JP7179343B2 - 新規な乳酸菌株およびそれを含む免疫賦活剤 - Google Patents
新規な乳酸菌株およびそれを含む免疫賦活剤 Download PDFInfo
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- JP7179343B2 JP7179343B2 JP2019222687A JP2019222687A JP7179343B2 JP 7179343 B2 JP7179343 B2 JP 7179343B2 JP 2019222687 A JP2019222687 A JP 2019222687A JP 2019222687 A JP2019222687 A JP 2019222687A JP 7179343 B2 JP7179343 B2 JP 7179343B2
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- HTJNEBVCZXHBNJ-XCTPRCOBSA-H trimagnesium;(2r)-2-[(1s)-1,2-dihydroxyethyl]-3,4-dihydroxy-2h-furan-5-one;diphosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.OC[C@H](O)[C@H]1OC(=O)C(O)=C1O HTJNEBVCZXHBNJ-XCTPRCOBSA-H 0.000 description 1
- 229960005066 trisodium edetate Drugs 0.000 description 1
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- 150000003669 ubiquinones Chemical class 0.000 description 1
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- 239000004474 valine Substances 0.000 description 1
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- 229930007845 β-thujaplicin Natural products 0.000 description 1
Images
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Description
本発明の他の実施形態はこの単離された菌株またはその培養物を含む組成物である。
さらに他の実施形態では、上記の単離された菌株または組成物を有効成分として含むIgA産生促進剤または免疫賦活剤である。これらの組成物は、飲食品、医薬品、外用剤または飼料の形態で利用され、これらを投与された対象者の粘膜免疫を賦活するために用いることが好ましい。
本発明の他の側面において、上記の単離された菌株を高糖濃度、かつD-フルクトースを含む培地に接種して培養するステップを含む免疫賦活剤の製造方法が提供される。
(I)新規乳酸菌
(II)免疫賦活作用
(III)各種組成物およびその用途
(IV)免疫賦活剤の製造方法
本発明の好ましい実施形態によれば、野菜黒糖発酵液から単離された新規な乳酸菌株およびその変異株が提供される。より好ましくは、商品名「ジオリナ(登録商標)酵素」の培養液から得られるラクトバチルス属に属する菌であり、さらに好ましくは、ラクトバチルス・コーソイ(Lactobacillus kosoi)10H株(受託番号NITE BP-02811)またはその変異株である。「変異株」とは、特定の菌株に対し、当業者に周知の方法により当業者がその主要な性質に変化を及ぼさない範囲で変異させたもの、あるいは、それと同等であると当業者が確認できるものを包含する意味である。
10H株は、グラム陽性、カタラーゼ陰性の桿菌で、おおよそ0.7~0.8×1.5~2.2μmの形態を示す。本菌株は好フルクトース増殖特性を有し、図1に示すように、D-フルクトースを添加しないMRS培地のみでは生育しない。10H株の生育には少なくとも5%のD-フルクトースを必要とするが、その濃度を20%まで上昇させても良好に生育可能である。D-フルクトースを5~10%添加したMRS寒天培地ではコロニーを形成するが、4%以下のD-フルクトース濃度ではほとんどコロニー形成が認められない。この性質は、本菌株が難培養菌株として、これまで通常の方法では単離できなかった原因であると考えられる。コロニーは、好気的、微好気的および嫌気的条件下で形成され、直径約1~3mmの白色または不透明の環状形態を示す。その他の菌学的特性は以下のとおりである。
生育温度:18~39℃(至適温度は27℃)
ガス発生:有
NaCl耐性:2%(w/v)以上で増殖抑制
炭素源の利用可能性について、ビオメリュー社のAPI50CHLキットを用いて調べたところ、10H株はすべての炭素源の利用が認められなかった。そこで、20種類の炭素源をそれぞれMRSブロスに添加して27℃で48時間培養し、培養上清をHPLCで分析したところ、D-フルクトースおよびスクロースのみが利用されていた。10H株は、D-フルクトースを代謝して乳酸および酢酸を産生したが、エタノールは産生しない。乳酸および酢酸の比率はおよそ3:2である。
