JP5144085B2 - Intestinal immunity enhancing agent containing lactic acid bacteria having IgA antibody production improving action - Google Patents
Intestinal immunity enhancing agent containing lactic acid bacteria having IgA antibody production improving action Download PDFInfo
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- JP5144085B2 JP5144085B2 JP2007039019A JP2007039019A JP5144085B2 JP 5144085 B2 JP5144085 B2 JP 5144085B2 JP 2007039019 A JP2007039019 A JP 2007039019A JP 2007039019 A JP2007039019 A JP 2007039019A JP 5144085 B2 JP5144085 B2 JP 5144085B2
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- lactic acid
- acid bacteria
- iga antibody
- antibody production
- intestinal immunity
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Description
本発明は、腸管でのＩｇＡ抗体産生を向上させることにより感染抵抗性を高めるなどの腸管免疫力増強作用を有する乳酸菌ラクトバチルス・プランタラム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）を含有する腸管免疫力増強剤に関するものである。 The present invention relates to an intestinal immunity enhancing agent containing a lactic acid bacterium Lactobacillus plantarum having an intestinal immunity enhancing action such as enhancing infection resistance by improving IgA antibody production in the intestinal tract. is there.
腸管粘膜は、常に無数のウイルス、細菌、寄生虫、病原性抗原や食物抗原にさらされており、これらの異物抗原から生体を守るシステムとして腸管免疫系は発達してきた。しかし、大きな手術を行なった後や病後などは腸管免疫力が一時的に低下し、外部からの異物侵入に対する抵抗力が弱り、感染症にかかるリスクが増大することが知られている。加齢と共に腸管免疫力が徐々に低下した老人や腸管免疫系の発達が不十分な幼児も同様のリスクを背負っている。 The intestinal mucosa is constantly exposed to a myriad of viruses, bacteria, parasites, pathogenic antigens and food antigens, and the intestinal tract immune system has been developed as a system for protecting living bodies from these foreign antigens. However, it is known that intestinal immunity temporarily decreases after a large operation or after illness, resistance to entry of foreign substances from the outside is weakened, and the risk of infection is increased. Older people whose intestinal immunity gradually declines with age and infants with inadequate development of the intestinal immunity system have the same risks.
腸管免疫力を評価する指標としては、Ｔ細胞の増殖能、免疫グロブリンＡ（以下、ＩｇＡ抗体という）の産生量、細胞が生産する種々の働きをもつペプチド、サイトカイン量などが知られている。これらの中でもＩｇＡ抗体は、細菌やウイルスの中和、組織への細菌の付着の抑制などに重要な役割を果たしている。従って、 ＩｇＡ抗体量の増加は腸管免疫力向上の有力な指標となる。上記した感染予防や治療において、ＩｇＡ抗体量を高く保つ作用を有する製剤、ＩｇＡ抗体の生産力を高めることができる製剤の開発が強く望まれている。 As indexes for evaluating intestinal immunity, known are T cell proliferative ability, production amount of immunoglobulin A (hereinafter referred to as IgA antibody), peptides having various functions produced by cells, cytokine amounts, and the like. Among these, the IgA antibody plays an important role in neutralizing bacteria and viruses and suppressing the adhesion of bacteria to tissues. Therefore, an increase in the amount of IgA antibody is an effective index for improving intestinal immunity. In the above-described infection prevention and treatment, there is a strong demand for the development of a preparation capable of keeping the IgA antibody amount high and a preparation capable of increasing the productivity of IgA antibody.
