JP4015611B2 - 高濃度オメガ三系高度不飽和脂肪酸を含有する微生物を生育する方法 - Google Patents
高濃度オメガ三系高度不飽和脂肪酸を含有する微生物を生育する方法 Download PDFInfo
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- JP4015611B2 JP4015611B2 JP2003388954A JP2003388954A JP4015611B2 JP 4015611 B2 JP4015611 B2 JP 4015611B2 JP 2003388954 A JP2003388954 A JP 2003388954A JP 2003388954 A JP2003388954 A JP 2003388954A JP 4015611 B2 JP4015611 B2 JP 4015611B2
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Description
請求項4に記載の発明では、請求項3に記載の方法において、培地が培地1リットル当たり約250ミリグラム以下の塩化物含量を有していることをその要旨とする。
請求項7に記載の発明では、請求項5に記載の食品において、同食品は押出し成形物であることをその要旨とする。
請求項11に記載の発明では、請求項10に記載の方法において、硫酸ナトリウムの濃度は約2g/lから約25g/lの間であることをその要旨とする。請求項12に記載の発明では、請求項1に記載の方法において、微生物が塩性環境より得られることをその要旨とする。
本発明の一実施例は、微生物相のトラウストキトリウム属、シゾキトリウム属及びこれらの混合物を生育する新規の方法に関する。この方法には、特に硫酸ナトリウム等の非塩化物ナトリウム塩を有する培地において微生物相を生育することが含まれる。より詳細には、発酵に必要なナトリウムの大部分は非塩化物ナトリウム塩として供給される。本発明の方法は、培地における塩化物含有量が大幅に低減され、よって塩化物が発酵装置を腐食することを回避できるために、特に市販向けの製造に有用である。加えて、本発明は水産養殖に利用される食品の製造に特に有用である。それは、この種の培地において培養されるトラウストキトリウム属及びシゾキトリウム属は高塩化物の培地において培養される場合よりも遥かに小さなクランプを形成し、よって仔エビの餌料源としてより得やすくなっているためである。特に、硫酸ナトリウム含有の培地において培養されるトラウストキトリウム属及びシゾキトリウム属は平均寸法が直径約150ミクロン以下の細胞集合体を有し得る。
アメリカン・タイプ・カルチャ・コレクション(ATCC)における約20,500株の内、分離する時に10株が同一の分類群に属すると後に確認された。この保存機関にてまだ生存可能な菌株を入手し、開示した処理によって分離かつ培養された菌株と比較するのに使用した。この比較結果を以下の例4,5に提示している。
シゾキトリウム属 S31 20888 8/8/88
シゾキトリウム属 S8 20889 8/8/88
本発明は特定の微生物株に関して開示されているが、開示した示唆に基づいて得られる有用な方法及び菌株の全てを包含するものであり、当業者に可能な手段である代替、改変及び最適化の全てを包含している。
(例)
(例1.収集及び選別)
内陸の浅い塩水池から150mlの試料水を収集し、無菌ポリエチレン瓶に保存した。試料水とともに生きている植物の素材及び自然に生じた有機堆積物(腐敗動植物物質)も幾分含有するように特に注意を払った。このサンプルを実験室に戻すまで氷の上に置いた。実験室で試水を15〜30秒間振り動かし、2種類のフィルタを有するフィルタ装置の中にこのサンプルを1〜10ml、ピペットで移し入れ、即ち注入した。これらのフィルタは、1)上部における、孔寸法が約25μmの無菌47mm径ワットマン第4番フィルタ及び2)同ワットマンフィルタの下部における、孔寸法が約1.0μmである47mm径ポリカーボネートフィルタである。フィルタの称呼孔寸法に僅かな変動があると、ポリカーボネートフィルタ上に収集される細胞の寸法は約1.0〜25μmの範囲になる。