酵素活性は、ビオメリュー社の酵素活性研究用システム「アピザイム(API ZYM)」を用いて調べた。10H株は、酸性フォスファターゼおよびナフトール-AS-BI-フォスフォヒドロラーゼの産生が認められ、アルカリフォスファターゼ、リパーゼ、ロイシンアリルアミダーゼおよびバリンアリルアミダーゼについて弱い活性を示す。
10H株の細胞壁のアミノ酸組成は、主に、アスパラギン酸(Asp)、グルタミン酸(Glu)、アラニン(Ala)およびリジン(Lys)で構成されていた。メソ-ジアミノピメリン酸およびオルニチンのいずれも細胞壁のペプチドグリカンには存在せず、上記アミノ酸のモル比は、Asp/Glu/Ala/Lys=1.0/1.9/4.1/1.1であった。これは、10H株の細胞壁ペプチドグリカンが、L-Lys-D-Asp(A4α)型であることを示唆する。なお、A4α型は、ラクトバチルス属の細胞壁を構成するペプチドグリカンであることが報告されている。
10H株の16S rRNA遺伝子配列は、1474bpの連続した配列であり、GenBankの受入番号LC318484として登録されている。この16S rRNA遺伝子配列の相同性をBLASTにより解析した結果、10H株と最もよく似た菌株は、ラクトバチルス・クンケイ(L.kunkeei)YH-15、ラクトバチルス・オーゼンシス(L.ozensis)Mizu2-1およびラクトバチルス・アピノーラム(L.apinorum)Fhon13Nであり、それぞれ、95.5%、95.4%および95.3%の配列同一性を有する。これらの配列同一性は、原核生物の種の分化として推奨されている閾値である98.65%より有意に低い。最尤法(maximum-likelihood method)を用いて構築した系統発生樹によれば、10H株は、上記3種の近縁種とともに1つの区別できる系統発生クラスターを形成する(図2参照)。
また、本明細書に記載した新規乳酸菌の分類学的性質は、本発明者らにより公表された論文(Chiou T-Y et al, Antonie van Leeuwenhoek(2018)、111:1149-1156)に記載されており、その全体は参照により本明細書に組み込まれるものとする。
本明細書において、「免疫賦活剤」とは、ラクトバチルス・コーソイ(Lactobacillus kosoi)10H株(受託番号NITE BP-02811)の菌体またはその培養物を有効成分として含有する組成物であって、口腔、鼻腔、呼吸器官、消化管などの粘膜上皮におけるIgAの分泌を促進し、宿主の免疫機構を賦活するために有効な組成物を意味する。本発明の免疫賦活剤は、以下に詳述するように、飲食品、医薬品、外用剤または飼料の形態等を含む。また、これらの中でも健康食品が好ましく、特に、免疫力が低下した対象者の健康を維持増進するための食品組成物が好ましい。
〔各種組成物〕
本実施形態の新規乳酸菌を飲食品、医薬品、外用剤(外用医薬品、化粧品等)、飼料等の各種組成物の形態で用いる場合、当該乳酸菌の菌体を、乳酸菌培養の常法に従って培養し、得られた培養物から遠心分離等の集菌手段によって分離されたものをそのまま用いることのみならず、当該培養・発酵液(培養上清)、その培養物の粗精製品あるいは精製品、それらの凍結乾燥品、或いは菌体を酵素や物理的手段を用いて処理した細胞質や細胞壁画分も用いることができる。
(飲食品)
本実施形態の組成物を飲食品とする場合は、例えば発酵乳、乳酸菌飲料、発酵野菜飲料、発酵果実飲料、発酵豆乳飲料等を挙げることができる。「発酵乳」とは、乳又は乳製品を乳酸菌、ビフィズス菌又は酵母で発酵させた糊状又は液状にしたものをいう。従って該発酵乳には飲料形態と共にヨーグルト形態が包含される。また「乳酸菌飲料」とは、乳又は乳製品を乳酸菌、ビフィズス菌又は酵母で発酵させた糊状又は液状にしたものを主原料としてこれを水に薄めた飲料をいう。
本実施形態の組成物を医薬品とする場合は、10H株と共に製剤学的に許容される適当な製剤担体を用いて、一般的な医薬組成物の形態に調製されて実用される。該製剤担体としては、通常、この分野で使用されることの知られている充填剤、増量剤、結合剤、付湿剤、崩壊剤、表面活性剤、滑沢剤等の希釈剤あるいは賦形剤を例示できる。これらは得られる製剤の投与単位形態に応じて適宜選択使用される。
本実施形態の組成物を化粧品、外用医薬品、医薬部外品等の外用剤組成物とする場合は、10H株と共に、製剤学的に許容される適当な製剤担体を用いて、一般的な外用剤組成物の形態に調製されて実用される。
本実施形態の組成物を飼料とする場合は、例えば、鶏の非抗生剤投与時期、豚、牛等の離乳期における感染症予防用として、経口投与用製剤形態(水溶液、乳化液、顆粒、粉末、カプセル、錠剤等)を挙げることができる。
