JP4064481B2 - Immunostimulator - Google Patents

Description

【０００８】
以上の菌学的性質、および細胞壁のペプチドグリカンタイプがメゾ−ジアミノピメリン酸（meso-diaminopimelic acid）であること、さらに、Ｌ−１３７菌株と２２タイプの乳酸菌基準株との間での DNA-DNA交雑実験法を行ったところ、Ｌ−１３７菌株はラクトバチルス・プランタラムにのみ強くDNAの相同性が得られたことにより、本発明に用いられる微生物はラクトバチルス・プランタラムと同定された。
本発明の微生物については、Journal of Fermentation and Bioengineering, Vol.73, No.3, 193-197(1992)及び Vol. 80, No.2, 124-130(1995)にも報告されている。
本発明の免疫賦活剤は、ラクトバチルス属に属し、Ｔリンパ球共刺激作用とＢリンパ球活性化抑制作用を有する菌を天然培地、合成培地、半合成培地などの培地に培養することにより得ることができる。
培地としては、窒素源および炭素源を含有するものが用いられる。窒素源としてはたとえば、肉エキス、ペプトン、グルテン、カゼイン、酵母エキス、アミノ酸等であり、炭素源としては、たとえば、グルコース、キシロース、フラクトース、イノシトール、水アメ、麹汁、澱粉、バカス、フスマ、糖蜜、グリセリン等が用いられる。
このほか、無機質として、たとえば硫酸アンモニウム、リン酸カリウム、塩化マグネシウム、食塩、鉄、マンガン、モリブデン更に各種ビタミン類その他を添加することができる。
【０００９】
培養温度は２５〜４０℃、好ましくは２７〜３５℃であり、培養時間は１２〜４８時間程度であり、通気振盪してもよい。
培地のｐＨは３〜６、好ましくは４〜６である。培養終了後菌体を採取し蒸留水を加え、遠心分離などの手段により上清を除き、必要によりその操作を繰り返し、遠心分離や濾過等により菌体を採取する。
採取された菌体は生菌のまま、またはたとえば過熱、紫外線照射、ホルマリン処理などにより不活性化して投与に適した剤型にすることもできる。分離された生菌体、死菌体はさらに摩砕や破砕処理をし、得られた処理物を必要により加熱滅菌、無菌濾過し、濾液を凍結乾燥して製品とすることもできる。
菌体の処理物にはたとえば、上記摩砕物、破砕物、それらからの抽出液、凍結乾燥品が含まれる。
また、本発明に用いられる乳酸菌の一種、ラクトバチルス・プランタラム Ｌ−１３７株は、元々発酵食品であるブロングイスダから分離されたものであり、食品、たとえば果菜類、穀類から選択された少なくとも１種または、果菜類や穀類を発酵可能な形態に処理したもの、たとえば切断物、粉砕物、摩砕物、搾汁、搾汁濃縮物を本発明において用いられる菌により発酵させた菌を含む発酵物をそのまま用いることができ、これも本発明の好ましい態様の１つである。
前記の発酵法を利用すると、野菜汁に対して、フレッシュなニンジン汁などを得るためのフレッシュスクイーズ法などの特別な前処理を施す必要がなく、また乳成分を添加する必要がなく、乳酸菌の果菜類などの処理物（野菜汁など）に対する高い発酵能により、乳酸菌を多量に含み味覚的にも極めて優れた発酵物を得ることができる。また、被発酵処理物が臭いのきつい果菜類や穀類を含んでいても、野菜類などの特有の不快臭や加熱による不快臭を顕著に低減できるとともに、風味を改善でき、極めて容易に食することができる発酵食品が得られる。しかも、サイレージと異なり発酵食品から分離された食習慣のある乳酸菌であるため、本発明の微生物は安全性も高い。
【００１０】
上述の乳酸菌発酵物は、果菜類及び穀類から選択された少なくとも一種の処理物を前記乳酸菌により発酵させることにより得られる。前記果菜類には、種々の可食性植物、例えば、野菜類（例えば、ニンジン、トマト、ホウレン草、ぱせり、シソ葉、大葉、芽キャベツ、小松菜、カボチャ、大根葉、ピーマン、ケール、カンショ葉、春菊、セリなどの緑黄色野菜類、セロリ、キャベツ、アスパラガス、キュウリ、スイカなどの他の野菜類）、リンゴ、バナナ、パパイヤ、アボガド、ミカン、グレープフルーツ、レモン、パイナップル、ピーチ、柿、イチゴ、ブドウ、メロン、ココナッツなどの果物類などが含まれる。穀類には、米、トウモロコシ、大豆、小麦、ライ麦などが含まれる。これらの果菜類などは単独で又は二種以上組み合わせて使用でき、必要に応じて、これらの果菜類などは、ジャガイモ、サツマイモなどのイモデンプン類と併用してもよい。好ましい果菜類には、ニンジンなどの緑黄色野菜類、バナナなどの果物類、米などの穀類などが含まれる。
また、処理物としては、切断物、粉砕物、摩砕物、搾汁、搾汁濃縮物などが単独又は二種以上組み合わせて使用できる。好ましい処理物には、野菜汁、果汁などの搾汁や搾汁濃縮物などの搾汁類が含まれる。この搾汁類において、ニンジンを用いる場合、ニンジン汁の濃度はBrix２〜３０程度の範囲から選択できる。
また、５０重量％以上のニンジン処理物を含む処理物は、発酵飲料などの風味を改善する上で有用である。
【００１１】
前記果菜類などの処理物は、通常、ブランチング処理及び／又は殺菌処理に供された後、前記乳酸菌による発酵に供される。ブランチング処理は、前記果菜類などやその処理物、特に果菜類やその切断物を加熱処理し、酵素活性を失活させることにより行うことができ、ブランチング処理の後、遠心分離やフィルタープレスなどの方法で搾汁しジュースを得る場合が多い。また、殺菌処理は、ブランチング処理された前記果菜類などやその処理物、特に搾汁類について行う場合が多い。なお、ブランチング処理および殺菌処理は、風味を損なわない範囲で選択でき、ブランチング処理は、慣用の方法、例えば、必要に応じてオートクレーブを用い、７０〜１００℃で短時間処理することにより行うことができる。殺菌処理は、慣用の方法、例えば、７０〜１２５℃程度の温度又は高温短時間で加熱殺菌する方法、紫外線などの光線を照射する方法などが採用できる。
