JP4942831B2 - 抗アレルギー用組成物 - Google Patents
抗アレルギー用組成物 Download PDFInfo
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- JP4942831B2 JP4942831B2 JP2010064451A JP2010064451A JP4942831B2 JP 4942831 B2 JP4942831 B2 JP 4942831B2 JP 2010064451 A JP2010064451 A JP 2010064451A JP 2010064451 A JP2010064451 A JP 2010064451A JP 4942831 B2 JP4942831 B2 JP 4942831B2
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Description
添加比率の違いによるラクトバチルス・クリスパタスKT−11株(FERM P−21457)の抗アレルギー効果の相乗作用について以下に示す。
ラクトバチルス・クリスパタスKT−11株またはラクトバチルス・プランタラムJCM1149TをMRS液体培地にそれぞれ接種し、37℃、24時間培養した。培養後、遠心分離(2,000rpm、10分間)で菌体を回収し、蒸留水で3回洗浄した。洗浄した菌体は凍結乾燥処理後、滅菌0.15M塩化ナトリウム−0.01Mリン酸緩衝液(PBS、pH7.2)に懸濁して熱処理(65℃、30分間)したものを菌体試料液とした。
6週齢オスのC3H/HeN系マウスから腸管パイエル板を摘出し、5%ウシ胎児血清(FBS)、100U/mLペニシリンGナトリウムおよび100μg/mLストレプトマイシン硫酸塩を含むRPMI−1640培地中で細胞を懸濁した。同上の培地で2回遠心洗浄(4℃、1,300rpm、15分間)した後、生細胞数が1.0×106個/mLになるようにパイエル板細胞懸濁液を調製した。
培養したパイエル板細胞懸濁液を、1mMEDTA、5%FBSを含むHank’s Balanced Salt Solution(HBSS)で遠心洗浄(4℃、12,000rpm、3秒間)した。洗浄したパイエル板細胞1.0×106個を活性化培地(10%FBSを含むRPMI−1640培地にブレフェルジンA20μg/mL、イオノマイシン2μg/mLおよびフォルボール12−ミリステート13−アセテート20ng/mLを含む)1mLに懸濁し、37℃、5%CO2存在下で4時間培養し、細胞内にサイトカインを産生および蓄積させた。
ラクトバチルス・クリスパタスKT−11株の抗アレルギー効果の相乗作用について以下に示す。
表1に示す供試菌を実施例1と同様の方法でそれぞれ調製し、ラクトバチルス・クリスパタスKT−11株と各供試菌を75:25の割合で混合して菌体試料液とした。
パイエル板細胞懸濁液の調製と培養は実施例1と同様の方法で調製後、パイエル板細胞懸濁液1mLを各穴に分注した48穴平底マイクロプレートに、各濃度に調製した菌体試料液100μLを各穴に添加し、37℃、5%CO2存在下で48時間培養した。IFN−γ産生ヘルパーT細胞数の変化を実施例1と同様の方法で測定した。
表1に示す供試菌を実施例1と同様の方法でそれぞれ調製し、菌体試料液とした。
マウスマクロファージ様株化細胞であるJ774.1細胞は、5%FBS、100U/mLペニシリンGナトリウムおよび100μg/mLストレプトマイシン硫酸塩を含むRPMI−1640培地に懸濁後、滅菌プラスチックシャーレ内で37℃、5%CO2存在下でコンフルの状態まで培養したものを用いた。1×106個/mLに調製したJ774.1細胞懸濁液1mLを分注した48穴平底マイクロプレートに、最終濃度が0または1μg/mLに調製したラクトバチルス・クリスパタスKT−11株の菌体試料液とPBSに溶解させた市販のラクトバチルス・アシドフィルス由来のペプチドグリカン溶液(和光純薬工業株式会社製「ペプチドグリカンタイプII」)をそれぞれ100μLずつを各穴に添加し、37℃、5%CO2存在下で48時間培養した。培養後、4℃、2000rpmで15分間遠心分離を行い、培養上清液を回収した。