腸管免疫力を向上させる食品成分として、乳酸菌、麹カビ或いは酵母などの食用微生物やそれらの細胞壁成分、又は、シイタケなど担子菌類の多糖類、特にβグルカン類などが知られている。これらの中で最近プロバイオティックスとして注目される乳酸菌による免疫賦活力に注目した食品素材も数多く提案されている。乳酸菌の属も多岐に渡り、ラクトコッカス属に着目したもの（特許文献１）、エンテロコッカス属に着目したもの（特許文献２）、ラクトバチルス属に着目したもの（特許文献３、特許文献４）など様々である。しかしながら、特許文献１では、ＩｇＡ抗体産生量の増加が重要な指標になると指摘しておきながら直接的な定量を実施していないのでその効果が明確でない。また、特許文献２では、ｉｎ ｖｉｔｒｏでのＩＬ−１２産生しか評価していないし、特許文献３では、ＩｇＡ抗体産生能を評価しているもののパイエル板を用いたｉｎ ｖｉｔｒｏ試験を行なっているに過ぎず、特許文献４では、ＩＬ−１２に加えてＩＦＮγ、亜硝酸イオン濃度を指標としているが、生体内で免疫賦活効果を示すのかどうかは各文献では明らかにされていない。ｉｎ ｖｉｔｒｏで効果を示したものは生体で必ず効果を示すと仮定しても、特許文献４では、乳酸菌全般の効果として免疫力増強作用があるとしているのにガセリ菌とカゼイ菌各１菌株しか試験していない、カゼイ菌全般に効果があるとしているが、寄託した菌株での評価しかしていない、さらに、イネ科穀物との組み合わせによる相乗効果についてもオートミール以外の素材との組み合わせについては一切示していないなど、不備な点が多い。そこで、ＩｇＡ抗体産生能などを指標としたｉｎ ｖｉｖｏでの試験結果に基づいた腸管免疫力増強作用を有する安全な機能性素材、特に乳酸菌由来製剤の開発が切望されている。 Known food components for improving intestinal immunity include edible microorganisms such as lactic acid bacteria, mold fungi, and yeast, cell wall components thereof, and polysaccharides of basidiomycetes such as shiitake mushrooms, particularly β-glucans. Among these, many food materials have been proposed that have focused on the immunostimulatory activity of lactic acid bacteria, which has recently attracted attention as probiotics. The genus of lactic acid bacteria is also wide-ranging. Those focused on the genus Lactococcus (Patent Document 1), those focused on the genus Enterococcus (Patent Document 2), those focused on the genus Lactobacillus (Patent Document 3, Patent Document 4), etc. There are various. However, in Patent Document 1, it is pointed out that an increase in the amount of IgA antibody produced is an important index, but the effect is not clear because direct quantification is not performed. In Patent Document 2, only IL-12 production is evaluated in vitro. In Patent Document 3, although IgA antibody production ability is evaluated, only an in vitro test using a Peyer's board is performed. In addition, in Patent Document 4, in addition to IL-12, IFNγ and nitrite ion concentration are used as indices, but it is not clarified in each document whether an immunostimulatory effect is shown in vivo. Even if it is assumed that what has shown an effect in vitro necessarily shows an effect in a living body, Patent Document 4 states that there is an immunity enhancing action as an effect of lactic acid bacteria in general, but only one strain of gasseri and casei Although not tested, it is said that it is effective in all cases of the casei, but it has only been evaluated with the deposited strain, and the synergistic effect in combination with gramineous grains is also shown for all combinations with ingredients other than oatmeal There are many deficiencies such as not. Therefore, development of a safe functional material having an intestinal immunity enhancing action based on the in vivo test results using IgA antibody production ability as an index, in particular, a preparation derived from lactic acid bacteria is eagerly desired.
本発明の目的は、ＩｇＡ抗体産生向上による腸管免疫力増強作用を有する、安全な天然由来の乳酸菌（植物性乳酸菌）を有効成分として含有する腸管免疫力増強剤を提供することである。 An object of the present invention is to provide an intestinal immunity enhancing agent containing a safe naturally-derived lactic acid bacterium (plant lactic acid bacterium) as an active ingredient, which has an intestinal immunity enhancement effect by improving IgA antibody production.
本発明者らは、上記目的を達成すべく種々検討した結果、パン酵母から分離したラクトバチルス・プランタラム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）に属する乳酸菌を含有する製剤が、高いＩｇＡ抗体産生向上作用を有し、上記目的を達成するものであることを見出し、本発明に到達した。
本発明は、高いＩｇＡ抗体産生向上作用を有し、腸管免疫力増強作用を有するラクトバチルス・プランタラムに属する乳酸菌を有効成分として含有する腸管免疫力増強剤であって、上記乳酸菌がラクトバチルス・プランタラムＡＹＡ株（受託番号ＦＥＲＭ Ｐ−２１１０６）である腸管免疫力増強剤を提供するものである。
As a result of various studies to achieve the above object, the present inventors have found that a preparation containing lactic acid bacteria belonging to Lactobacillus plantarum isolated from baker's yeast has a high IgA antibody production improving action, It has been found that the above object can be achieved, and the present invention has been achieved.
The present invention is higher have IgA antibody production enhancing action, an intestinal immunity enhancing agent containing as an active ingredient a lactic acid bacterium belonging to Lactobacillus plantarum with intestinal immunity enhancing action, the upper Symbol lactic acid bacteria Lactobacillus A plantaram AYA strain (Accession No. FERM P-21106) is provided.