前記微生物による発酵は、前記乳酸菌を搾汁類などの処理物に直接接種して行ってもよいが、通常、適当な培地や前記処理物を用いて馴化培養した前記乳酸菌をスターターとして搾汁類などの処理物に接種して行う場合が多い。発酵は、慣用の方法、例えば、処理物に対して０.５〜３重量％程度のスターターを接種し、２５〜４０℃（例えば、２５〜３８℃）、好ましくは２７〜３８℃（例えば、２７〜３５℃）程度で行うことができる。発酵時間は、果菜類などの処理物の種類などに応じて、例えば、数時間〜数日間程度の範囲から選択できる。
本発明の好ましい態様には、ニンジンなどの緑黄色野菜、果物及び穀類のうちの少なくとも一種の処理物（特に野菜および果物のうち少なくとも一種から得られた搾汁類）を前記乳酸菌で発酵させ、乳酸菌発酵飲料（緑黄色野菜ジュース、果物ジュースなど）やその加工品（緑黄色野菜ゼリー，スプレットなど）として得る方法が含まれる。
【００１２】
なお、発酵に際しては、必要に応じて、他の微生物、例えば、乳酸菌（ラクトバチルス・プランタラム、ラクトバチルス・カゼイ、ラクトバチルス・ラムノサスなど）やエンテロコッカス属微生物（エンテロコッカス・フェカーリスなど）、酵母などを併用してもよい。
さらに、必要に応じて、前記処理物に、種々の添加剤、例えば、ビタミン、アミノ酸、ミネラル、植物繊維、糖類、蜂蜜などの甘味料、香料、牛乳、脱脂粉乳などの乳成分、果汁などを添加して発酵させてもよく、得られた乳酸菌発酵物に前記添加剤を添加してもよい。
このようにして、果菜類などの処理物を前記乳酸菌により発酵させると、得られる乳酸菌発酵物の風味を改善できる。この方法により得られた乳酸菌発酵物は、被発酵処理物が臭いのきつい果菜類や穀類を含んでいても、不快臭を顕著に低減できるとともに、加熱による不快臭も抑制できる。また、乳酸菌の発酵により適度な酸味（例えば、ｐＨ４〜５程度）を呈するとともに、官能的に優れた風味を有しており、極めて容易に食することができる。
【００１３】
本発明の免疫賦活剤は、自体公知の食品あるいは、食品成分、医薬担体または賦形剤と自体公知の方法で合して、免疫力を高める食品や医薬剤としても利用可能である。用いる食品あるいは、食品成分、医薬担体または賦形剤は特に限定するものではなく、当該免疫賦活剤の具体的用途に応じて当業者が適宜選択できる。また免疫賦活剤の形態も特に限定する物ではなく、具体的用途に応じて種々の固体や液体の形態とすることができる。本発明の免疫賦活剤は、医薬として用いる場合、経口投与あるいは、非経口投与が考えられるが、一般的に請求項１に記載の有効成分のいずれか、あるいは組み合わせて用いることが可能であり、その投与量は、投与形態にもよるが、経口投与の場合有効成分として成人１日当たり４０ｍｇ〜４０ｇであり、静注の場合は０.１ｍｇ〜１ｇである。本発明の免疫賦活剤を食品として用いる場合、調味料、畜肉加工品、水産加工品、農産加工品、ステープル、調味食品、調味済食品、デザート類、乳油製品、菓子、スナック菓子等の形態で提供することも可能である。
本発明の免疫賦活剤は、たとえば、ウイルス、バクテリヤ等の微生物による感染症や各種悪性腫瘍などの予防・治療に有効である。
【００１４】
【実施例】
以下に実施例および試験例をあげて本発明をさらに具体的に説明するが、本発明はそれらによって限定されるものではない。
実施例１
ラクトバチルス・プランタラムＬ−１３７乾燥菌体の製造方法
乳酸菌培養培地であるＧＹＰ培地のグルコースの代わりにスターチを加えた培地２００ｍｌにラクトバチルス・プランタラムＬ−１３７をスターターとして１重量％接種し、３２℃で２４時間前培養を行った。その後、６ＬのＧＹＰ培地にその前培養した培養液をスターターとして１重量％接種し、３２℃にて２４時間静地培養した。培養後、５０００ｒｐｍで３５分間遠心分離した。そして、上清を除き、菌体を集めた。さらに、集めた菌体ペーストを生理食塩水に良く分散し、５０００ｒｐｍで３５分遠心分離したのち、上清を除き菌体を集めた。これを３回繰り返したのち、蒸留水に分散した。そして７０℃で１０分間殺菌した。これを凍結乾燥し、乾燥菌体を７.０７ｇ得た。
【００１５】
実施例２
４倍濃縮ニンジン汁のラクトバチルス・プランタラムＬ−１３７発酵物の製造方法
Ｌ−１３７乾燥菌体の作成方法と同様の方法で６リットルのＧＹＰ培地で３２℃、２４時間培養したのち生理食塩水中に分散、遠心分離することにより集めた菌体ペーストを４倍濃縮ニンジン汁（人参１／６濃縮搾汁 宮崎農協製を蒸留水ににより希釈したもの）３００ｍｌに添加し、３２℃で２４時間培養した。そして７０℃で１０分間殺菌した。こうして得られたニンジン発酵液を適当量の蒸留水で希釈し、凍結乾燥した。凍結乾燥物として約７７.４ｇを得た。
【００１６】
試験例１
本試験例では、実施例１で得たラクトバチルス・プランタラムＬ−１３７菌体を用いて、マウス脾臓リンパ球の増殖反応に対するラクトバチルス・プランタラムＬ−１３７菌体のＴリンパ球共刺激効果およびＢリンパ球抑制効果を検証した。
マウス（B10．A、雌、8週令）から無菌的に脾臓を摘出し、ＲＰＭＩ １６４０培地中で脾臓を押し潰し、＃２００メッシュに通し脾臓細胞浮遊液を得た。脾臓細胞浮遊液の細胞数を自動血球計測装置により測定した後、細胞数を５×１０⁶／ｍｌの濃度にＲＰＭＩ １６４０培地で調製し、９６穴組織培養プレートに１穴あたり１００μｌを播種した。Ｂリンパ球増殖刺激物質のリポポリサッカロイド（Difco 社製）を２００μｇ／ｍｌの濃度でＲＰＭＩ １６４０培地に溶解した液、Ｂリンパ球増殖刺激物質の抗マウスイムノグロブリンＭ（Cappel 社製）を２００μｇ／ｍｌの濃度でＲＰＭＩ １６４０培地に溶解した液、Ｔリンパ球増殖刺激物質のフィトヘマグルチニン（Difco 社製）をＲＰＭＩ １６４０培地で４００倍希釈した液、およびＲＰＭＩ １６４０培地を、それぞれ１穴当たり５０μｌ播種した脾臓細胞浮遊液に加えて、Ｂリンパ球刺激群（１）、Ｂリンパ球刺激群（２）、Ｔリンパ球刺激群（１）、無刺激群とした。Ｔリンパ球刺激群（２）として、細胞播種前にＴリンパ球増殖刺激物質の抗マウスＣＤ３抗体（Cedarlane 社製）を１０μｇ／ｍｌの濃度でホウ酸緩衝液に溶解した液を１穴当たり１００μｌ加え、３７℃で３時間放置し、抗マウスＣＤ３抗体を各穴に付着させ、３時間後にＲＰＭＩ １６４０培地で洗浄後、ＲＰＭＩ １６４０培地を１穴当たり５０μｌ加えた穴に細胞を播種した。これらの５群にＲＰＭＩ １６４０培地（対照）あるいはラクトバチルス・プランタラムＬ−１３７菌体を５０μｇ／ｍｌ、１２,５μｇ／ｍｌおよび３.１３μｇ／ｍｌの濃度でＲＰＭＩ １６４０培地に溶解した液をそれぞれ１穴当たり５０μｌ加え、３７℃の５％炭酸ガス培養器内で２日間培養し、培養後代謝活性を調べた。