本発明の他の実施形態では、ラクトバチルス・コーソイ10H株を、高糖濃度、かつD-フルクトースを含む培地に接種して培養するステップを含む免疫賦活剤の製造方法が提供される。本実施形態において、「高糖濃度」とは、糖濃度を高くした培地を用いて培養することを意味し、例えば、グルコース、フルクトース、マンノースなどの単糖、ショ糖、ラクトースなどの二糖、オリゴ糖、多糖類、糖アルコール類などの糖溶液の添加により調整することができる。高糖濃度培地とは、通常の培養培地の浸透圧よりも高い浸透圧を有する培地である。高糖濃度培地は、例えば、添加する糖類の最終濃度が少なくとも10質量%、好ましくは10~40質量%であり、40質量%以上の糖濃度でも生育可能である。さらに、10H株の炭素源としてD-フルクトースを5~20質量%、好ましくは5~10質量%含む培地である。D-フルクトースは、単糖として添加されるだけでなく、ショ糖やオリゴ糖あるいは多糖類(野菜やキノコなどを含む)として添加された培地成分が培養中に分解されて供給されてもよい。従って、高糖濃度の培地としては、D-フルクトース(果糖)をその構成成分として含むショ糖や、黒砂糖または黒糖などの天然物由来の糖類が好ましい。かかる高糖濃度、かつD-フルクトースを含む培地で培養することにより、他の乳酸菌株等の存在下においても、10H株を優勢菌株として生育させることができる。
ラクトバチルス・コーソイ(Lactobacillus kosoi)10H株は、日本国長野県佐久市のアルソア佐久ファクトリィで製造された野菜黒糖発酵液から得られた。この発酵液には、黒砂糖(10重量%)、ガラクトオリゴ糖(7重量%)および赤砂糖(33重量%)を含む、重量換算で約50%以上の糖が存在していた。限界希釈法(Button et al、1993、Simu and Hagstrom 2004)を参考に、ポリスチレン製96ウェルマイクロタイタープレートを用いた方法に改変し、この菌株を単離した。
Proteose Peptone No.3 10g/L
Beef extract 10g/L
Yeast extract 5g/L
Dextrose 20g/L
Polysorbate 80 1g/L
クエン酸アンモニウム 2g/L
酢酸ナトリウム 5g/L
硫酸マグネシウム 0.1g/L
硫酸マンガン 0.05g/L
リン酸二カリウム 2g/L
を用いて発酵液を1000倍希釈したものを出発試料とした。マイクロタイタープレートにて、2倍階段希釈(20~218倍)した後、各ウェルを滅菌した50容量%の酵素液で満たした。このマイクロタイタープレートは、静置培養のため30℃にて大気圧中で静置した。5日後、1つのウェルに存在する1つのコロニーを抜き取り、10%(w/v)D-フルクトースを追加したMRSブロス中に継代培養した。10%(w/v)D-フルクトースを追加したラクトバチリMRS寒天(ディフコラボラトリ社)を用いたペトリ皿中でコロニー形成を観察した。単離された菌株が純粋であることを確認し、10%(w/v)グリセロールおよび10%(w/v)スキムミルク中に懸濁し-80℃で保存した。
10H株の増殖特性を調べるため、MRSブロスのみ、MRSブロスにD-フルクトースを5%、10%、15%または20%(w/v)の濃度で添加したもの、またはピルビン酸を0.5容量%の濃度で添加したMRSブロスを用いて、30℃で培養を行った。0~110時間の間に、660nmの波長における培養液の吸光度(OD660)を、JASCO社製、V-730UV-可視スペクトロフォトメーターを用いて測定した結果を図1に示す。図1に示したように、10H株は、MRSブロスのみおよび0.5%ピルビン酸を添加したMRSブロスではほとんど増殖しなかったが、5%~20%のD-フルクトースを添加したMRSブロス中で良く増殖した。
単離したラクトバチルス・コーソイ(Lactobacillus kosoi)10H株の培養菌体から常法によりゲノムDNAを抽出し、PCR増幅シーケンシングにより、アプライドバイオシステムズ社の3130ジェネティックアナライザを用いて、16S rRNA遺伝子の配列を決定した。近縁菌株との16S rRNA遺伝子の塩基配列の相同性に基づき、最尤法(maximum-likelihood method)を用いて系統発生樹を構築した。その結果を図2に示す。50%以上のブートストラップ値を分岐点に示した。また、GenBankの受入番号をカッコ内に示す。
(パイエル板細胞の調製)
6週齢オスBALB/cA マウス(CREA Japanより購入)をAIN-76A DIET(Research Dietsより購入)にて1週間飼育した後、炭酸ガスにて安楽死させ、パイエル板を摘出した。RPMI10培地[RPMI1640培地(Gibco BRL)に、100U/mlのペニシリン、100μg/mlのストレプトマイシン、55μmol/lの2-メルカプトエタノールおよび10%牛胎児血清(FBS;GibcoBRL)]にてパイエル板を洗浄後、25mmol/lのHEPES、5mmol/lのEDTA(pH8.0)および1mmol/lのジチオスレイトールを加えたRPMI10培地にて45分、37℃、5%CO2条件下で培養した。再度RPMI10培地で洗浄した後、400U/mlのタイプIコラゲナーゼ(Sigma)と、30U/mlのDNaseI(Takara)とを加えたRPMI10培地にて50分、37℃、5%CO2条件下で培養した。反応液を45μmフィルターにて濾過し、RPMI10培地に置換後、5.0×106cells/mlに細胞数を調製した。