本発明の腸管免疫力増強剤を構成するラクトバチルス・プランタラムに属する乳酸菌は、高いＩｇＡ抗体産生向上作用を有し、該作用によって腸管免疫力増強作用をもたらす。それゆえ、この乳酸菌を含有する製剤を摂取することにより、免疫力の低い幼児期や加齢や病後の免疫力が低下している状態での腸管免疫力を増強し、細菌の感染に対する抵抗力をつけることが可能になる。本発明の腸管免疫力増強剤は、食経験のある天然食品素材から分離した乳酸菌由来であるので安全性にも優れている。 Lactic acid bacteria belonging to Lactobacillus plantarum constituting the intestinal tract immunity enhancing agent of the present invention have a high IgA antibody production improving action, and this action brings about an intestinal immunity enhancing action. Therefore, by ingesting a preparation containing this lactic acid bacterium, intestinal immunity is enhanced in low-immunity childhood, aging and post-disease immunity, and resistance to bacterial infection. It becomes possible to turn on. The intestinal immunity enhancing agent of the present invention is derived from a lactic acid bacterium isolated from a natural food material having a food experience, and thus is excellent in safety.
前記乳酸菌ラクトバチルス・プランタラムＡＹＡ株は、独立行政法人産業技術総合研究所、特許生物寄託センターに寄託されており、その受託番号はＦＥＲＭ Ｐ−２１１０６である。 The lactic acid bacterium Lactobacillus plantarum AYA strain has been deposited at the National Institute of Advanced Industrial Science and Technology and the Patent Organism Depositary, and the deposit number is FERM P-21106.
乳酸菌ラクトバチルス・プランタラムＡＹＡ株の菌学的性質を下記に示す。
ＭＲＳ液体培地（ＤＩＦＣＯ社）を用いて、３０℃、１８時間培養したときの菌の形態
（１）菌の形態 桿菌
（２）グラム染色 陽性
（３）運動性 なし
（４）胞子 なし
（５）カタラーゼ なし
（６）通性嫌気性
（７）ブドウ糖の代謝 ５０％以上乳酸に転換する
（８）生育温度範囲 １５℃、３０℃および３５℃では生育を認めるが、４５℃では生育を認めない
（９）乳酸発酵 ホモ型
（１０）乳酸の旋光性 ＤＬ
（１１）炭水化物の発酵性 グリセロールは陽性、Ｄ-アラビノースは陰性、Ｌ-アラビノースは陽性、リボースは陽性、Ｄ-キシロースは陰性、ガラクトースは陽性、グルコース
は陽性、フルクトースは陽性、マンノースは陽性、ラムノースは陽性、マンニトールは陽性、ソルビトールは陽性、αメチルＤグルコシドは陰性、アミグダリンは陽性、エスクリンは陽性、サリシンは陽性、セロビオースは陽性、マルトースは陽性、ラクトースは陽性、メリビオースは陽性、シュクロースは陽性、トレハロースは陽性、イヌリンは陰性、メレジトースは陽性、ラフィノースは陽性、スターチは陰性、グルコン酸は陽性。
The mycological properties of the lactic acid bacterium Lactobacillus plantarum AYA are shown below.
Bacteria morphology when cultured for 18 hours at 30 ° C. using MRS liquid medium (DIFCO) (1) Bacteria morphology Bacilli (2) Gram staining Positive (3) Motility None (4) Spore None (5) Catalase None (6) Facultative anaerobic (7) Glucose metabolism Convert to 50% or more lactic acid (8) Growth temperature range Growth is observed at 15 ° C, 30 ° C and 35 ° C, but growth is not observed at 45 ° C ( 9) Lactic acid fermentation Homo type (10) Optical rotation of lactic acid DL
(11) Fermentation of carbohydrates: glycerol is positive, D-arabinose is negative, L-arabinose is positive, ribose is positive, D-xylose is negative, galactose is positive, glucose is positive, fructose is positive, mannose is positive, rhamnose Is positive, mannitol is positive, sorbitol is positive, α-methyl D glucoside is negative, amygdalin is positive, esculin is positive, salicin is positive, cellobiose is positive, maltose is positive, lactose is positive, melibiose is positive, sucrose is positive Positive for trehalose, negative for inulin, positive for melezitose, positive for raffinose, negative for starch, positive for gluconic acid.
乳酸菌ラクトバチルス・プランタラムＡＹＡ株は、食経験が豊富な素材（パン酵母）から分離したものであるため、腸管免疫力を増強させる製剤に安全に利用することができる。 Since the lactic acid bacteria Lactobacillus plantarum AYA strain is isolated from a raw material (baker's yeast) with abundant dietary experience, it can be safely used in a preparation that enhances intestinal immunity.
乳酸菌ラクトバチルス・プランタラムＡＹＡ株は、そのままあるいは必要に応じて薬学的に許容される種々の担体、賦形剤、その他の添加剤、その他の成分を配合して製剤化することによって、腸管免疫力増強剤とすることができる。また、該腸管免疫力増強剤は、乳酸菌ラクトバチルス・プランタラムＡＹＡ株を用いて発酵させた種々の動植物性物質をベースとしてもよい。 Lactic acid bacteria Lactobacillus plantarum AYA can be prepared by combining various pharmaceutically acceptable carriers, excipients, other additives, and other ingredients as they are or as necessary. It can be a force enhancer. The intestinal immunity enhancing agent may be based on various animal and plant substances fermented using the lactic acid bacterium Lactobacillus plantarum AYA.