細胞代謝活性は、培養の終わる３時間前に臭化３−（４，５−ジメチル−２−チアゾリル）−２，５−ジフェニル−２Ｈテトラゾリウムを５ｍｇ／ｍｌの濃度でＲＰＭＩ １６４０培地に溶解した液を１穴当たり１０μｌ加え、培養終了時に２０％ドデシル硫酸ナトリウム溶液を１穴当たり５０μｌ加え、３７℃で１日放置後、マイクロプレートリーダーで培養液の吸光度５５０ｎｍを測定することにより細胞代謝活性を求めた。〔表２〕にその結果を示す。
【００１７】
【表２】

〔表２〕から明らかなごとく、ラクトバチルス・プランタラムＬ−１３７菌体は、脾臓細胞が刺激を受けていない無刺激群では、わずかに細胞代謝活性を上昇させたにすぎなかったが、抗原受容体を介するＴリンパ球刺激群（２）および抗原受容体以外の経路のＴリンパ球刺激群（１）では顕著に細胞代謝活性を上昇させ、ラクトバチルス・プランタラムＬ−１３７菌体のＴリンパ球共刺激効果が認められた。一方、ラクトバチルス・プランタラムＬ−１３７菌体は、抗原受容体を介するＢリンパ球刺激群（２）および抗原受容体以外の経路のＢリンパ球刺激群（１）では細胞代謝活性の上昇を抑制する作用が認められた。
【００１８】
試験例２
本試験例では、実施例１で得たラクトバチルス・プランタラムＬ−１３７菌体を用いて、マウス脾臓リンパ球のインターフェロン−γ産生反応に対するラクトバチルス・プランタラムＬ−１３７菌体の増強効果を検証した。
マウス（B10．A、雌、7週齢）から無菌的に脾臓を摘出し、ＲＰＭＩ １６４０培地中で脾臓を押し潰し、＃２００メッシュに通し脾臓細胞浮遊液を得た。
脾臓細胞浮遊液の細胞数を自動血球計測装置で測定した後、細胞数を５×１０⁶／ｍｌの濃度にＲＰＭＩ １６４０培地で調製し、９６穴組織培養プレートに１穴当たり１００μｌを播種した。
Ｔリンパ球増殖刺激物質のフィトヘマグルチニン（Difco 社製）をＲＰＭＩ １６４０培地で４００倍希釈した液を１穴当たり５０μｌ播種した脾臓細胞浮遊液に加えた。これにＲＰＭＩ １６４０培地（対照）あるいはラクトバチルス・プランタラムＬ−１３７菌体を２００μｇ／ｍｌおよび１００μｇ／ｍｌの濃度でＲＰＭＩ １６４０培地に溶解した液をそれぞれ１穴当たり５０μｌ加え、３７℃の５％炭酸ガス培養器内で３日間培養し、培養後の培養上清のインターフェロン−γをエンザイムイムノアッセイで測定した。
エンザイムイムノアッセイは、ハムスター抗マウスインターフェロン−γ抗体（Genzyme 社製）をホウ酸緩衝液で３μｇ／ｍｌに調製した溶液を、９６穴組織培養プレート１穴当たり１００μｌ加え、５℃で３日間放置しハムスター抗マウスインターフェロン−γ抗体を各穴に付着させたプレートを用いて行った。培養上清を１穴当たり５０μｌ加え室温で９０分間放置し、培養上清のインターフェロン−γをプレートに付着したハムスター抗マウスインターフェロン−γ抗体と結合させた。洗浄後ラット抗マウスインターフェロン−γ抗体を加え、プレートに結合させたインターフェロン−γに結合させた。洗浄後ペルオキシダーゼで標識した抗ラットＩｇＧ抗体を加え、プレートに結合させたラット抗マウスインターフェロン−γ抗体に結合させた。洗浄後、過酸化水素０.００６％とオルトフェニレンジアミン０.１％を含有するリン酸緩衝液を１穴当たり１００μｌ加え、室温で２０分間反応させ、反応を１.５Ｎ硫酸で停止し、マイクロプレートリーダーで吸光度４９２ｎｍを測定し、リコンビナントマウスインターフェロン−γで作成した標識曲線から、培養上清中のインターフェロン−γの濃度を求めた。〔表３〕にその結果を示す。
【００１９】
【表３】

〔表３〕から明らかなごとくラクトバチルス・プランタラムＬ−１３７菌体はフィトヘマグルチニンにより誘導されるインターフェロン−γの産生を大幅に上昇させた。
【００２０】
試験例３
本試験例では、実施例１で得たラクトバチリス・プランタラムＬ−１３７菌体を用いて、マウス腹腔マクロファージのインターロイキン１２産生性に対するラクトバチリス・プランタラムＬ−１３７菌体の促進効果を検証した。
マウス（Ｃ５７ＢＬ／６、雌、１５週齢）の腹腔に無菌的にＲＰＭＩ １６４０培地を注入し、腹部を良く揉んだ後、注入したＲＰＭＩ １６４０培地を回収し腹腔細胞浮遊液を得た。腹腔細胞浮遊液の細胞数とそれに含まれるマクロファージの割合を自動血球計測装置で測定した後、マクロファージとして１×１０⁶／ｍｌの細胞数にＲＰＭＩ １６４０培地で調製し、９６穴組織培養プレートに１穴当たり１００μｌを播種した。３７℃の５％炭酸ガス培養器内に２時間放置し、腹腔マクロファージを各穴に付着させ、２時間後にＲＰＭＩ １６４０培地で洗浄後、ＲＰＭＩ １６４０培地を１穴当たり１００μｌ加えた。
これにＲＰＭＩ １６４０培地（対照）あるいはマクロファージ活性化物質のリポポリサッカライド（Difico 社製）を０.２μｇ／ｍｌの濃度でＲＰＭＩ １６４０培地に溶解した液、あるいはラクトバチリス・プランタラムＬ−１３７菌体を０.２μｇ／ｍｌの濃度でＲＰＭＩ １６４０培地に溶解した液をそれぞれ１穴当たり１００μｌ加え、３７℃の５％炭酸ガス培養器内で１５時間培養し、培養後の培養上清のインターロイキン１２と腫瘍壊死因子αをエンザイムイムノアッセイで測定した。
