JP5066091B2 - 抗アレルギー作用を有する菌株並びにその菌体を含有する飲料、食品及び抗アレルギー剤 - Google Patents
抗アレルギー作用を有する菌株並びにその菌体を含有する飲料、食品及び抗アレルギー剤 Download PDFInfo
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- JP5066091B2 JP5066091B2 JP2008530894A JP2008530894A JP5066091B2 JP 5066091 B2 JP5066091 B2 JP 5066091B2 JP 2008530894 A JP2008530894 A JP 2008530894A JP 2008530894 A JP2008530894 A JP 2008530894A JP 5066091 B2 JP5066091 B2 JP 5066091B2
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Images
Classifications
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- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C12/00—Processes specially adapted for making special kinds of beer
- C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
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- 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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Description
ラクトバチラス・ブレビス亜種ブレビス(Lactobacillus brevis subspecies brevis)に属する13菌株(SBC8803及び菌株a〜l)は、発明者が分離し、実験に用いるまで凍結乾燥菌体として4℃で保管した。また、ラクトバチラス・ラムノサス(Lactobacillus rhamnosus)に属する菌株Xは市販のヨーグルトから分離し、実験に用いるまで凍結乾燥菌体として4℃で保管した。
上記のラクトバチラス・ブレビス亜種ブレビスに属する13菌株及びラクトバチラス・ラムノサスに属する菌株Xについて、ビール中での増殖能を選抜した。選抜方法は、特開2003−250557号公報に記載された方法に従って行なった。すなわち、各乳酸菌株のゲノムDNAを鋳型として所定のプライマーセット(特開2003−250557号公報の配列表の配列番号1及び配列番号2に記載の核酸配列からなるオリゴヌクレオチドからなるプライマーセット)でPCRを行い、増幅されたDNAジャイレースサブユニットB遺伝子断片を制限酵素で切断し、アクリルアミドゲル電気泳動後の制限酵素切断パターンを解析することにより判定した。この方法では、制限酵素切断パターンは、大きく4つのグループに分類でき、ビール中で増殖可能である菌株は、グループIIbに属することが判明している。
i)菌体懸濁液の調製
上記の各菌株の抗アレルギー作用を評価するために、各菌株の菌体懸濁液を調製した。まず、各菌株を嫌気的条件下(N2:CO2:H2=90:5:5のガス組成)、MRS液体培地(ディフコ社製、組成:1%プロテオースペプトン、1%牛肉エキス、0.5%酵母エキス、2%ブドウ糖、0.1%Tween 80、0.5%クエン酸アンモニウム、0.01%硫酸マグネシウム、0.005%硫酸マンガン、0.2%リン酸二カリウム)で3日間静置培養し、その培養液を1,500回転で10分間遠心分離して、各菌株の菌体を回収した。得られた菌体は、PBSで洗浄した後に凍結乾燥し、1mg/mLとなるようにPBSに懸濁した。こうして得られた菌体懸濁液は、オートクレーブ滅菌(121℃、15分)して以下の実験に使用した。
マウス脾臓細胞にラクトバチラス・ブレビス亜種ブレビスに属する13菌株の菌体縣濁液を加えて一定時間培養し、脾臓細胞から分泌されるインターフェロンγの量を調べることにより、各菌株のインターフェロンγ産生促進作用を評価した。