本発明の腸管免疫力増強剤は、高いＩｇＡ抗体産生向上作用を有するラクトバチルス・プランタラムに属する乳酸菌、特に好ましくは上記の本発明のラクトバチルス・プランタラムＡＹＡ株を有効成分として含有するものである。 The intestinal immunity enhancing agent of the present invention contains a lactic acid bacterium belonging to Lactobacillus plantarum having a high IgA antibody production improving action, particularly preferably the aforementioned Lactobacillus plantarum AYA strain of the present invention as an active ingredient. is there.
本発明の腸管免疫力増強剤で用いられる上記の高いＩｇＡ抗体産生向上作用を有するラクトバチルス・プランタラムに属する乳酸菌は、その作用メカニズムは限定されず、結果的にＩｇＡ抗体産生が向上し、該ＩｇＡ抗体産生向上による腸管免疫力増強作用を有するものであればよい。
ここで、「高いＩｇＡ抗体産生向上作用を有する」とは、好ましくは、腸管免疫力増強剤を摂取する前後において、統計的に有意水準５％でＩｇＡ抗体産生が向上される状態をいう。
The lactic acid bacteria belonging to Lactobacillus plantarum having the high IgA antibody production improving action used in the intestinal immunity enhancing agent of the present invention is not limited in its action mechanism, and as a result, IgA antibody production is improved, Any substance may be used as long as it has an action to enhance intestinal immunity by improving IgA antibody production.
Here, “having a high IgA antibody production improving action” preferably means a state in which IgA antibody production is statistically improved at a statistically significant level of 5% before and after taking the intestinal immunity enhancing agent.
乳酸菌のＩｇＡ抗体産生向上作用の評価は、以下の方法により評価することが好ましい。
乳酸菌をＭＲＳ培地等で培養し、この培養した乳酸菌を０．１〜２０質量％含む飼料をマウスに一定期間摂取させる。摂取後、マウス（乳酸菌添加群）を解剖し、小腸パイエル板細胞を取り出す。この小腸パイエル板細胞を培養し、培養上清を回収する。回収した培養上清中のＩｇＡ抗体産生量をＥＬＩＳＡ法により測定する。同様にして、乳酸菌を含まない飼料を摂取させたマウス（乳酸菌無添加群）のＩｇＡ抗体産生量を測定する。乳酸菌添加群のＩｇＡ抗体産生量と乳酸菌無添加群のＩｇＡ抗体産生量とを比較して、乳酸菌のＩｇＡ抗体産生向上作用を評価する。
It is preferable to evaluate the effect of improving the production of IgA antibody by lactic acid bacteria by the following method.
Lactic acid bacteria are cultured in an MRS medium or the like, and the mouse is fed with a feed containing 0.1 to 20% by mass of the cultured lactic acid bacteria for a certain period. After ingestion, the mouse (lactic acid bacteria added group) is dissected and the small intestine Peyer's patch cells are removed. The small intestinal Peyer's patch cells are cultured, and the culture supernatant is collected. The amount of IgA antibody produced in the collected culture supernatant is measured by ELISA. Similarly, the amount of IgA antibody produced in mice fed with a feed containing no lactic acid bacteria (group without lactic acid bacteria) is measured. The IgA antibody production improvement effect of lactic acid bacteria is evaluated by comparing the IgA antibody production amount of the lactic acid bacteria addition group and the IgA antibody production amount of the lactic acid bacteria non-addition group.
本発明の腸管免疫力増強剤中の乳酸菌の含有量は、ＩｇＡ抗体産生を向上しうる量であればいかなる量であってもよく、使用形態、腸管免疫力増強剤の剤形、投与又は摂取する者の症状や年齢性別などによって適宜変化させることができる。本発明の腸管免疫力増強剤を経口投与又は摂取させる場合には、１人１日当たりの投与量又は摂取量が１ｍｇ〜２０ｇとなるように含有させることが好ましい。 The content of the lactic acid bacterium in the intestinal immunity enhancing agent of the present invention may be any amount as long as it can improve IgA antibody production, and the usage form, the dosage form of the intestinal immunity enhancing agent, administration or ingestion Can be changed as appropriate according to the symptoms and age and gender of the person. When the intestinal immunity enhancing agent of the present invention is orally administered or ingested, it is preferably contained so that the daily dose or intake per person is 1 mg to 20 g.