エンザイムイムノアッセイは、ラット抗マウスインターロイキン１２ ＩｇＧ２ａ抗体（Genzyme 社製）あるいはハムスター抗マウス腫瘍壊死因子α＆β抗体（Genzyme 社製）をホウ酸緩衝液で６μｇ／ｍｌに調製した溶液を、９６穴組織培養プレート１穴当たり１００μｌ加え３７℃で１日間放置し、ラット抗マウスインターロイキン１２ ＩｇＧ２ａ抗体あるいはハムスター抗マウス腫瘍壊死因子α＆β抗体を各穴に付着させたプレートを用いて行った。培養上清を１穴当たり５０μｌ加え室温で９０分間放置し、培養上清のインターロイキン１２あるいは腫瘍壊死因子αをプレートに付着したラット抗マウスインターロイキン１２ ＩｇＧ２ａ抗体あるいはハムスター抗マウス腫瘍壊死因子α＆β抗体と結合させた。洗浄後ラット抗マウスインターロイキン１２ ＩｇＧ １抗体（Genzyme 社製）あるいはラビット抗マウス腫瘍壊死因子α抗体（Genzyme 社製）を加え、プレートに結合させたインターロイキン１２あるいは腫瘍壊死因子αに結合させた。洗浄後ペルオキシダーゼで標識した抗ラットＩｇＧ １抗体あるいは抗ラビットＩｇＧ抗体を加え、プレートに結合させたラット抗マウスインターロイキン１２ＩｇＧ １抗体あるいはラビット抗マウス腫瘍壊死因子α抗体に結合させた。洗浄後、過酸化水素０.００６％とオルトフェニレンジアミン０.１％を含有するリン酸緩衝液を１穴当たり１００μｌ加え、室温で２０分間反応させ、反応を１.５Ｎ硫酸で停止し、マイクロプレートリーダーで吸光度４９２ｎｍを測定し、リコンビナントマウスインターロイキン１２あるいは腫瘍壊死因子αで作成した標準曲線から、培養上清中のインターロイキン１２あるいは腫瘍壊死因子αの濃度を求めた。
〔表４〕にその結果を示す・
【００２１】
【表４】

〔表４〕から明らかなごとくラクトバチリス・プランタラムＬ−１３７菌体は、マクロファージからのインターロイキン１２の産生を大幅に上昇させたが、腫瘍壊死因子αの産生は軽度にしか上昇させなかった。強力なマクロファージ活性化剤であるリポポリサッカライドのサイトカイン産生促進作用と比較すると、ラクトバチリス・プランタラムＬ−１３７菌体が選択的にマクロファージのインターロイキン１２の産生を上昇させることが明らかとなった。
【００２２】
【発明の効果】
本発明の免疫賦活剤は、有効成分の生産性が容易で且つ高く、また得られた免疫賦活剤は人体に投与した場合安全性が高く且つ免疫賦活効果も高く単独または他の免疫賦活剤と併用して、ウイルス、バクテリヤ等による感染症、悪性腫瘍等の予防・治療に用いることができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an immunostimulant comprising a bacterial body of Lactobacillus belonging to lactic acid bacteria or a processed product thereof.
[0002]
[Prior art]
The in vivo immune system plays an important role in defense against microorganisms such as bacteria, yeast, mold and virus, and defense against tumors, and the center of the defense mechanism is T lymphocytes. T lymphocytes are stimulated by recognizing these microorganisms and tumors via antigen receptors, are activated specifically for antigens, and increase the ability to eliminate these foreign substances. In living organisms that are constantly exposed to microorganisms and whose cells are mutated, these T lymphocytes are always activated through antigen receptors, while they are also non-antigen-specifically activated in pathways other than antigen receptors. Has been. In both antigen-specific and non-antigen-specific activation, the activation of T lymphocytes is further promoted by the addition of other stimuli, ie, costimulation. Currently used immunostimulants enhance the defense mechanism of the body against microbial infection and tumors by nonspecifically activating immunocompetent cells, but there are few that can be satisfied with effectiveness, Moreover, since these immunostimulants generally have low specificity of action, there are concerns about side effects such as autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis caused by activation of B lymphocytes.
[0003]
[Problems to be solved by the invention]
The present invention has an immunostimulatory effect itself, and further exhibits a stronger immunostimulatory effect when used in combination with other immunostimulatory substances, and has substantially no side effects, and can be administered in combination with food, for example. An object is to provide an activator.