まず、6週齢のBALB/cマウス(雌)から脾臓を無菌的に摘出し、10%FBS含有RPMI1640培地に浸漬した。その後、脾臓をシャーレに移して乳棒ですり潰し、マウス脾臓細胞の縣濁液を、目開き70μm、線径39μmのナイロンフィルターネット(日本理化学機器株式会社)に通した。このナイロンフィルターネットを通過したマウス脾臓細胞の縣濁液は、1,500回転で10分間遠心分離し、上清を捨てた後に赤血球溶血試薬(0.16M塩化アンモニウム、トリス・HCl、pH7.2)を沈殿したマウス脾臓細胞に加え、5分間、室温で静置した。その後、新しい10%FBS含有RPMI1640培地を加えてマウス脾臓細胞を洗浄し、1,500回転で10分間遠心分離し、上清を捨てた後に細胞数が5×106個/mLになるように10%FBS含有RPMI1640培地を加えた。こうして得られたマウス脾臓細胞を、以下の実験に用いた。
細胞密度が2.5×106個/mLになるように96ウェルプレートにマウス脾臓細胞を播種し、37℃、5%CO2の条件下、10%FBS含有RPMI1640培地で培養した。マウス脾臓細胞を培養している各ウェルに、各菌株の菌体縣濁液(最終濃度:10μg/mL)を添加し、72時間経過後に培養上清中に分泌されたインターフェロンγの量をELISAで定量した。ポジティブコントロールには、マウスの脾臓細胞に対してインターフェロンγ産生促進作用を有するリポポリサッカライド(以下、LPS)(SIGMA社、最終濃度:10μg/mL)を、ネガティブコントロールには、PBSを使用した。
マウス脾臓細胞に対してインターフェロンγ産生促進作用を示したSBC8803について、OVA免疫マウスの脾臓細胞に対するTh1サイトカイン(インターフェロンγ及びインターロイキン12)産生促進作用、Th2サイトカイン(インターロイキン4)産生抑制作用及びIgE産生抑制作用を評価した。その際、ラクトバチラス・ブレビス亜種ブレビスに属する菌株b及び菌株c並びにラクトバチラス・ラムノサスに属する菌株Xの作用についても同時に調べ、SBC8803の作用と比較した。
OVA免疫マウスは、6週齢のBALB/cマウス(雌)にOVAを腹腔内投与し、人為的にアレルギーを惹起して作成した。具体的には、まず、100μgのOVA(Ovalbumin、Eggwhite、Purified;Worthington Biochemical Corporation)及び10mgの水酸化アルミニウムを1mLのPBSに溶解してOVA抗原溶液を調製し、その200μLをマウスの腹腔内に投与して初回免疫を行なった。その1週間後、上記と同じようにOVA抗原溶液を調製し、再度、その200μLをマウスの腹腔内に投与して追加免疫を行なった。追加免疫がされたマウスは、約1週間後にアレルギーが惹起され、このマウスをOVA免疫マウスとして以下の実験に用いた。
追加免疫から2週間後のOVA免疫マウスから脾臓を無菌的に摘出し、そこから上記と同じ手順で脾臓細胞を調製した。
インターフェロンγ、インターロイキン12及びインターロイキン4を測定するために、2.5×105個のOVA免疫マウスの脾臓細胞を96ウェルプレートに播種し(細胞密度は、2.5×106 cells/mL)、37℃、5%CO2の条件下、10%FBS含有RPMI1640培地で培養した。OVA免疫マウスの脾臓細胞を培養している各ウェルにOVA(最終濃度:100μg/mL)と各菌株の菌体縣濁液(最終濃度:1μg/mL)をそれぞれ添加し、72時間経過後に培養上清中に分泌された各サイトカインの量をELISAで定量した。その際、菌体縣濁液の代わりにPBSを添加したコントロールを設けた。
総IgEを測定するために、96ウェルプレートに2.5×105個のOVA免疫マウスの脾臓細胞を播種し(細胞密度は、2.5×106 cells/mL)、37℃、5%CO2の条件下、10%FBS含有RPMI1640培地で培養した。OVA免疫マウスの脾臓細胞を培養している各ウェルにOVA(最終濃度:100μg/mL)及び各菌株の菌体縣濁液(最終濃度:1μg/mL)をそれぞれ添加し、14日経過後に培養上清中に分泌された総IgEの量をELISAで定量した。コントロールには、菌体縣濁液の代わりにPBSを添加し、同様にELISAで定量した。
in vitroの実験で強い抗アレルギー作用を有することが判明したSBC8803について、OVA免疫マウスに対するIgE産生抑制作用をin vivoで評価した。その際、ラクトバチラス・ラムノサスに属する菌株Xの作用についても同時に調べ、SBC8803の作用と比較した。
NC/Ngaマウスは、2,4,6−トリニトロクロロベンゼン(塩化ピクリル)の塗布によってアトピー様皮膚炎を発症する病態モデルマウスであり、NC/Ngaマウスにおける血清中IgE抗体値の上昇は、アトピー様皮膚炎の発症と関連することが報告されている。そこで、SBC8803を0.05%又は0.5%含有している混餌飼料をNC/Ngaマウスに摂取させながら塩化ピクリルの塗布を行い、NC/Ngaマウスのアトピー様皮膚炎の発症に及ぼすSBC8803の経口投与の効果を調べた。
