JP2005323603A - 高濃度オメガ三系高度不飽和脂肪酸を含有する微生物相バイオマス - 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相バイオマス Download PDFInfo
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- JP2005323603A JP2005323603A JP2005168232A JP2005168232A JP2005323603A JP 2005323603 A JP2005323603 A JP 2005323603A JP 2005168232 A JP2005168232 A JP 2005168232A JP 2005168232 A JP2005168232 A JP 2005168232A JP 2005323603 A JP2005323603 A JP 2005323603A
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Abstract
【解決手段】非塩化物ナトリウム塩、特に、硫酸ナトリウムを含有する発酵培地において生育することにより、高濃度オメガ三系高度不飽和脂肪酸を含有する、トラウストキトリウム、シゾキトリウム及びそれらの混合物からなる微生物相バイオマスが生成される。更に、得られた微生物相バイオマスは、水産養殖用の食品を生成するのに有用な寸法の細胞集合体を含むものである。
【選択図】 図1
Description
菌株 ATCC No. 寄託年月日
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。
トラウストキトリッドの4種の菌株、即ちシゾキトリウム属sp.S31(ATCC第20888番)、トラウストキトリウム属sp.U42−2(ATCC第20891番)(双方とも例1の方法で分離かつ選別)並びにS.アグリゲータム(ATCC第28209番)及びT.アウレウム(ATCC第28210番)(アメリカン・タイプ・カルチャ・コレクションから入手)を固体F−1培地から摘採し、回転振とう培養機(200rpm)上で27℃にて3〜4日間、保温培養した。以下のようにM培地塩(25g/lのNaCl、5g/lのMgSO4・7H2O、1g/lのKCl、200mg/lのCaCl2)を希釈することによって、異なる塩度域の培地を調製した。それは、1)100%(w/v)のM培地塩、2)80%(v/v)のM培地、20%(v/v)の蒸留水、3)60%(v/v)のM培地、40%(v/v)の蒸留水、4)40%(v/v)のM培地、60%(v/v)の蒸留水、5)20%(v/v)のM培地、80%(v/v)の蒸留水、6)15%(v/v)のM培地、85%(v/v)の蒸留水、7)10%(v/v)のM培地、90%(v/v)の蒸留水、8)7%(v/v)のM培地、93%(v/v)の蒸留水、9)3%(v/v)のM培地、97%(v/v)の蒸留水、10)1.5%(v/v)のM培地、98.5%(v/v)の蒸留水である。以下の栄養素、即ち5gのブドウ糖、5gのグルタミン酸塩、1gの酵母エキス、200mgの(NH4)2SO4、200mgのNaHCO3、5mlのPII金属、1mlのAビタミン溶液及び2mlの抗生物質溶液を処理剤に添加した(1リットル当り)。これら処理剤の各々の50mlにF−1培地にて生育する細胞の1mlを接種した。これら培養株を回転振とう培養機(200rpm)上に置き、48時間、27℃で維持した。遠心分離により細胞を採収し、ガスクロマトグラフィーにより全脂肪酸を測定した。この結果を図6に示している。例1の方法で分離されたトラウストキトリウム属sp.U42−2(ATCC第20891番)はT.アウレウム(ATCC第28211番)が生成する脂肪酸の量のほぼ2倍、S.アグリゲータム(ATCC第28209番)が生成する脂肪酸の量の8倍以上を生成できる。加えて、U42−2は評価塩度域の上端において、より広範囲の耐塩性を有しているように思われる。やはり例1の方法で分離されたシゾキトリウム属sp.S31(ATCC第20888番)は脂肪酸収率が多く(周知のATCC株の2.5〜10倍)、ATCC株よりも遥かに広範囲の耐塩性を示した。加えて、シゾキトリウム属sp.S31(ATCC第20888番)は非常に低い塩度で最大に生育している。市販性を考慮した場合、この特性は経済的に大きな効果となる。それは、塩水が金属反応装置に腐食効果を及ぼし、塩水処理に関連して問題が発生するためである。
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。
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 (16)
- 約150ミクロン以下の寸法の細胞集合体を有する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とからなる群から選択される微生物相バイオマス。
- 前記シゾキトリウム属はATCC第20888番、第20889番及びこれらの変異体からなる群から選択されるとともにオメガ三系高度不飽和脂肪酸を産生できるという特徴を備え、前記シゾキトリウムは少なくとも約0.5%の乾燥重量のオメガ三系高度不飽和脂肪酸含量を有している請求項1に記載の微生物相バイオマス。
- 前記トラウストキトリウム属はATCC第20890番、第20891番、第20892番及びこれらの変異体からなる群から選択されるとともにオメガ三系高度不飽和脂肪酸を産生できるという特徴を備え、前記トラウストキトリウム属が少なくとも約0.5%の乾燥重量のオメガ三系高度不飽和脂肪酸含量を有している請求項1に記載の微生物相バイオマス。
- 培地1リットル当り約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、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でトラウストキトリウム、シゾキ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微生物を生育することを含む請求項1に記載の微生物相を生成する方法。
- 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4に記載の方法。
- 前記シゾキトリウム属はATCC第20888番、第20889番及びこれらの変異体からなる群から選択されるとともにオメガ三系高度不飽和脂肪酸を産生できるという特徴を備え、前記シゾキトリウムは少なくとも約0.5%の乾燥重量のオメガ三系高度不飽和脂肪酸含量を有している請求項4に記載の方法。
- 前記トラウストキトリウム属はATCC第20890番、第20891番、第20892番及びこれらの変異体からなる群から選択されるとともにオメガ三系高度不飽和脂肪酸を産生できるという特徴を備え、前記トラウストキトリウム属が少なくとも約0.5%の乾燥重量のオメガ三系高度不飽和脂肪酸含量を有している請求項4に記載の方法。
- 前記培地が培地1リットル当り約250ミリグラム以下の塩化物含量を有している請求項4に記載の方法。
- a)非塩化物ナトリウム塩を含んだ培地中で生育される請求項1に記載の微生物相バイオマスと、
b)亜麻仁、菜種、大豆、アボカドミール及びこれらの混合物からなる群から選択される物質とからなる食品。 - 前記食品の組成には約5〜95重量%の前記物質が含まれる請求項9に記載の食品。
- 前記食品は押出し成形物である請求項9に記載の食品。
- 請求項1に記載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混合物からなる群から選択される微生物相を、仔エビ、塩水エビ、クルマムシ及び軟体動物からなる群から選択された生体に給餌することからなる水産養殖の方法。
- 培地1リットル当り約500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、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からなる群から選択された微生物を生育することを含む請求項1に記載の微生物相バイオマスを生育する方法。
- 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で請求項1に記載の微生物相バイオマスを生育する方法。
- 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14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14に記載の方法。
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1995
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1997
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1999
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2000
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2003
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2005
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2006
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2007
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2009
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