シゾキトリウムアグリゲータム(Schizochytrium Aggregatum)(ATCC 28209)の細胞を固体F−1培地から摘採し、50mlのFFM培地に接種した(フラー(Fuller)ら、1964年)。この培地は海水を1,000ml、ブドウ糖を1.0g、ゼラチン加水分解物を1.0g、肝臓エキスを0.01g、酵母エキスを0.1g、PII金属を5ml、1mlのB−ビタミン溶液(ゴールドスタイン(Goldstein)ら、1969年)及び1mlの抗生物質溶液(25g/lの硫酸ストレプトマイシン及びペニシリン−G)を含有している。1.0mlのビタミン配合物(pH7.2)はチアミンHClを200μg、ビオチンを0.5μg、シアノコバラミンを0.05μg、ニコチン酸を100μg、パントテン酸カルシウムを100μg、リボフラビンを5.0μg、ピリドキシンHClを40.0μg、ピリドキサミン2HClを20.0μg、p−アミノ安息香酸を10μg、塩素HClを500μg、イノシトールを1.0mg、チミンを0.8g、オロチン酸を0.26mg、ホリニン酸を0.2μg及び葉酸を2.5μg含有している。27℃にして回転振とう培養機(200rpm)上に培養株を置いた。3〜4日後、この培養株の1mlを各々50mlの以下の処理剤に移し変えた。1)FFM培地(対照標準として)及び2)250mg/lのKH2PO4及び250mg/lの酵母エキスを添加したFFM培地である。これら培養株を48時間、27℃にして回転振とう培養機(200rpm)上に置いた。細胞を採収し、細胞の収率を定量化した。第1処理において、無灰乾燥重量ベースでの細胞の最終濃度は616mg/lであった。第2処理において、細胞の最終濃度は1,675mg/lであり、培地においてPO4及び酵母エキスの濃度を高くするという効果が強調されたことを示している。
シゾキトリウム属sp.S31(ATCC No.20888)の細胞を固体F−1培地から摘採し、50mlのM−5培地の中に置いた。この培地は(1リットルベースで)1gの酵母エキス、25gのNaCl、5gのMgSO4・7H2O、1gのKCl、200mgのCaCl2、5gのブドウ糖、5gのグルタミン酸塩、1gのKH2PO4、5mlのPII金属、1mlのA−ビタミン溶液及び1mlの抗生物質溶液からなっている。この溶液のpHを7.0に調節し、溶液をろ過殺菌した。コーンスティープリカー(4g/40ml;pH7.0)及び酵母エキス(1g/40ml;pH7.0)の無菌溶液を調製した。一組のM−5培地フラスコに以下の量の酵母エキス溶液を添加した。1)2ml;2)1.5ml;3)1ml;4)0.5ml及び5)0.25mlである。別の一組のM−5培地フラスコに酵母エキス及びコーンスティープリカー溶液を以下の濃度で添加した。1)2mlの酵母エキス;2)1.5mlの酵母エキス及び0.5mlのコーンスティープリカー;3)1.0mlの酵母エキス及び1.0mlのコーンスティープリカー;4)0.5mlの酵母エキス及び1.5mlのコーンスティープリカー及び5)2mlのコーンスティープリカーである。各フラスコに接種するのにF−1培地における培養株を1mlだけ使用した。これら培養株を48時間、27℃にして回転振とう培養機上に置いた。遠心分離法によって細胞を採収し、(無灰乾燥重量として)細胞の収率を測定した。表1に結果を示している。この結果は0.8g/lの培地まで酵母エキスを添加することによって細胞の収率を高めることができるということを示している。しかし、酵母エキスとともにコーンスティープリカーを添加すると更に効果的であり、処理剤の収率が2倍になる。コーンスティープリカーは酵母エキスよりも遥かに安価であるため、細胞を経済的に生成するにはこれは非常に有利である。
例1に記載の方法に基づいて選択され、新たに分離された一群の151個の菌株を後期指数増殖期中にサンプルとし、ガスクロマトグラフィーによってHUFA含有量を分析した。全ての菌株はM1培地又は液体FFM培地のいずれかにおいて生育され、いずれにしても細胞の収率が最大であることを示した。M1培地はM5培地と同一の組成であり、ブドウ糖及びグルタミン酸塩の濃度が1g/lである点が例外である。加えて、以前に分離されたトラウストキトリウム属又はシゾキトリウム属を5種、アメリカン・タイプ・カルチャ・コレクションから得た。この保存機関では保存物から生存できる形態で得られる全ての菌株を表示している。これらの菌株とはT.アウレウム(aureum)(ATCC第28211番)、T.アウレウム(ATCC第34304番)、T.ロゼウム(roseum)(ATCC第28210番)、T.ストレータム(straitum)(ATCC第34473番)及びS.アグリゲータム(ATCC第28209番)であった。従来の培地において菌株は全て生育が短縮されることを示し、本発明のM5培地及びFFM培地等の培地においては概して生育が向上することを示した。本発明の培地において菌株の生育が向上したことに基づき、各周知株の脂肪酸生成量を上記のように測定した。