体重を指標に1群10匹となるように、8週齢のNC/Ngaマウス(雄)を3群に分け、各マウスの剃毛した腹部及びフットパットに、150μLの5%塩化ピクリル溶液(エタノール:アセトン=4:1の溶液に溶解)を塗布して一次感作を行った。その4日後からは1週間に1回ずつ(合計9回)、オリーブオイルに溶解した15μLの1%塩化ピクリル溶液を両耳介に塗布することによって二次感作を行い、NC/Ngaマウスにアトピー様皮膚炎を発症させた。
各群のNC/Ngaマウスには、それぞれSBC8803を含有していないコントロール飼料、SBC8803を0.05%含有している混餌飼料、又はSBC8803を0.5%含有している混餌飼料を、一次感作の2週間前から試験終了日まで自由に摂取させた。
ラクトバチラス・ブレビス亜種ブレビスに属するSBC8803のγ−アミノ酪酸(以下、GABA)の産生能について試験した。まず、SBC8803を、100mLの液体培地(3%麦芽エキス(DIFCO社)、2%酵母エキス(DIFCO社)、0.2%グルタミン酸ナトリウム、pH6.0)に植菌し、4日間静置培養した。その後、SBC8803の培養液を1,500回転で10分間遠心分離して培養上清を回収し、その培養上清に含まれるGABAの量をHPLCで定量した。使用したHPLCの条件は以下の通りである。
・HPLC装置:Agilent HPLC 1100
・使用カラム:ZORBAX eclipse AAA(4.6×150mm、3.5μm)(島津ジーエルシー社)
・カラムオーブン:40℃
・流量:1.0mL/min
・蛍光検出器:Ex.340nm、Em.450nm
・HPLC試薬:10mg/mL Agilent OPA試薬(0.4M ホウ酸バッファー、pH10.2)(島津ジーエルシー社)
・溶離液:A液:40mM NaH2PO4(pH7.8)
B液:45体積%アセトニトリル、45体積%MeOH、10%H2O
・ タイムテーブル(グラジェント):表1を参照
抗アレルギー作用を有するSBC8803を用いて果汁乳酸発酵液を製造し、得られた果汁乳酸発酵液の香りと風味について官能検査を行なった。
Claims (4)
- ラクトバチラス・ブレビス亜種ブレビス(Lactobacillus brevis subspecies brevis)に属するSBC8803(FERM BP−10632)菌株。
- 請求項1記載の菌株の菌体を含有する飲料。
- 請求項1記載の菌株の菌体を含有する食品。
- 請求項1記載の菌株の菌体を有効成分として含有する抗アレルギー剤。
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PCT/JP2007/066121 WO2008023663A1 (fr) | 2006-08-21 | 2007-08-20 | Souche bactérienne possédant une activité anti-allergique, et boisson, aliment et agent anti-allergique comprenant une cellule de la souche bactérienne |
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JP5660508B2 (ja) * | 2010-04-08 | 2015-01-28 | 国立大学法人旭川医科大学 | 腸管保護剤 |
JP5943342B2 (ja) * | 2012-03-02 | 2016-07-05 | 国立研究開発法人産業技術総合研究所 | 概日リズム改善剤 |
CN102626138B (zh) * | 2012-04-06 | 2013-10-02 | 光明乳业股份有限公司 | 含γ-氨基丁酸的褐色发酵乳基料、乳饮料及其制备方法 |
JP6381869B2 (ja) * | 2012-12-12 | 2018-08-29 | 島根県 | 肝臓中性脂肪低減作用を有する津田かぶ由来の乳酸菌 |
JP6671652B2 (ja) * | 2013-06-13 | 2020-03-25 | 国立研究開発法人産業技術総合研究所 | 体温上昇剤 |
JP5990209B2 (ja) * | 2014-03-12 | 2016-09-07 | サッポロビール株式会社 | レモン果汁発酵液の製造方法 |
JP6456997B2 (ja) * | 2017-03-16 | 2019-01-23 | 公益財団法人 佐賀県地域産業支援センター | アスパラガス抽出物の製造方法 |
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