JP4515157B2 - 免疫調節作用を有する乳酸菌 - Google Patents
免疫調節作用を有する乳酸菌 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Description
京都の伝統的な漬物であるしば漬けから、Lactobacillus plantarum を4種、Lactobacillus pentosus を12種分離し、Th1型免疫賦活能の高い菌株を選択するためインターロイキン12(IL−12)誘導能を比較する実験に供した。使用菌株については、表2に株名、種名およびEPSの有無を示した。IL−12誘導能を比較した結果、DS84C株(S−PT84)に特に強い活性を見出したため、以後の実験はS−PT84のみを用いて行った。
BALB/c マウス(7週齢・雄)に4.05%チオグリコレートを腹腔内投与し、4日後にPBSを用いて腹腔内マクロファージを回収し、10%FBSを含むRPMI培地を用いて2×106cells/ mLに調整した後、24wellプレートに0.5mLずつ播き込んだ。各wellに各菌株の加熱死菌(10μg/mL)を添加し24時間培養後の培養上清中のIL−12濃度を測定した。IL−12の活性型はp35とp40のサブユニットが結合したp70であることから、IL−12(p70)を測定した。なおIL−12の測定にはOptEIA mouse IL-12測定キット(BD Pharmingen社製)を使用した。
BALB/c マウス(7週齢・雄)に各菌株の加熱死菌(500μg/0.2mL/mouse)の懸濁液(溶媒:生理的食塩水)を腹腔内投与し、6時間後に頚椎脱臼後、心臓より採血した。なお、controlのマウスには生理的食塩水を等量投与した。採血を投与後6時間としたのは、S−PT84を用いた予備検討の結果、死菌投与後の血清IL-12濃度のピークが投与後6時間であったことによる(図2参照)。採血後、遠心分離により血清を採取し、血清中のIL−12濃度をOptEIA mouse IL-12測定キット(BD Pharmingen社製)を用いて測定した。
BALB/cマウス(7週齢・雄)から脾臓を摘出し、常法に従い脾細胞を調製した。上記S−PT84の加熱死菌(1μg/mL)を含む培地で24時間培養した。対照として、培地に何も添加せずに培養したもの(control)と、コンカナバリンA(2.5μg/mL)を添加して培養したもの(ConA)とを設けた。S−PT84死菌の刺激により、どのようなサイトカインが産生されるかを調べるため、各培養上清中のサイトカイン濃度をCBAkit(BD Pharmingen社製)を用いて測定した。また、脾細胞を回収し、蛍光ラベルされた抗CD4抗体(CY-CHCROMETMラベル、BD bioscience社製)、抗CD8抗体(FITCラベル、Immunotech社製)、抗CD69抗体(PEラベル、BD bioscience社製)を用いて細胞を標識し、CD4、CD8およびCD69陽性細胞の割合をフローサイトメトリー(Beckman Coulter社製)を用いて測定した。
C57BL/6マウス(7週齢・雄)に、S−PT84の加熱死菌(500μg/0.2mL/mouse)の懸濁液(溶媒:生理的食塩水)を腹腔内投与し、24時間後に肝臓を摘出し、遠心法により肝リンパ球を調製した。対照(control)は生理的食塩水のみを腹腔内投与した。調製した肝リンパ球のNK活性をPINK法を用いて測定した。PINK法とは、標的細胞であるYac-1を疎水性膜系蛍光色素3,3’-dioctadecyloxacarbocyanine perchlorate (Dio)で標識し、さらに、死細胞の核を膜不透過性核酸結合蛍光色素propidium iodide (PI)により二重染色し、傷害されなかったYac-1はDio単染色、傷害されたYac-1は二重染色でフローサイトメトリーで検出することにより、マウスリンパ球の細胞障害活性を算出する方法である。また、蛍光ラベルされた抗CD4抗体(CY-CHCROMETMラベル、BD bioscience社製)、抗CD8抗体(FITCラベル、Immunotech社製)、抗CD69抗体(PEラベル、BD bioscience社製)を用いて細胞を標識し、CD4、CD8およびCD69陽性細胞の割合をフローサイトメトリー(Beckman Coulter社製)を用いて測定した。