[0004]
[Means for Solving the Problems]
The present inventors have this filtrate and overlaid research on adjuvants for solving the above problem, Lactobacillus plantarum is a kind of lactic acid bacterium (Lactobacillus plantarum) cells of L-137 strain T-lymphocytes co It was found to show a stimulating effect. In addition, the activity of T lymphocytes activated by stimulation through the antigen receptor is further increased, and the activity of T lymphocytes activated nonspecifically by a pathway other than the antigen receptor is also increased. I found it to rise. With the activation of these T lymphocytes, the production of interferon-γ of T lymphocytes is enhanced, while the stimulation by B via the antigen receptor and the activation by pathways other than the antigen receptor are suppressed. The fact was also found. Apart from this, it was also found that the cells have an action of selectively promoting the production of macrophage interleukin 12. In other words, Lactobacillus plantarum L-137 cells increase the activation of T lymphocytes via antigen receptors, thereby enhancing the elimination reaction against microorganisms and tumor cells that are constantly occurring in the living body. -Enhance production against viruses and tumors by enhancing γ production. However, since it hardly activates lymphocytes alone, it does not induce an immune response unfavorable for the living body, and suppresses activation of B lymphocytes through antigen receptors and non-specific activation of antigens. Thus, autoimmune diseases induced by polyclonal activation of B lymphocytes predicted with immune activation are not exacerbated. In addition, Lactobacillus plantarum L-137 cells increase the production of interleukin 12, which is a cytokine that activates natural killer cells having tumor cytotoxicity, from macrophages, and as a result, increase the protective ability against tumors. It is also useful for preventing the onset of acquired immune deficiency syndrome (AIDS). However, tumor necrosis factor-α production is only slightly increased and does not induce side effects such as fever, weight loss, increased sensitivity to endotoxin shock caused by tumor necrosis factor-α, which is increased by normal macrophage activators . Thus, Lactobacillus plantarum L-137 has no side effects and is an immunostimulant suitable for daily use, and the cell increases the activation of non-antigen-specific T lymphocytes. since it promotes the production of interleukin-12 by macrophages and have completed the present invention found to be also effective in combination with other adjuvants. That is, the present invention includes a bacterial body of Lactobacillus plantarum L-137 strain (FERM P-15317) having T lymphocyte co-stimulatory action and B lymphocyte inhibitory action or / and interleukin 12 production promoting action. An immunostimulator.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The bacterium used in the present invention belongs to the genus Lactobacillus, and has any T lymphocyte costimulatory action and B lymphocyte activation inhibitory action or / and an action of promoting macrophage interleukin 12 production. It may be a fungus.
For example, mouse spleen cells are cultured in a medium containing a T lymphocyte proliferation stimulating substance such as phytohemagglutinin or a B lymphocyte proliferation stimulating substance such as lipopolysaccharide. Occasionally, a bacterium belonging to the genus Lactobacillus is added and cultured for a certain period of time to determine the mitochondrial metabolic activity of the cells in the medium. 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide It can be easily determined by a colorimetric method of measuring using
The interleukin 12 production promoting action of fungal macrophages is measured, for example, by culturing mouse peritoneal macrophages on a tissue culture plate, adding bacteria belonging to the genus Lactobacillus, culturing for a certain period, and measuring the concentration of interleukin 12 in the medium by enzyme immunoassay. This can be easily determined.
Examples of substances that increase the activity of T lymphocytes by co-stimulation with the immunostimulant of the present invention, that is, immunostimulants that can be used in combination with the immunostimulant of the present invention include cytokines such as interleukin 2 and phytohemagglutinin. And anti-T lymphocyte surface antigen antibodies such as anti-CD3 antibodies.
[0006]
Lactobacillus plantarum L-137 may be mentioned as a representative of the bacteria belonging to the genus Lactobacillus used in the present invention. It has been deposited on the 30th as accession number FERM P-15317, Microtechnical Laboratories No. 15317.
Lactobacillus plantarum L-137 is a microorganism isolated from Philippine fermented food Burong isda, and has an assimilability to specific saccharides (gluconic acid, arabinose, rhamnose and starch). It is different from the reference strain of Lactobacillus plantarum JCM 1149 and Lactobacillus plantarum L-051 (Mikken Kenkyu No. 11912). That is, it shows the following assimilation properties for the following saccharides.
Gluconic acid −
Arabinose −
Rhamnose −
Starch +
Furthermore, the bacteriological properties of Lactobacillus plantarum L-137 and the aforementioned Lactobacillus plantarum JCM 1149 reference strain and Lactobacillus plantarum L-051 strain are as shown in [Table 1].
[0007]
[Table 1]

[0008]
The above bacteriological properties, and that the peptidoglycan type of the cell wall is meso-diaminopimelic acid, and further, a DNA-DNA hybridization experiment between L-137 strain and 22 types of lactic acid bacteria reference strain As a result of the method, the L-137 strain was found to have strong DNA homology only to Lactobacillus plantarum, and thus the microorganism used in the present invention was identified as Lactobacillus plantarum.
The microorganism of the present invention is also reported in Journal of Fermentation and Bioengineering, Vol. 73, No. 3, 193-197 (1992) and Vol. 80, No. 2, 124-130 (1995).
The immunostimulant of the present invention is obtained by culturing a bacterium belonging to the genus Lactobacillus and having T lymphocyte costimulatory action and B lymphocyte activation inhibitory action in a medium such as a natural medium, a synthetic medium, or a semi-synthetic medium. be able to.
A medium containing a nitrogen source and a carbon source is used as the medium. Examples of the nitrogen source include meat extract, peptone, gluten, casein, yeast extract, amino acid and the like, and examples of the carbon source include glucose, xylose, fructose, inositol, water candy, soup, starch, bacus, bran, Molasses, glycerin and the like are used.
In addition, for example, ammonium sulfate, potassium phosphate, magnesium chloride, sodium chloride, iron, manganese, molybdenum, and various vitamins can be added as inorganic substances.
[0009]
The culture temperature is 25 to 40 ° C., preferably 27 to 35 ° C., the culture time is about 12 to 48 hours, and aeration shaking may be performed.
The pH of the medium is 3-6, preferably 4-6. After completion of the culture, the cells are collected, distilled water is added, the supernatant is removed by means such as centrifugation, the operation is repeated as necessary, and the cells are collected by centrifugation or filtration.
The collected microbial cells can be made into live dosage forms or inactivated by, for example, overheating, ultraviolet irradiation, formalin treatment, etc., to give a dosage form suitable for administration. The separated live cells and dead cells can be further ground or crushed, and the obtained processed product can be heat-sterilized and aseptically filtered if necessary, and the filtrate can be freeze-dried to obtain a product.
Examples of the processed product of the microbial cell include the above-mentioned ground product, crushed product, extract from them, and lyophilized product.