培養上清液中のIL−12p70量は酵素免疫測定法(ELISA)を用いて測定した。すなわち、100μg/mLの抗マウスIL−12p70抗体と4%ウシ血清アルブミン(BSA)を含む0.1M炭酸緩衝液(pH9.6)100μLを96穴マイクロプレートの各穴に分注し、4℃で一晩静置した。0.05%Tween20を含むPBS(PBS−T)で洗浄した後、0.4%BSAを含む0.1M炭酸緩衝液300μLを加え、25℃で120分間静置した。再びPBS−Tで洗浄した後、0.4%BSAと2%ポリビニルピロリドン(PVP)を含むPBS−Tで最適な倍率に希釈した培養上清液100μLを各穴に分注し、25℃で120分間反応させた。さらに、PBS−Tで洗浄した後、2%PVPを含むPBS−Tで最適な倍率に希釈したビオチン標識抗マウスIL−12p70抗体溶液100μLを各ウェルに分注し、25℃で60分間反応させた。次いで、PBS−Tで洗浄した後、2%PVPを含むPBS−Tで最適な倍率に希釈した西洋ワサビペルオキシダーゼ(HRP)標識ストレプトアビジン溶液100μLを各穴に分注し、25℃で30分間反応させた。TMB溶液100μLを各穴に分注し、完全に遮光して25℃で30分間反応させた後、4N硫酸100μLを各穴に分注して反応を停止させ、直ちにBio−Radモデル550マイクロプレートリーダーを用いて、450nmにおける吸光度を測定した。なお,IL−12p70量は既知濃度のIL−12p70から得たスタンダード曲線より算出した。
Claims (12)
- ラクトバチルス・クリスパタス(Lactobacillus crispatus)KT−11株(FERM P−21457)の菌株の生菌、死菌又はその菌体処理物と、他の乳酸菌の生菌、死菌又はその菌体処理物とを、菌体量換算で、99:1〜50:50の割合で含有する抗アレルギー用組成物であって、前記他の乳酸菌が、ラクトバチルス(Lactobacillus)属、ビフィドバクテリウム(Bifidobacterium)属、ロイコノストック(Leuconostoc)属、及びエンテロコッカス(Enterococcus)属の乳酸菌からなる群から選ばれる1又は2以上の乳酸菌であり、前記菌体処理物が、菌体破砕物、菌体溶解物、又は乳酸菌由来のペプチドグリカンであることを特徴とする抗アレルギー用組成物。
- 菌体量換算で、99:1〜75:25の割合で含有することを特徴とする請求項1記載の抗アレルギー用組成物。
- ラクトバチルス属の乳酸菌が、ラクトバチルス・パラカゼイ(L.paracasei)、ラクトバチルス・クリスパタス、ラクトバチルス・ブレビス(L. brevis)、ラクトバチルス・ブルガリカス(L.bulgaricus)、ラクトバチルス・ラムノーサス(L.rhamnosus)、ラクトバチルス・プランタラム(L.plantarum)からなる群から選ばれる1又は2以上の乳酸菌であることを特徴とする請求項1又は2記載の抗アレルギー用組成物。
- 死菌が、緩衝液中で加熱処理した菌体であることを特徴とする請求項1〜3のいずれか記載の抗アレルギー用組成物。
- インターフェロン−ガンマ(IFN−γ)産生能を促進する作用を有することを特徴とする請求項1〜4のいずれか記載の抗アレルギー用組成物。
- IL−12p70産生能を促進する作用を有することを特徴とする請求項1〜5のいずれか記載の抗アレルギー用組成物。
- 請求項1〜6のいずれか記載の抗アレルギー用組成物を含有する医薬品。
- 請求項1〜6のいずれか記載の抗アレルギー用組成物を含有する飲食品又はサプリメント。
- 請求項1〜6のいずれか記載の抗アレルギー用組成物を含有するペットフード又はペット用サプリメント。
- 請求項1〜6のいずれか記載の抗アレルギー用組成物を含有する飼料。
- 以下の工程(a)〜(c)を備えた抗アレルギー用組成物の製造方法。
(a)ラクトバチルス・クリスパタスKT−11株(FERM P−21457)の菌株の生菌、死菌、菌体破砕物、菌体溶解物、又はペプチドグリカンを調製する工程;
(b)他の乳酸菌であって、ラクトバチルス属、ビフィドバクテリウム属、ロイコノストック属、及びエンテロコッカス属の乳酸菌からなる群から選ばれる1又は2以上の乳酸菌の生菌、死菌、菌体破砕物、菌体溶解物、又はペプチドグリカンを調製する工程;
(c)工程(a)で調製された生菌、死菌、菌体破砕物、菌体溶解物、又はペプチドグリカンと、工程(b)で調製された生菌、死菌、菌体破砕物、菌体溶解物、又はペプチドグリカンとを、菌体量換算で、99:1〜50:50の割合で配合する工程; - ラクトバチルス・クリスパタスKT−11株(FERM P−21457)の菌株の生菌、死菌、菌体破砕物、菌体溶解物、又はペプチドグリカンと、他の乳酸菌であって、ラクトバチルス属、ビフィドバクテリウム属、ロイコノストック属、及びエンテロコッカス属の乳酸菌からなる群から選ばれる1又は2以上の乳酸菌の生菌、死菌、菌体破砕物、菌体溶解物、又はペプチドグリカンとを、菌体量換算で、99:1〜50:50の割合で含有する組成物を、抗アレルギー剤の調製に使用する方法。
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