次に本発明をさらに具体的に説明するために実施例を挙げるが、本発明は、以下の実施例に制限されるものではない。 EXAMPLES Next, examples are given to describe the present invention more specifically, but the present invention is not limited to the following examples.
実施例１
（使用した乳酸菌）
鉄砲漬け、ゴーヤ漬け、キムチなどの漬物やイーストなど、日本人の食経験が豊富な植物性素材から単離された植物性乳酸菌の中から桿菌であること、同様の性状をもたらすものが複数菌株見つかること、通常の培地で増殖性が高いことなどを基準に、表１に示す代表的な３１菌株を選抜した。表１に示すＡＹＡは、本発明のラクトバチルス・プランタラムＡＹＡ株である。
Example 1
(Lactic acid bacteria used)
Several strains that are gonococci among plant lactic acid bacteria isolated from plant materials with abundant Japanese dietary experience, such as pickled pickles, pickled bitter gourd, pickles such as kimchi, yeast, etc. 31 typical strains shown in Table 1 were selected on the basis of finding and high growth ability in a normal medium. AYA shown in Table 1 is the Lactobacillus plantarum AYA strain of the present invention.
（乳酸菌試料の調製）
１０μｇ／ｍｌシクロヘキシミドを含むＭＲＳ（ｄｅ Ｍａｎ−Ｒｏｇｏｓａ−Ｓｈａｒｐｅ）培地を用い、表１に示した各乳酸菌株を３７℃で４８時間培養した。その後、遠心分離によって集菌し、滅菌水で３回洗浄した後、滅菌水に懸濁し、１２１℃で３０分間オートクレーブ処理し、これらを凍結乾燥して乳酸菌試料をそれぞれ得た。
(Preparation of lactic acid bacteria sample)
Each lactic acid bacterial strain shown in Table 1 was cultured at 37 ° C. for 48 hours using MRS (de Man-Rogosa-Sharp) medium containing 10 μg / ml cycloheximide. Thereafter, the cells were collected by centrifugation, washed with sterilized water three times, suspended in sterilized water, autoclaved at 121 ° C. for 30 minutes, and freeze-dried to obtain lactic acid bacteria samples, respectively.
（乳酸菌の一次スクリーニング：ｉｎ ｖｉｔｒｏ試験によるＩｇＡ抗体産生向上作用の評価）
１．パイエル板細胞の調製
ＢＡＬＢ／ｃマウスから小腸を採取し、パイエル板細胞を回収する。このパイエル板を磨り潰し、セルストレイナーを通し細胞のみを回収する。回収した細胞を、日本水産製のウシ胎児血清：ＦＣＳ（Ｆｏｅｔａｌ Ｃａｌｆ Ｓｅｒｕｍ）を５質量％添加したＲＰＭＩ培地１０ｍｌで２回洗浄し、最終的に５質量％ＦＣＳ入りＲＰＭＩ培地１ｍｌに懸濁する。細胞数を計測し３×１０6ｃｅｌｌｓ／ｍｌになるように希釈し、各ウエル１００μｌずつ９６穴プレートにまく。
２．乳酸菌との共培養
５質量％ＦＣＳ入りＲＰＭＩ培地で各乳酸菌試料を２００μｇ／ｍｌになるように調製し、その内１００μｌを上で準備した９６穴プレートに加え、ＣＯ2インキュベーターで７日間培養する。対照区として５質量％ＦＣＳ入りＲＰＭＩ培地１００μｌのみを対照ウエルに加え、同時に処理する。
３．ＩｇＡ定量
培養上清を回収し、ＥＬＩＳＡ法により上清中のＩｇＡ抗体量を定量し、乳酸菌フリーの対照区の値と比較した。
４．実験結果
実験結果を図１に示した。一連の実験を３回繰り返し、ＡＹＢ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）、ＡＹＡ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）、ＯＹＣ−９４（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｈｉｌｇａｒｄｉｉ）が強いＩｇＡ抗体産生向上作用を示すことがわかった。
(Primary screening of lactic acid bacteria: Evaluation of IgA antibody production improving effect by in vitro test)
1. Preparation of Peyer's Patch Cells Collect small intestine from BALB / c mice and collect Peyer's patch cells. The Peyer's board is crushed and only the cells are collected through a cell strainer. The collected cells are washed twice with 10 ml of RPMI medium supplemented with 5% by mass of fetal bovine serum: FCS (Foetal Calf Serum) manufactured by Nihon Suisan, and finally suspended in 1 ml of RPMI medium containing 5% by mass FCS. Count the number of cells, dilute to 3 × 10 6 cells / ml, and place 100 μl of each well in a 96-well plate.