In addition, Lactobacillus plantarum L-137 strain, a kind of lactic acid bacteria used in the present invention, was originally isolated from Bron Isda, which is a fermented food, and is at least one selected from foods such as fruit vegetables and cereals. Alternatively, a fermented product containing bacteria obtained by fermenting fruit vegetables and cereals into a form that can be fermented, such as a cut product, a pulverized product, a milled product, a squeezed juice, and a squeezed concentrate by the bacterium used in the present invention It can be used as it is and is also one of the preferred embodiments of the present invention.
Using the above fermentation method, it is not necessary to apply special pretreatment such as a fresh squeeze method for obtaining a fresh carrot juice to vegetable juice, and it is not necessary to add milk components. Due to the high fermentability with respect to processed products such as fruit vegetables (vegetable juice, etc.), a fermented material that contains a large amount of lactic acid bacteria and that is extremely excellent in taste can be obtained. In addition, even if the processed food contains pungent fruits and vegetables and cereals, it is possible to remarkably reduce peculiar unpleasant odors such as vegetables and unpleasant odors caused by heating, improve the flavor, and eat very easily A fermented food product is obtained. In addition, unlike the silage, since it is a lactic acid bacterium having a dietary habit isolated from fermented food, the microorganism of the present invention is also highly safe.
[0010]
The above-mentioned fermented lactic acid bacteria can be obtained by fermenting at least one processed product selected from fruit vegetables and cereals with the lactic acid bacteria. The fruits and vegetables include various edible plants such as vegetables (for example, carrots, tomatoes, spinach, parsley, perilla leaves, large leaves, Brussels sprouts, komatsuna, pumpkin, radish leaves, peppers, kale, sweet potato leaves, spring chrysanthemum, Other vegetables such as celery, celery, cabbage, asparagus, cucumber, watermelon), apple, banana, papaya, avocado, mandarin, grapefruit, lemon, pineapple, peach, strawberry, strawberry, grape, melon , Fruits such as coconut. Cereals include rice, corn, soybeans, wheat, rye and the like. These fruit vegetables etc. can be used individually or in combination of 2 or more types, and these fruit vegetables etc. may be used together with potato starches, such as a potato and a sweet potato, as needed. Preferred fruit vegetables include green and yellow vegetables such as carrots, fruits such as bananas, and grains such as rice.
Moreover, as a processed material, a cut material, a ground material, a ground material, squeezed juice, a squeezed concentrate etc. can be used individually or in combination of 2 or more types. Preferred processed products include juices such as vegetable juice and fruit juice, and juices such as juice concentrate. In this squeezed juice, when carrot is used, the concentration of carrot juice can be selected from a range of about Brix 2 to 30.
Moreover, the processed material containing 50 weight% or more of carrot processed materials is useful when improving flavors, such as fermented drinks.
[0011]
Processed products such as fruit vegetables are usually subjected to blanching treatment and / or sterilization treatment and then subjected to fermentation by the lactic acid bacteria. The blanching treatment can be carried out by heat-treating the fruit vegetables and the processed products thereof, in particular the fruit vegetables and the cut products thereof, to deactivate the enzyme activity. In many cases, juice is obtained by juice extraction. In addition, the sterilization treatment is often performed on the blanched fruit and vegetables and the processed products thereof, particularly juices. The blanching treatment and the sterilization treatment can be selected within a range that does not impair the flavor, and the blanching treatment is performed by a conventional method, for example, by using an autoclave as necessary for a short time at 70 to 100 ° C. be able to. As the sterilization treatment, a conventional method, for example, a method of sterilizing by heating at a temperature of about 70 to 125 ° C. or a high temperature in a short time, a method of irradiating light such as ultraviolet rays, or the like can be adopted.
Fermentation with the microorganisms may be performed by directly inoculating the processed product such as squeezed lactic acid bacteria, but usually the lactic acid bacterium conditioned and cultured using a suitable medium or the processed product is used as a starter. This is often done by inoculating processed materials such as Fermentation is a conventional method, for example, inoculating a processed product with about 0.5 to 3% by weight of a starter, 25 to 40 ° C. (for example, 25 to 38 ° C.), preferably 27 to 38 ° C. (for example, 27 to 35 ° C.). The fermentation time can be selected from a range of several hours to several days, for example, depending on the type of processed product such as fruit vegetables.
In a preferred embodiment of the present invention, at least one processed product of green-yellow vegetables such as carrots, fruits and cereals (especially juices obtained from at least one of vegetables and fruits) is fermented with the lactic acid bacteria, and the lactic acid bacteria The methods include obtaining fermented beverages (green-yellow vegetable juice, fruit juice, etc.) and processed products (green-yellow vegetable jelly, spreads, etc.).
[0012]
During fermentation, if necessary, other microorganisms such as lactic acid bacteria (Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus etc.), Enterococcus genus microorganisms (Enterococcus faecalis etc.), yeast, etc. You may use together.
Furthermore, if necessary, the processed product may contain various additives such as vitamins, amino acids, minerals, vegetable fibers, sugars, sweeteners such as honey, fragrances, milk components such as milk and skim milk powder, fruit juices, and the like. You may add and ferment, and you may add the said additive to the obtained lactic-acid-bacteria fermented material.
Thus, when processed products, such as fruit vegetables, are fermented with the said lactic acid bacteria, the flavor of the obtained lactic acid bacteria fermented material can be improved. The fermented lactic acid bacteria obtained by this method can remarkably reduce unpleasant odors and suppress unpleasant odors due to heating even if the processed product contains pungent fruit vegetables and grains. Moreover, while exhibiting moderate acidity (for example, about pH 4-5) by fermentation of lactic acid bacteria, it has the taste excellent in sensory and can eat very easily.
[0013]
The immunostimulant of the present invention can be used as a food or a pharmaceutical agent for enhancing immunity by combining with a per se known food or a food ingredient, a pharmaceutical carrier or an excipient by a per se known method. The food to be used or food ingredient, pharmaceutical carrier or excipient is not particularly limited, and can be appropriately selected by those skilled in the art depending on the specific use of the immunostimulator. Also, the form of the immunostimulant is not particularly limited, and various solid and liquid forms can be used depending on the specific application. When the immunostimulant of the present invention is used as a medicine, oral administration or parenteral administration can be considered, but in general, any of the active ingredients according to claim 1 or a combination thereof can be used, The dose varies depending on the administration form, but it is 40 mg to 40 g per day as an active ingredient in the case of oral administration, and 0.1 mg to 1 g in the case of intravenous injection. When the immunostimulant of the present invention is used as food, it is provided in the form of seasonings, processed meat products, processed fishery products, processed agricultural products, staples, seasoned foods, seasoned foods, desserts, milk oil products, confectionery, snack confectionery, etc. It is also possible to do.