BALB/cマウス(7週齢・雄)6匹に、S−PT84(死菌体)を1週間飲水(2mg/day
相当)にて自由に摂取させた。また、S−PT84を摂取させない対照群(6匹)を設けた。1週間後に心臓より採血し、さらに脾臓を摘出した。血液から遠心分離により血清を採取し、血清中のIL−12濃度をOptEIA mouse IL-12測定キット(BD Biosciences社製)を用いて測定した。また、摘出した脾臓から常法に従い脾細胞を調製し、脾リンパ球のCD4+、CD8+およびCD3+細胞数の計測(それぞれの標識抗体を用いてフローサイトメトリーにより計測した)、PINK法によるNK活性の測定、およびTh1/Th2の比を測定した(マウス脾細胞5×106個にコンカナバリンA2.5μg/mLを24時間作用させ、上清中に産生されるIL−4およびIFN−γ濃度を測定した。Th1/Th2比はIFN−γ濃度をIL−4濃度で除した値を用いた。)。
BALB/cマウス(7週齢・雄)20匹を各群の平均体重がほぼ同じになるように2群に分け、S−PT84摂取群とS−PT84非摂取群(対照群)とした。S−PT84摂取群にはS−PT84(死菌体)を22日間飲水(2mg/day 相当)にて自由に摂取させた。摂取開始8日目に、全個体に制癌化学療法剤でアルキル化剤のシクロフォスファミド(Cyclophosphamide:CY)を200mg/kgの用量で腹腔内投与した。CY投与後1、2、3、5、8、12および15日目に各個体の体重を測定した。
BALB/c マウス(7週齢・雄)を5群に分けた。群構成は、無処置群(5匹)、S−PT84非摂取・CY非投与群(10匹)、S−PT84非摂取・CY投与群(10匹)、S−PT84摂取・CY非投与群(10匹)およびS−PT84摂取・CY投与群(11匹)とした。S−PT84摂取の2群にはS−PT84(死菌体)を12日間飲水(2mg/day 相当)にて自由に摂取させた。摂取開始8日目に、CY投与群のマウスに制癌化学療法剤でアルキル化剤のシクロフォスファミド(Cyclophosphamide:CY)を200mg/kgの用量で腹腔内投与した。CY投与後5日目に、無処置群を除く4群のマウスにS−PT84の加熱死菌(500μg/0.2mL/mouse)の懸濁液(溶媒:生理的食塩水)を腹腔内投与した。S−PT84の死菌投与後6時間目に無処置群を含む全個体の心臓から採血した。採血後、遠心分離により血清を採取し、血清中のIL−12濃度をOptEIA mouse IL-12測定キット(BD Pharmingen社製)を用いて測定した。
BALB/cマウス(7週齢・雄)36匹を4群に分けた。群構成は、無処置群(5匹)、対照群(10匹)、S−PT84群(11匹)、Dex投与群(10匹)とし、S−PT84群にはS−PT84(死菌体)を7週間飲水(2mg/day 相当)にて自由に摂取させ、Dex投与群には、0.5mg/kgの用量で7週間強制経口投与を行った。無処置群および対照群には水道水を7週間自由に摂取させた。なお、Dexは抗炎症、抗アレルギー作用を有するステロイド剤であり、陽性対照薬として用いた。S−PT84摂取またはDex投与開始から1週間後および2週間後に、20μgの卵白アルブミン(OVA)と2mgの水酸化アルミニウムゲルを混和して、無処置群を除く3群のマウスに腹腔内投与した。2回目の投与日(0週目)から1週間ごとに5週目まで全個体について合計6回の採血を行い、OVA特異的IgE濃度を測定した。OVA特異的IgE濃度の測定はOptEIA mouse IgE測定キット(BD Pharmingen社製)を改良し、補足用抗体の代わりにOVAをコーティングしたELISA法にて測定した。また、3週目の血液サンプルを用いて、総IgEを測定した。総IgEの測定には、OptEIA mouse IgE測定キット(BD Pharmingen社製)を用いELISA法にて測定した。
C57BL/6マウス(6週齢・雄)9匹を各群の平均体重がほぼ同じになるように3群に分けた。群構成は、対照群(3匹)、ストレス負荷群(3匹)およびS−PT84摂取+ストレス負荷群(3匹)とした。S−PT84摂取群にはS−PT84(死菌体)を7日間飲水(2mg/day 相当)にて自由に摂取させた。摂取開始8日目に、ストレス負荷群及びS−PT84摂取+ストレス負荷群6匹を1度水浸させた後、先端に空気穴を施した50mLのポリエチレンチューブに入れ、24時間拘束した。対照群は24時間、絶水、絶食とした。その後動物から脾臓を摘出、脾リンパ球を調製し、NK活性を測定した。