2. Co-culture with lactic acid bacteria Each lactic acid bacterium sample is prepared to 200 μg / ml in RPMI medium containing 5% by mass FCS, 100 μl of which is added to the 96-well plate prepared above, and cultured in a CO 2 incubator for 7 days. As a control, only 100 μl of RPMI medium containing 5% by mass FCS is added to the control well and processed simultaneously.
3. IgA quantification The culture supernatant was collected, and the amount of IgA antibody in the supernatant was quantified by ELISA, and compared with the value of the control group free of lactic acid bacteria.
4). Experimental results The experimental results are shown in FIG. A series of experiments was repeated three times, and it was found that AYB (Lactobacillus plantarum), AYA (Lactobacillus plantarum), and OYC-94 (Lactobacillus hillardii) showed a strong IgA antibody production improving effect.
実施例２及び比較例１〜４
表２に示す、ＡＹＡ（実施例２）、ＡＹＢ（比較例１）、ＯＹＣ−９４（比較例２）、ＯＹＣ−１０５（比較例３）及びＬ．ＧＧ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｒｈａｍｎｏｓｕｓ、入手先：ＡＴＣＣ）（比較例４）について、これらをマウスに経口投与した場合のＩｇＡ抗体産生向上作用を次のように評価した。
Example 2 and Comparative Examples 1 to 4
AYA (Example 2), AYB (Comparative Example 1), OYC-94 (Comparative Example 2), OYC-105 (Comparative Example 3) and L. Regarding GG (Lactobacillus rhamnosus, source: ATCC) (Comparative Example 4), the effect of improving IgA antibody production when these were orally administered to mice was evaluated as follows.
１．飼料の調製
各乳酸菌について、実施例１の（乳酸菌試料の調製）で示す方法と同様にして乳酸菌試料をそれぞれ調製した。この乳酸菌試料を下記に示す配合の基礎飼料（ＡＩＮ−９３（米国国立栄養研究所によるマウス・ラットを用いた栄養研究のための標準精製飼料）をベースとした飼料）に５質量％になるように混合し、乳酸菌入り飼料を調製した。対照群として乳酸菌無添加の基礎飼料も調製した。基礎飼料の原料は、全てオリエンタル酵母工業株式会社製のものを使用した。飼料は、乳酸菌試料の混合後、加水し筒状に成形することで得た。
２．動物実験スケジュール
５週齢のＢＡＬＢ／ｃマウスの体重を測定し、体重を指標として層別連続無作為化法を用いて各群６匹ずつの群分けを行なった。群分け後、上記１で調製した基礎飼料及び乳酸菌入り飼料の摂取を開始した。なお、体重測定に先立ち順化処理（１週間）を行ったが、この間にはいずれのマウスにも基礎飼料を自由摂取させた。
乳酸菌入り飼料の摂取開始後２８日目にマウスを解剖し、小腸パイエル板細胞を回収した。その後、これを５質量％ＦＣＳ入りＲＰＭＩ培地で７日間培養し、培養上清を回収した。上清中のＩｇＡ抗体量をＥＬＩＳＡ法により定量した。
一方、回収した小腸パイエル板細胞をＦＡＣＳ（フローサイトメーター）で解析し、ＩｇＡ陽性細胞の割合を算出した。
３．実験結果
摘出した小腸パイエル板細胞を培養した上清のＩｇＡ抗体量を測定した結果を図２に示す。また、小腸パイエル板細胞をＦＡＣＳにより分離し、ＩｇＡ陽性細胞存在率を算出した結果を図３に示す。ダネット法により統計解析を行ったところ、ＩｇＡ抗体量については、いずれの群でも対照群（無添加）に対する有意差はつかなかった（有意水準５％）が、ＡＹＡを含む飼料を摂食した群（実施例２）で非常に高いＩｇＡ抗体産生能が認められた。また、ＡＹＡを含む飼料を摂食した群（実施例２）及びＯＹＣ−１０５を摂食した群（比較例３）で、ＩｇＡ陽性細胞の割合が対照群（無添加）に比べて有意に高い値を示した。このことから、乳酸菌ラクトバチルス・プランタラムＡＹＡ株（ＦＥＲＭ Ｐ−２１１０６）を含有する腸管免疫力増強剤が、高いＩｇＡ抗体産生向上作用を有することが証明された。
1. Preparation of feed For each lactic acid bacterium, a lactic acid bacterium sample was prepared in the same manner as in Example 1 (Preparation of lactic acid bacterium sample). The lactic acid bacteria sample is 5% by mass in a basic feed (AIN-93 (a standard purified feed for nutritional research using mice and rats by the National Institute of Nutrition) based on the following formula) To prepare a feed containing lactic acid bacteria. As a control group, a basic feed without lactic acid bacteria was also prepared. The raw materials for the basic feed were all manufactured by Oriental Yeast Co., Ltd. After mixing the lactic acid bacteria sample, the feed was obtained by adding water and forming into a cylindrical shape.