The immunostimulant of the present invention is effective for the prevention and treatment of infections caused by microorganisms such as viruses and bacteria and various malignant tumors.
[0014]
【Example】
The present invention will be described more specifically with reference to examples and test examples below, but the present invention is not limited thereto.
Example 1
Method for producing Lactobacillus plantarum L-137 dry cells Inoculate 1% by weight of Lactobacillus plantarum L-137 as a starter in 200 ml of a medium in which starch is added instead of glucose in the GYP medium which is a lactic acid bacteria culture medium. Pre-culture was performed at 32 ° C. for 24 hours. Thereafter, 6% GYP medium was inoculated with 1% by weight of the pre-cultured culture solution as a starter and cultured at 32 ° C. for 24 hours. After incubation, the mixture was centrifuged at 5000 rpm for 35 minutes. The supernatant was removed and the cells were collected. Further, the collected cell paste was well dispersed in physiological saline and centrifuged at 5000 rpm for 35 minutes, and then the supernatant was removed to collect the cells. This was repeated three times and then dispersed in distilled water. And it sterilized for 10 minutes at 70 degreeC. This was freeze-dried to obtain 7.07 g of dried cells.
[0015]
Example 2
Production method of Lactobacillus plantarum L-137 fermented 4 times concentrated carrot juice L-137 In the same manner as the method for preparing dry cells, cultured in 6 liters of GYP medium at 32 ° C. for 24 hours and then in physiological saline The cell paste collected by dispersing and centrifuging is added to 300 ml of 4-fold concentrated carrot juice (concentrated ginseng 1/6 ginseng diluted with distilled water from distilled water) and cultured at 32 ° C. for 24 hours did. And it sterilized for 10 minutes at 70 degreeC. The carrot fermentation broth thus obtained was diluted with an appropriate amount of distilled water and freeze-dried. About 77.4 g was obtained as a lyophilized product.
[0016]
Test example 1
In this test example, using the Lactobacillus plantarum L-137 cells obtained in Example 1, the T lymphocyte costimulatory effect of Lactobacillus plantarum L-137 cells on the proliferation reaction of mouse spleen lymphocytes And the B lymphocyte inhibitory effect was verified.
The spleen was aseptically removed from a mouse (B10.A, female, 8 weeks old), spleen was crushed in RPMI 1640 medium, and passed through # 200 mesh to obtain a spleen cell suspension. After the number of cells in the spleen cell suspension was measured with an automatic hemocytometer, the number of cells was adjusted to a concentration of 5 × 10 ⁶ / ml with RPMI 1640 medium, and 100 μl per well was seeded in a 96-well tissue culture plate. A solution obtained by dissolving lipopolysaccharide (Bifco), a B lymphocyte proliferation stimulating substance, in RPMI 1640 medium at a concentration of 200 μg / ml, and 200 μg anti-mouse immunoglobulin M (Cappel), a B lymphocyte proliferation stimulating substance. 50 μl / well of a solution dissolved in RPMI 1640 medium at a concentration of / ml, a solution obtained by diluting a T lymphocyte proliferation stimulating substance phytohemagglutinin (Difco) 400 times with RPMI 1640 medium, and RPMI 1640 medium In addition to the spleen cell suspension, the B lymphocyte stimulation group (1), the B lymphocyte stimulation group (2), the T lymphocyte stimulation group (1), and the non-stimulation group were used. As a T lymphocyte stimulation group (2), 100 μl per well of a T lymphocyte proliferation stimulating substance anti-mouse CD3 antibody (Cedarlane) dissolved in borate buffer at a concentration of 10 μg / ml before cell seeding. In addition, the mixture was allowed to stand at 37 ° C. for 3 hours to allow the anti-mouse CD3 antibody to adhere to each well, and after 3 hours, washed with RPMI 1640 medium, cells were seeded in a hole to which 50 μl of RPMI 1640 medium was added per well. In these 5 groups, RPMI 1640 medium (control) or Lactobacillus plantarum L-137 cells dissolved in RPMI 1640 medium at concentrations of 50 μg / ml, 12.5 μg / ml and 3.13 μg / ml, respectively 50 μl was added per well and cultured in a 5% carbon dioxide incubator at 37 ° C. for 2 days. After the culture, metabolic activity was examined.
Cell metabolic activity was determined by dissolving 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide in RPMI 1640 medium at a concentration of 5 mg / ml 3 hours before the end of the culture. 10 μl per well, 50 μl of 20% sodium dodecyl sulfate solution per well at the end of the culture, left at 37 ° C. for 1 day, and then measured the absorbance at 550 nm of the culture with a microplate reader to determine cell metabolic activity It was. The results are shown in [Table 2].
[0017]
[Table 2]

As apparent from [Table 2], Lactobacillus plantarum L-137 cells only slightly increased cell metabolic activity in the non-stimulated group in which the spleen cells were not stimulated. In the T lymphocyte stimulation group (2) through the receptor and the T lymphocyte stimulation group (1) of pathways other than the antigen receptor, the cell metabolic activity is remarkably increased, and the T of the Lactobacillus plantarum L-137 cell body is increased. A lymphocyte costimulatory effect was observed. On the other hand, Lactobacillus plantarum L-137 cells show increased cell metabolic activity in the B lymphocyte stimulation group (2) via the antigen receptor and the B lymphocyte stimulation group (1) of pathways other than the antigen receptor. An inhibitory effect was observed.
[0018]
Test example 2
In this test example, the Lactobacillus plantarum L-137 cells obtained in Example 1 were used to enhance the effect of Lactobacillus plantarum L-137 cells on the interferon-γ production reaction of mouse spleen lymphocytes. Verified.
The spleen was aseptically removed from a mouse (B10.A, female, 7 weeks old), spleen was crushed in RPMI 1640 medium, and passed through # 200 mesh to obtain a spleen cell suspension.
After measuring the number of cells in the spleen cell suspension with an automatic hemocytometer, the number of cells was adjusted to a concentration of 5 × 10 ⁶ / ml with RPMI 1640 medium, and 100 μl per well was seeded in a 96-well tissue culture plate.