シゾキトリウム属sp.S31(ATCC第20888番)、シゾキトリウム属sp.S8(ATCC第20889番)、トラウストキトリウム属sp.S42、トラウストキトリウム属sp.U42−2、トラウストキトリウム属sp.U42及びU30並びにトラウストキトリウムアウレウム(ATCC第28211番)及びシゾキトリウムアグリゲータム(ATCC第28209番)(周知の菌株)の細胞を固体F−1培地から摘採し、50mlのM−5培地に置いた。この溶液のpHを7.0に調節し、溶液をろ過殺菌した。回転振とう培養機(200rpm、27℃)上で3日間生育した後、各培養菌株の1〜2mlをM−5培地の他のフラスコに移し換え、2日間、同振とう培養機上に置いた。次に、これら培養株(1〜2ml)をM−5培地の他のフラスコに移し換え、1日間、振とう培養機上に置いた。この工程により全ての培養株は指数増殖期中にあった。次に、これらの後期培養株を用いてM−5培地の2本の250mlフラスコに接種し、各々に株を育成することとした。次に、これらフラスコを25℃及び30℃にて振とう培養機上に置き、ベックマンDB−G分光測光器(660nm、1cmの路程)上でその光学濃度の変化を監視した。光学濃度の読取りは0,6,10,14,17.25,20.25,22.75時間で行った。次に、ソロキン((Sorokin)1973年)の手法によって光学密度データから指数増殖率(1日当りの倍加)を計算した。この結果を表8及び図4(25℃における菌株U30の生育が標準)に示している。例1における方法によって分離された菌株が25℃及び30℃の双方で周知のATCC菌株よりも遥かに高い生育率を有し、連続して生育するのに欠かせないリン酸塩濃度に最適化しても結果は同じであることをデータは示している。南極の冷水から分離されたトラウストキトリアレス株は30℃で生育する様子を示さなかった。
シゾキトリウム属sp.S31(ATCC第20888番)、シゾキトリウム属sp.S8(ATCC第20889番)(双方とも例1の方法で分離)並びにトラウストキトリウムアウレウム(ATCC第28211番)及びシゾキトリウムアグリゲータム(ATCC第28209番)(従来の菌株)の細胞を固体F−1培地から摘採し、50mlのM−5培地の中に置いた(例3を参照)。この溶液のpHを7.0に調節し、溶液をろ過殺菌した。回転振とう培養機(200rpm、27℃)上で3日間生育した後、各培養株の1〜2mlをM−5培地の他のフラスコに移し換え、2日間、同振とう培養機上に置いた。次に、これら培養株の各々の無灰乾燥重量を素早く測定し、50mlのM−5培地を含有する2本の250ml三角フラスコの中に各培養株を3.29mgだけピペットで移し換えた。これらフラスコを回転振とう培養機上に置いた(200rpm、27℃)。24時間後、各培養株の20ml分を遠心分離し、上澄みを廃棄し、グルタミン酸塩(N源)を全く含有しない50mlのM−5培地を有する250ml三角フラスコに細胞を移し換えた。フラスコを振とう培養機に置き直し、ルパージュ及びロイ(1984年)の手法によって12時間後に試料採取して無灰乾燥重量を測定し、脂肪酸分を定量化した。この結果を図5(周知の菌株であるATCC第28211番の収率が標準)に示している。この結果は例1の方法によって分離された菌株が指数増殖及び窒素制限下において、同時間内で従来のATCC株より2〜3培も多い無灰乾燥重量を生成したことを示している。加えて、本発明の菌株から得られる全脂肪酸及びオメガ三系脂肪酸の収率が多くなり、菌株S31(ATCC第20888番)は従来のATCC株より3〜4培も多いオメガ三系脂肪酸を生成している。
250ml三角フラスコ中のM/10−5培地の50mlに、寒天斜面から摘採したシゾキトリウム属sp.S31(ATCC第20888番)のコロニーを接種した。M/10−5培地は1000mlの脱イオン水、2.5gのNaCl、0.5gのMgSO4・7H2O、0.1gのKCl、0.02gのCaCl2、1.0gのKH2PO4、1.0gの酵母エキス、5.0gのブドウ糖、5.0gのグルタミン酸、0.2gのNaHCO3、5mlのPII微量金属、2mlのビタミン配合物及び2mlの抗生配合物を含有している。回転振とう培養機(200rpm)上で30℃にて培養株を保温培養した。2日後、培養株は適度の密度になり、活発に生育していた。この活発に生育する培養株を20ml用いて、同一の培地を1700ml含有する2リットル発酵そうに接種した。但し、この培地ではブドウ糖及びグルタミン酸塩の濃度は40g/lに増大していた(M/10−40培地)。発酵そうを30℃、1vol/minのエアレーション、300rpmの混合で維持した。48時間後、発酵そう中の細胞密度は21.7g/lであった。細胞を遠心分離によって採収し、凍結乾燥させ、N2下にて保存した。