2. Animal Experiment Schedule The body weights of 5-week-old BALB / c mice were measured, and grouping was performed for 6 animals in each group using the stratified continuous randomization method with the body weight as an index. After grouping, ingestion of the basic feed and feed containing lactic acid bacteria prepared in 1 above was started. In addition, acclimatization processing (one week) was performed prior to the body weight measurement, and during this period, any mouse was allowed to freely ingest the basal diet.
Mice were dissected on the 28th day after the start of ingestion of feed containing lactic acid bacteria, and small intestinal Peyer's patch cells were collected. Thereafter, this was cultured in RPMI medium containing 5% by mass FCS for 7 days, and the culture supernatant was recovered. The amount of IgA antibody in the supernatant was quantified by ELISA.
On the other hand, the collected small intestinal Peyer's patch cells were analyzed by FACS (flow cytometer), and the ratio of IgA positive cells was calculated.
3. Experimental Results FIG. 2 shows the result of measuring the amount of IgA antibody in the supernatant obtained by culturing the extracted small intestinal Peyer's patch cells. Moreover, the small intestine Peyer's patch cell was isolate | separated by FACS, and the result of having calculated the IgA positive cell presence rate is shown in FIG. Statistical analysis by Dunnett's method revealed that IgA antibody levels were not significantly different from the control group (no addition) in any group (significant level 5%), but were fed the diet containing AYA. In Example 2, a very high IgA antibody production ability was observed. Further, in the group fed with the feed containing AYA (Example 2) and the group fed with OYC-105 (Comparative Example 3), the proportion of IgA positive cells was significantly higher than that in the control group (no addition). The value is shown. From this, it was proved that the intestinal immunity enhancing agent containing the lactic acid bacterium Lactobacillus plantarum AYA strain (FERM P-21106) has a high IgA antibody production improving action.
（基礎飼料の配合）
カゼイン ２０．０質量％
コーンスターチ ５０．５質量％
シュークロース １０．０質量％
ラード １０．０質量％
セルロースパウダー ５．０質量％
ＡＩＮ−９３ミネラル混合 ３．５質量％
ＡＩＮ−９３ビタミン混合 １．０質量％
(Basic feed composition)
Casein 20.0 mass%
Corn starch 50.5% by mass
Sucrose 10.0% by mass
Lard 10.0% by mass
Cellulose powder 5.0% by mass
AIN-93 mineral mixture 3.5% by mass
AIN-93 vitamin mix 1.0% by mass
次に、本発明の腸管免疫力増強剤の実施例を示す。 Next, examples of the intestinal immunity enhancing agent of the present invention are shown.
実施例３（錠剤）
実施例１の（乳酸菌試料の調製）で示す ５ ｇ
方法で調製したＡＹＡの乳酸菌試料
トウモロコシデンプン １０ ｇ
乳糖 ４０ ｇ
カルボキシメチルセルロースカルシウム ８ ｇ
微結晶セルロース ２７ ｇ
ポリビニルピロリドン ７ ｇ
ステアリン酸マグネシウム ３ ｇ
合計 １００ ｇ
乳酸菌試料に微結晶セルロース、トウモロコシデンプン、乳糖、カルボキシメチルセルロースカルシウムを混合し、次いでポリビニルピロリドンの水溶液を結合剤として加えて常法により顆粒化する。これに滑沢剤としてステアリン酸マグネシウムを加えて混合した後、１錠１００ｍｇの錠剤に打錠する。
Example 3 (tablets)
5 g shown in (Preparation of lactic acid bacteria sample) in Example 1
Lactobacillus sample of AYA prepared by the method Corn starch 10 g
Lactose 40 g
Carboxymethylcellulose calcium 8 g
Microcrystalline cellulose 27 g
Polyvinylpyrrolidone 7 g
Magnesium stearate 3 g
Total 100 g
Microcrystalline cellulose, corn starch, lactose and carboxymethylcellulose calcium are mixed with the lactic acid bacteria sample, and then an aqueous solution of polyvinylpyrrolidone is added as a binder and granulated by a conventional method. To this, magnesium stearate as a lubricant is added and mixed, and then tableted into 100 mg tablets.