A solution obtained by diluting a T lymphocyte proliferation stimulating substance phytohemagglutinin (Difco) 400-fold with RPMI 1640 medium was added to a spleen cell suspension seeded with 50 μl per well. To this was added 50 μl of RPMI 1640 medium (control) or Lactobacillus plantarum L-137 cells dissolved in RPMI 1640 medium at concentrations of 200 μg / ml and 100 μg / ml, respectively, and 5% at 37 ° C. After culturing in a carbon dioxide incubator for 3 days, interferon-γ in the culture supernatant after the culture was measured by enzyme immunoassay.
Enzyme immunoassay was performed by adding 100 μl of a hamster anti-mouse interferon-γ antibody (Genzyme) adjusted to 3 μg / ml with borate buffer per well of 96-well tissue culture plate and leaving at 5 ° C. for 3 days. This was carried out using a plate with anti-mouse interferon-γ antibody attached to each hole. The culture supernatant was added in an amount of 50 μl per well and allowed to stand at room temperature for 90 minutes, and the interferon-γ of the culture supernatant was bound to the hamster anti-mouse interferon-γ antibody attached to the plate. After washing, a rat anti-mouse interferon-γ antibody was added and allowed to bind to interferon-γ bound to the plate. After washing, an anti-rat IgG antibody labeled with peroxidase was added and allowed to bind to the rat anti-mouse interferon-γ antibody bound to the plate. After washing, 100 μl of phosphate buffer containing 0.006% hydrogen peroxide and 0.1% orthophenylenediamine was added per well, reacted at room temperature for 20 minutes, and the reaction was stopped with 1.5N sulfuric acid. The absorbance at 492 nm was measured with a plate reader, and the concentration of interferon-γ in the culture supernatant was determined from the labeling curve prepared with recombinant mouse interferon-γ. The results are shown in [Table 3].
[0019]
[Table 3]

As apparent from [Table 3], Lactobacillus plantarum L-137 cells significantly increased the production of interferon-γ induced by phytohemagglutinin.
[0020]
Test example 3
In this test example, using Lactobacillus plantarum L-137 obtained in Example 1, the promoting effect of Lactobacillus plantarum L-137 on interleukin 12 productivity of mouse peritoneal macrophages was verified.
The RPMI 1640 medium was aseptically injected into the abdominal cavity of a mouse (C57BL / 6, female, 15 weeks old) and the abdomen was thoroughly massaged, and then the injected RPMI 1640 medium was recovered to obtain a peritoneal cell suspension. The number of cells in the peritoneal cell suspension and the ratio of macrophages contained therein were measured with an automatic hemocytometer, and then prepared as macrophages in 1 × 10 ⁶ / ml cells in RPMI 1640 medium. 100 μl was seeded per well. The peritoneal macrophages were allowed to adhere to each well for 2 hours in a 37 ° C. 5% carbon dioxide incubator, and after 2 hours, washed with RPMI 1640 medium, 100 μl of RPMI 1640 medium was added per well.
A solution prepared by dissolving RPMI 1640 medium (control) or macrophage activator lipopolysaccharide (Difico) in RPMI 1640 medium at a concentration of 0.2 μg / ml, or Lactobacillus plantarum L-137 cells was added thereto. 100 μl of each solution dissolved in RPMI 1640 medium at a concentration of 0.2 μg / ml was added to each well, and cultured in a 5% carbon dioxide incubator at 37 ° C. for 15 hours. Tumor necrosis factor α was measured by enzyme immunoassay.
In the enzyme immunoassay, a rat anti-mouse interleukin 12 IgG2a antibody (manufactured by Genzyme) or a hamster anti-mouse tumor necrosis factor α & β antibody (manufactured by Genzyme) prepared with borate buffer at 6 μg / ml was added to 96-well tissue culture. 100 μl per well of plate was added and allowed to stand at 37 ° C. for 1 day, using a plate with rat anti-mouse interleukin 12 IgG2a antibody or hamster anti-mouse tumor necrosis factor α & β antibody attached to each hole. Rat anti-mouse interleukin 12 IgG2a antibody or hamster anti-mouse tumor necrosis factor α & β antibody in which 50 μl of culture supernatant was added per well and allowed to stand at room temperature for 90 minutes and the culture supernatant interleukin 12 or tumor necrosis factor α was attached to the plate And combined. After washing, rat anti-mouse interleukin 12 IgG1 antibody (Genzyme) or rabbit anti-mouse tumor necrosis factor α antibody (Genzyme) was added and bound to interleukin 12 or tumor necrosis factor α bound to the plate. . After washing, an anti-rat IgG 1 antibody or anti-rabbit IgG antibody labeled with peroxidase was added, and bound to a rat anti-mouse interleukin 12 IgG 1 antibody or rabbit anti-mouse tumor necrosis factor α antibody bound to the plate. After washing, 100 μl of phosphate buffer containing 0.006% hydrogen peroxide and 0.1% orthophenylenediamine was added per well, reacted at room temperature for 20 minutes, and the reaction was stopped with 1.5N sulfuric acid. The absorbance at 492 nm was measured with a plate reader, and the concentration of interleukin 12 or tumor necrosis factor α in the culture supernatant was determined from a standard curve prepared with recombinant mouse interleukin 12 or tumor necrosis factor α.
The results are shown in [Table 4].
[0021]
[Table 4]

As apparent from [Table 4], Lactobacillus plantarum L-137 cells significantly increased the production of interleukin 12 from macrophages, but only slightly increased the production of tumor necrosis factor α. It was revealed that Lactobacillus plantarum L-137 cells selectively increased the production of interleukin 12 in macrophages when compared with the cytokine production promoting action of lipopolysaccharide, which is a powerful macrophage activator.
[0022]
【The invention's effect】
The immunostimulant of the present invention is easy and high in productivity of active ingredients, and the obtained immunostimulant has high safety and high immunostimulatory effect when administered to the human body alone or with other immunostimulators. In combination, it can be used for the prevention and treatment of infectious diseases such as viruses and bacteria, and malignant tumors.

Claims (1)

An immunostimulant comprising a cell of Lactobacillus plantarum L-137 strain (FERM P-15317) having a T lymphocyte co-stimulatory action and a B lymphocyte inhibitory action or / and an interleukin 12 production promoting action.