 6 casesi were orally inoculated per mouse. On the third day, using FIA (Freund's incomplete adjuvant), (1) PBS (phosphate buffered saline) + FIA, (2) to (5)
<実施例7> 抗原皮下投与における細胞性免疫のデキストランによる増強
8週齢のBALB/c雄マウス30匹を6匹ずつ5群に分けた。各群は、(1)コントロール群、(2)BSA群、(3)BSA+L.casei subsp.casei群、(4)BSA+デキストラン群、(5)BSA+L.casei subsp.casei+デキストラン群とした。実験開始より試験期間を通して、(1)、(2)と(3)には通常食を、(4)と(5)にはデキストランを通常食1kgあたり75mg添加して与えた。試験開始より3日間にわたり(3)と(5)にはL.casei subsp.caseiをマウス1匹あたり106個経口接種した。3日目にFIAを用いて、それぞれ、(1)にはPBS+FIA、(2)〜(5)にはBSA5mg/ml+FIAをそれぞれ1:1で乳化させ、1匹あたり200μlを足蹠の皮下に投与した。35日目に3日目と同様にして追加免疫を行い、その5日後、BSAをPBSに溶解し、マウス耳朶の皮内に投与し、24、48、72時間後に耳朶の厚みを測定した。その結果、(5)BSA+L.casei subsp.casei+デキストラン群の耳朶の厚みが有意に増加していた。(第5図参照)
<Example 7> Enhancement of cell-mediated immunity by dextran in subcutaneous administration of antigen Thirty 8-week-old BALB / c male mice were divided into 5 groups of 6 mice each. Each group consists of (1) control group, (2) BSA group, (3) BSA + L. casei subsp. casei group, (4) BSA + dextran group, (5) BSA + L. casei subsp. casei + dextran group. Throughout the test period from the start of the experiment, (1), (2) and (3) were given a normal diet, and (4) and (5) were given dextran added at 75 mg / kg of the normal diet. Over 3 days from the start of the test, L. casei subsp. 10 6 casesi were orally inoculated per mouse. On the third day, using FIA, emulsify PBS + FIA for (1) and
Claims (2)
ラクトバシラス・カゼイ 亜種 カゼイを選択的に増殖させるための、デキストランを含むラクトバシラス・カゼイ 亜種 カゼイ増殖促進用組成物。 Dextran and Lactobacillus casei sub-species Lactobacillus casei sub-species comprising casei are administered to an animal ingesting a composition for promoting casei growth,
Lactobacillus casei subspecies A composition for promoting the growth of Lactobacillus casei subspecies casei containing dextran for selectively growing casei.
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Free format text: JAPANESE INTERMEDIATE CODE: A761 Effective date: 20100414 |