実施例４（硬カプセル剤）
実施例１の（乳酸菌試料の調製）で示す １０ ｇ
方法で調製したＡＹＡの乳酸菌試料
微結晶セルロース ５５ ｇ
トウモロコシデンプン ２５ ｇ
乳糖 ３０ ｇ
ポリビニルピロリドン ４ ｇ
ステアリン酸マグネシウム １ ｇ
合計 １２５ ｇ
上記成分を常法により顆粒化した後、ゼラチン硬カプセルに充填する。
Example 4 (hard capsule)
10 g shown in Example 1 (Preparation of lactic acid bacteria sample)
AYA Lactic Acid Bacteria Sample Prepared by the Method Microcrystalline Cellulose 55 g
Corn starch 25 g
Lactose 30 g
Polyvinylpyrrolidone 4 g
Magnesium stearate 1 g
Total 125 g
The above ingredients are granulated by a conventional method and then filled into gelatin hard capsules.
実施例５（散剤）
実施例１の（乳酸菌試料の調製）で示す ５０ ｇ
方法で調製したＡＹＡの乳酸菌試料
微結晶セルロース ６００ ｇ
トウモロコシデンプン ３００ ｇ
ポリビニルピロリドン ５０ ｇ
合計 １０００ ｇ
上記成分を混合し、常法により散剤とする。
Example 5 (powder)
50 g shown in Example 1 (Preparation of lactic acid bacteria sample)
AYA Lactic Acid Bacteria Sample Prepared by the Method Microcrystalline Cellulose 600 g
Corn starch 300 g
Polyvinylpyrrolidone 50 g
Total 1000 g
The above ingredients are mixed and powdered by a conventional method.
実施例６（顆粒剤）
実施例１の（乳酸菌試料の調製）で示す １０ ｇ
方法で調製したＡＹＡの乳酸菌試料
乳糖 １３０ ｇ
トウモロコシデンプン ８７ ｇ
ポリビニルピロリドン ８ ｇ
Ｌ−メントール １５ ｇ
軽質無水ケイ酸 ５ ｇ
合計 ２５５ ｇ
上記の処方で、乳酸菌試料、乳糖、トウモロコシデンプン及びポリビニルピロリドン水溶液を混合し、造粒機にて攪拌下加熱造粒する。冷却後、粒度５００μｍ以下に篩分けし、Ｌ−メントールを加えた後、無水ケイ酸を加え、混合し分包（１. ０ｇ）して顆粒剤とする。
Example 6 (granule)
10 g shown in Example 1 (Preparation of lactic acid bacteria sample)
Lactic acid bacteria sample of AYA prepared by the method Lactose 130 g
Corn starch 87 g
Polyvinylpyrrolidone 8 g
L-menthol 15 g
Light anhydrous silicic acid 5 g
Total 255g
In the above formulation, a lactic acid bacterium sample, lactose, corn starch and a polyvinylpyrrolidone aqueous solution are mixed and heated and granulated with stirring in a granulator. After cooling, it is sieved to a particle size of 500 μm or less, L-menthol is added, silicic anhydride is added, mixed and packaged (1.0 g) to give granules.
Claims (1)
上記乳酸菌が、ラクトバチルス・プランタラムＡＹＡ株（受託番号ＦＥＲＭ Ｐ−２１１０６）である腸管免疫力増強剤。 An intestinal immunity enhancing agent containing a lactic acid bacterium belonging to Lactobacillus plantarum having a high IgA antibody production improving action and having an intestinal immunity enhancing action as an active ingredient ,
An intestinal immunity enhancing agent, wherein the lactic acid bacterium is Lactobacillus plantarum AYA strain (Accession No. FERM P-21106).
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| US8980289B2 (en) | 2010-08-31 | 2015-03-17 | Niigata University | Intestine immunomodulator |
| JP2013053089A (en) * | 2011-09-02 | 2013-03-21 | Univ Of Tokyo | Prophylactic/therapeutic agent for viral disease |
| WO2013099883A1 (en) | 2011-12-28 | 2013-07-04 | 株式会社山田養蜂場本社 | NOVEL LACTIC ACID BACTERIUM HAVING IgA PRODUCTION PROMOTING ACTIVITY, AND USE THEREOF |
| JP2014125446A (en) * | 2012-12-26 | 2014-07-07 | Hiroshima Univ | Intestinal immune system enhancer containing cell wall component of lactic acid bacteria |
| US9572844B2 (en) | 2013-02-22 | 2017-02-21 | Nitto Pharmaceutical Industries, Ltd. | Immunostimulation agent |
| US10093749B2 (en) | 2013-09-19 | 2018-10-09 | Nitto Pharmaceutical Industries, Ltd. | Exopolysaccharide produced by lactic acid bacterium |
| KR102500894B1 (en) | 2018-05-31 | 2023-02-20 | 베르텍스 가부시키가이샤 | Lactic acid bacteria and their uses |
| TWI689585B (en) * | 2018-12-10 | 2020-04-01 | 日商奧碧慧央集團股份有限公司 | Novel lactic acid strain and immune activating agent containing novel lactic acid strain |
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