MRS培地(Difco Laboratories)にて供試菌を一晩30℃で静置培養し、生理食塩水に置換後、吸光度(OD600)を0.02に調整した。必要により70℃または100℃で30分間、加熱処理を行った。Lactobacillus kosoi sp.nov.は研究室保管株を用いた。Bifidobacterium catenulatum(JCM1194)、Bifidobacterium breve(JCM1192)、Bifidobacterium animalis subsp. lactis(JCM10602)、Bifidobacterium adolescentis(JCM1275)、Bifidobacterium pseudolongum subsp.pseudolongum(JCM1205)、Bifidobacterium pseudocatenulatum(JCM1200)、Bifidobacterium longum subsp. longum(JCM1217)、Bifidobacterium longum subsp.infantis(JCM1222)、Lactobacillus plantarum subsp.plantarum(JCM1149)、Lactobacillus johnsonii(JCM2012)、Lactobacillus casei(JCM1134)、Lactobacillus reuteri(JCM1112)、Lactobacillus gasseri(JCM1131)、Lactococcus lactis subsp.lactis(JCM5805)は、RIKEN Japan Collection of Microorganisms(JCM)より購入した。Lactobacillus rhamnosus GG(ATCC53103)は、American Type Culture Collection(ATCC)より購入した。
調製したパイエル板細胞100μlと供試菌液100μlを96-well T-cell activation plate(Becton Dickinson)中で5日間、37℃、5%CO2条件下で共培養した。遠心後、得られた培養上清中のIgA量を、mouse IgA ELISA kit (Bethyl Laboratories)で測定した。
(乳酸菌粉末の調製)
実施例4と同様の方法で培養した乳酸菌10H株の培養液を遠心分離して集菌後、生理食塩水で2回洗浄した。これに、得られた菌体ペレットの2倍重量の10%(w/v)スクロース液を添加して凍結乾燥後、乳鉢で粉末化したものを生菌粉末とした(10.7×109cfu/g)。死菌粉末は、10%(w/v)スクロース液添加前に70℃、30分間の熱処理を行った。
BALB/cAマウス(6週齢、オス、日本クレア社)を購入後3週間、AIN-76(Research Diets)と水を自由摂取させ予備飼育を行った。5匹ずつ、コントロール群、生菌投与群、死菌投与群に群分けを行った。余った1匹のマウスはコントロール群に加えた。個別にゲージで飼育し、コントロール群にはAIN-76を、乳酸菌投与群にはAIN-76に乳酸菌粉末を0.38%添加した餌をそれぞれ与え、24日間飼育した。乳酸菌粉末入りの餌を与え始めた日を0日とし、適時体重、餌摂取量および糞中IgAを測定した。
乳酸菌粉末を摂取させてから0日目、10日、24日目の糞を回収し、凍結乾燥した。それぞれの糞重量を測定したのち、乳鉢を用いてそれぞれの糞を粉末にし、10 mg/200mL濃度で抽出バッファー[cOmplete MiniTM (プロテアーゼインヒビターカクテル錠、Roche社)1タブレットをPBS10mLに溶解]に懸濁した。ボルテックスでよく攪拌し、氷上で30分間放置した後、遠心分離し(15,000rpm、4℃、10min)、上清を回収した。上清中のIgA量は、Mouse IgA ELISA Quantitation Set(Bethyl Laboratories,Inc.)を用い、製品プロトコールに従って測定した。
Claims (10)
- 受託番号NITE BP-02811として寄託されているラクトバチルス・コーソイ(Lactobacillus kosoi)10Hの単離された菌株。
- 請求項1に記載の単離された菌株またはその培養物を含む組成物。
- 請求項1に記載の単離された菌株または請求項2に記載の組成物を有効成分として含むIgA産生促進剤。
- 請求項1に記載の単離された菌株または請求項2に記載の組成物を有効成分として含む免疫賦活剤。
- 飲食品、医薬品、外用剤または飼料の形態である請求項2に記載の組成物。
- 投与された対象者の粘膜免疫を賦活するための請求項2または5に記載の組成物。
- 請求項1に記載の単離された菌株を、少なくとも5%(w/v)のD-フルクトースを含む培地に接種して培養するステップを含む免疫賦活剤の製造方法。
- 前記菌株の加熱死菌体を含む請求項2に記載の組成物。
- 前記菌株の加熱死菌体を含む請求項3に記載のIgA産生促進剤。
- 前記菌株の菌体を、少なくとも70℃で30分間加熱処理する工程をさらに含む請求項7に記載の免疫賦活剤の製造方法。
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