例4に記載の種々の菌株によって生成される脂肪酸の生育及びガスクロマトグラフ分析によって、脂肪酸の多様性に相違があることが明らかになった。本発明の菌株は従来から得られる菌株よりも互いに異なる脂肪酸を合成することが少なかった。分離すべき不純物がより少なくなるため、脂肪酸の精製では脂肪酸の多様性が低いほうが効果的である。餌料補給を目的として、不要な脂肪酸を摂取する可能性が減じるため、互いに異なる脂肪酸の数は少ないほうが効果的である。
表9はATCC番号で示す周知の菌株及び本発明の種々の菌株において、総脂肪酸重量にして1%以上の濃度を占めて存在し、互いに異なっているHUFAsの数を示している。
250ml三角フラスコ中のM5培地の50mlに、寒天斜面から摘採したシゾキトリウム属sp.S31(ATCC第20888番)のコロニーを接種した。回転振とう培養機(200rpm)上で30℃にて培養株を保温培養した。2日後、培養株は適度の密度になり、活発に生育していた。この活発に生育する培養株の20mlを用いて、同一の培地を1000ml含有する1リットル発酵そうに接種した。但し、この培地ではブドウ糖及びグルタミン酸塩の濃度を40g/lに増大していた(M20培地)。発酵そうを30℃、pH7.4で維持し、エアレーションを1vol/min、混合を400rpmとした。48時間後、発酵そう中の細胞密度は18.5g/lであった。発酵そうにおけるエアレーション及び混合を止めて2〜4分以内に細胞は発酵そうの下部250mlにおいて凝集し、沈澱した。細胞のこの凝集領域は72g/lの細胞密度を有していた。
この細胞域は発酵そうからサイホンで吸収でき、更に(1)窒素制限時間中、別の反応装置に移され(例えば、幾つかの発酵そうの高密度生成を組み合わせる。
)或いは(2)遠心分離又はろ過によって直接採収できる。このように予め細胞を凝集させることによって、細胞を回収するために加工処理する必要がある水の量が60〜80%少なくなった。
250ml三角フラスコ中のM5培地の50mlに、寒天斜面から摘採したシゾキトリウム属sp.S31(ATCC第20888番)又はトラウストキトリウム属sp.U42−2(ATCC第20891番)のコロニーを接種した。M5培地については例3で記載し、その相違は2mlのビタミン配合物質及び2mlの抗生物質混合物を添加したことにある。回転振とう培養機(200rpm)上で30℃にて培養株を保温培養した。2日後、培養株は適度の密度になり、活発に生育していた。この培養株を用いてM5培地のフラスコにブドウ糖の代替としてデキストリン、ソルビトール、フルクトース、ラクトース、マルトース、スクロース、コーンスターチ、小麦でんぷん、ジャガイモでんぷん、グランドコーン(ground corn)の中の1つを接種し(5g/l)、或いはグルタミン酸塩の代替としてゲリセート(gelysate)、ペプトン、トリプトン、カゼイン、コーンスティープリカー、尿素、硝酸塩、アンモニウム、ホエー又はコーングルテンミールの中の1つを接種した(5g/l)。回転振とう培養機(200rpm、27℃)上で48時間、培養株を保温培養した。互いに異なる有機物上での生育を比較した培養密度を表10,11に示している。
M−5培地のシェーク(shake)フラスコにおいてトラウストキトリウム属sp.12B(ATCC第20890番)の細胞バイオマスを25℃で生成した(例3を参照)。M/10−5培地のシェークフラスコにおいてトラウストキトリウム属sp.S31(ATCC第20888番)の細胞バイオマスを27℃で生成した(例8を参照)。遠心分離によって各菌株の細胞を採収した。このペレットを一度蒸留水で洗浄し、再度遠心分離させ、50%の固形ペーストを生成した。生じたペーストを海水中に再度懸濁させ、次に給餌補給物として成体塩水エビ用の培養物に添加した。塩水エビは老廃農産物上にて予め飼養され、この結果、塩水エビのオメガ三系HUFA含有量は非常に少なく、全脂肪酸の僅か1.3〜2.3%であった(野生塩水エビのオメガ三系HUFAの平均含有量は全脂肪酸の6〜8%である。)。海水で満たした1リットルビーカー中に塩水エビ(2〜3/ml)を保持し、エアーストーン(airstone)を用いてこの培養物を曝気かつ混合した。