以下に示す方法により、S−PT84を含有する医薬品(錠剤)を製造した。
乳固形分21%のS−PT84発酵乳を、市販の牛乳に添加し3日間静置しヨーグルトを調製した。得られたヨーグルトは良好な香味を示した。
S−PT84を用いて表4に示した組成で乳酸菌飲料を調整した。得られた乳酸菌飲料は良好な香味を示した。
Claims (1)
- ラクトバチルス・ペントーサスS−PT84株(受託番号:FERM BP−10028、受領番号:FERM ABP−10028)である、乳酸菌。
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CA002568014A CA2568014A1 (en) | 2004-05-28 | 2005-03-30 | Lactic acid bacteria with immunoregulating activities |
PCT/JP2005/006709 WO2005116188A1 (en) | 2004-05-28 | 2005-03-30 | Lactic acid bacteria with immunoregulating activities |
US11/094,576 US7501274B2 (en) | 2004-05-28 | 2005-03-31 | Lactic acid bacteria with immunoregulating activities |
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JP2007308419A (ja) * | 2006-05-18 | 2007-11-29 | Shinshu Univ | 腸管免疫活性化剤及びIgA抗体産生促進剤、並びにこれらが含まれた食品、動物飼料及び医薬品 |
WO2008079009A1 (en) * | 2006-12-22 | 2008-07-03 | Campina Nederland Holding B.V. | Immunomodulating probiotic lactic acid bacteria |
JP5394628B2 (ja) * | 2007-02-09 | 2014-01-22 | クロスフィールドバイオ株式会社 | 新規なラクトバチルス属微生物および乳酸菌製剤 |
JP5128183B2 (ja) * | 2007-05-31 | 2013-01-23 | カゴメ株式会社 | 新規乳酸菌株、発酵飲食品および発酵飲食品の製造方法 |
JP5933162B2 (ja) * | 2007-05-31 | 2016-06-08 | カゴメ株式会社 | 発酵飲食品の製造方法 |
WO2010110045A1 (ja) * | 2009-03-24 | 2010-09-30 | サントリーホールディングス株式会社 | 免疫調節作用の増強された乳酸菌の製造方法 |
US20120034199A1 (en) * | 2009-04-09 | 2012-02-09 | Teck Chwen Loh | Monogastric animal feed |
NZ712617A (en) * | 2013-04-17 | 2020-02-28 | Suntory Holdings Ltd | Bacterium belonging to genus lactobacillus |
NZ712603A (en) * | 2013-04-17 | 2020-02-28 | Suntory Holdings Ltd | Composition containing bacterium belonging to genus lactobacillus |
JP6298912B1 (ja) * | 2017-04-10 | 2018-03-20 | 有限会社バイオ研 | 乳酸菌の製造方法、及び免疫調節用組成物 |
JP6916042B2 (ja) * | 2017-06-09 | 2021-08-11 | 株式会社明治 | 抗原特異的インターフェロンγ産生促進用組成物 |
JP7337878B2 (ja) * | 2021-05-21 | 2023-09-04 | 国立大学法人東京工業大学 | 免疫賦活化作用を有する乳酸菌 |
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