給餌補給物の添加後、塩水エビのサンプルを時々採収して洗浄し、ガスクロマトグラフィーによって脂肪酸含有量を測定した。この結果を図7及び8に示している。最後の給餌としてトラウストキトリッドベースの給餌補給物を給餌すると、塩水エビのオメガ三系含有量は菌株12Bを給餌する場合には5時間以内に、或いはS31を給餌する場合には11時間以内に野生塩水エビのオメガ三系含有量にまで増加可能である。塩水エビのオメガ三系HUFA含有量は、これら給餌補給物を24時間まで給餌すると、野生塩水エビのオメガ三系含有量よりも大幅に増加可能である。加えて、これら給餌補給物によって塩水エビのDHA含有量が大幅に増加する。通常、DHAは野生塩水エビでは微量にしか報告されていない。
この例では、発酵培地におけるナトリウム塩として塩化ナトリウムの代わりに硫酸ナトリウムを用いた時、オメガ三系生成及び総脂肪酸分が損なわれず、同等か或いは優れていることを示している。培地1リットル当り2.36gのナトリウム、1.5〜3.0gの窒素源及び3.0gのブドウ糖を含有し、pHが7.0の培地において、シゾキトリウム属 ATCC第20888番を生育した。200rpmにて28℃で48時間、細胞を保温培養した。この結果を表12に示している。
この例では、低塩度の培地でありながらバイオマス収率並びにオメガ三系及び脂肪酸の生成を高度に維持したシゾキトリウム属の発酵を示している。
(例15.塩化物含有量が少ない培地におけるシゾキトリウム属の培養)
この例では、最低限の塩化物濃度でありながら初期糖濃度に基づいてバイオマス収率を増大させた、本発明の微生物相の発酵を示している。
この例では、低塩化物濃度での発酵における様々な硫酸ナトリウム濃度の効果を示している。
Claims (11)
- 培地1リットル当り約120ミリグラム以下の塩化物と、炭素源及び窒素源と、微量養分と、1リットル当たり1グラム以上の非塩化物ナトリウム塩を含有する培地において約5〜48℃の温度及びpH約5.0〜11.0で、次の特性を備える微生物を生育する方法:
a)従属栄養生育が可能であること、
b)オメガ三系高度不飽和脂肪酸を生産可能であること、
c)30℃以上の温度にて生育可能であること、
d)広塩性であること。 - 培地1リットル当り3グラム以下の塩化物と、炭素源及び窒素源と、微量養分と、非塩化物ナトリウム塩とを含有する培地において、約5〜48℃の温度及びpH約5.0〜11.0で、次の特性を備える微生物を生育する方法であって;
ナトリウム濃度(前記培地1リットル当たりのナトリウムのグラム数で表す)が2.0g/l〜25g/lである、方法;
a)従属栄養生育が可能であること、
b)オメガ三系高度不飽和脂肪酸を生産可能であること、
c)30℃以上の温度にて生育可能であること、
d)広塩性であること。 - 培地1リットル当り3グラム以下の塩化物と、炭素源及び窒素源と、微量養分と、非塩化物ナトリウム塩とを含有する培地において、約5〜48℃の温度及びpH約5.0〜11.0で、次の特性を備える微生物を生育する方法であって;
前記培地中のナトリウムの50%以下を塩化ナトリウムとして供給する、方法;
a)従属栄養生育が可能であること、
b)オメガ三系高度不飽和脂肪酸を生産可能であること、
c)30℃以上の温度にて生育可能であること、
d)広塩性であること。 - 前記培地中のナトリウムの25%以下を塩化ナトリウムとして供給する、請求項3に記載の方法。
- 前記培地が培地1リットル当たり120ミリグラム以下の塩化物を含有する、請求項2又は3に記載の方法。
- 前記培地が培地1リットル当たり60mg〜120mgの塩化物を含有する、請求項1、2または3に記載の方法。
- 前記ナトリウム塩は硫酸ナトリウム、ソーダ灰、酸化ナトリウム、炭酸ナトリウム、重炭酸ナトリウム及びこれらの混合物からなる群から選択されたものである請求項1、2または3に記載の方法。
- 炭素源、窒素源、微量養分、及び硫酸ナトリウムを含有する培地において、約5〜48℃の温度及びpH約5.0〜11.0である、請求項1、2または3に記載の方法。
- 前記硫酸ナトリウムの濃度は約1g/lから約50g/lの間である請求項8に記載の方法。
- 前記硫酸ナトリウムの濃度は約2g/lから約25g/lの間である請求項9に記載の方法。
- 前記微生物は塩を含んだ環境より得られる請求項1、2または3に記載の方法。
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