JP6807329B2 - ω−7脂肪酸合成物、及び黄緑色藻を培養して該合成物を生産する方法と応用 - Google Patents
ω−7脂肪酸合成物、及び黄緑色藻を培養して該合成物を生産する方法と応用 Download PDFInfo
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Description
特記のない限り、本発明における「従属栄養培養」とは、光照射を提供せずに有機炭素源を提供する条件下で黄緑色藻を培養することを意味するため、「発酵」として理解でき、具体的には、黄緑色藻培養時に光照射を提供しないが、培地に適量の有機炭素源、窒素源、リン酸塩及びその他の栄養素を含むことを意味する。従属栄養培養条件において、黄緑色藻は有機炭素源をエネルギー源及びバイオマス合成用の炭素源とする。
前記「混合栄養培養」とは、光照射を提供すると同時に有機炭素源を提供する条件下で黄緑色藻を培養することを意味し、従って、「適切な光照射下での発酵」として理解でき、具体的には、黄緑色藻培養時、適量の光照射を提供すると同時に、培地に適量の有機炭素源、窒素源、リン酸塩及びその他の栄養素を含むことを意味する。混合栄養培養条件において、黄緑色藻は、光エネルギー及び/又は有機炭素源をエネルギー源として、有機炭素源及び/又は二酸化炭素をバイオマス合成用の炭素源とする。
好ましくは、本発明の前記黄緑色藻はトリボネマ・アケール(Tribonema aequale)、トリボネマ・ブルガレ(Tribonema vulgare)、トリボネマ・マイナス(Tribonema minus)、トリボネマ・ウロトリコイデス(Tribonema ulotrichoides)及びトリボネマ・ウリクラツムTribonema utriculosumを含むが、それらに制限されない。
黄緑色藻の従属栄養及び/又は混合栄養培養方法は、具体的には、
(1)バイオ技術分野の公知の技術的手段により、適量の抗生物質を黄緑色藻の藻液に添加して、2日間培養後、黄緑色藻の藻体を無菌水に移し替えて再懸濁させた。バイオ技術分野の公知の検査手段により再懸濁液が無菌であるか否かをテストした。本ステップを、無菌の黄緑色藻の藻株が得られるまで繰り返した。前記適量の抗生物質とは、ワーキング溶液の濃度が20mg/L−200mg/Lのクロロマイセチン又はカナマイシン又はストレプトマイシン又はアンピシリン又はその他抗生物質である。
(2)有機炭素源、窒素源、リン酸塩及びその他の栄養素を含有する培地を培養装置に投入して、115℃で20分間滅菌した。
(3)ステップ(2)における培地の温度が室温に下げられた後、ステップ(1)で得られた無菌黄緑色藻の藻株を培養装置に接種し、従属栄養培養及び/又は混合栄養培養を行った。従属栄養培養の条件としては、培地においてグルコース濃度が10g/Lで且つ光照射を提供せず、混合栄養培養の条件としては、培地においてグルコース濃度が10g/Lで且つ50μmol photons m−2 s−1の光照射を提供し、培養温度は25℃、シェーカーの回転数は180rpmである。
(4)培養終了後、バイオ技術分野の公知の技術的手段、例えば濾過、空気浮上等を用いて、黄緑色藻の培養液を収集して、黄緑色藻バイオマスを得た。
黄緑色藻の、従属栄養培養、混合栄養培養及び光合独立栄養培養条件での成長、油脂含有量及び脂肪酸組成
各種の有機炭素源による黄緑色藻成長への影響
混合栄養培養条件において、グルコース、グリセリン及び酢酸ナトリウムをそれぞれ有機炭素源として黄緑色藻を培養した。前記混合栄養培養条件としては、有機炭素源の濃度は10g/L、ペプトン濃度は2g/L、その他の栄養塩の濃度はBG11培地と同様であり、光照射強度は50μmol photons m−2s−1、初期バイオマスは0.3g/Lであった。250mL三角フラスコを培養容器として、単一の培養体積を100mLとした。前記三角フラスコをシェーカーに入れて、温度とシェーカー回転数をそれぞれ25℃と180rpmに設定して、振とう培養を行った。培養過程において、毎日、所定時刻にサンプリングしてそのバイオマス(乾燥重量)を測定した。
グルコース初期濃度による黄緑色藻成長への影響
混合栄養培養条件において、グルコースを有機炭素源として黄緑色藻を培養した。前記混合栄養培養条件としては、グルコース濃度は5g/L〜60g/L、ペプトン濃度は2g/L、その他の栄養塩の濃度はBG11培地と同様であり、光照射強度は50μmol photons m−2s−1、初期バイオマスは0.3g/Lであった。250mL三角フラスコを培養容器として、単一の培養体積を100mLとした。前記三角フラスコをシェーカーに入れて、温度とシェーカーの回転数をそれぞれ25℃と180rpmとして設定して、振とう培養を行った。培養過程において、毎日、所定時刻にサンプリングしてそのバイオマス(乾燥重量)を測定した。
撹拌速度による黄緑色藻成長への影響
混合栄養培養条件において、グルコースを有機炭素源として黄緑色藻を培養した。前記混合栄養培養条件としては、グルコース濃度は10g/L、ペプトン濃度は2g/L、その他の栄養塩の濃度はBG11培地として同様であり、光照射強度は50μmol photons m−2s−1、初期バイオマスは0.3g/Lであった。250mL三角フラスコを培養容器として、単一の培養体積を100mLとした。前記三角フラスコをシェーカーに入れて、温度設定を25℃、撹拌速度を90〜270rpmに設定して、振とう培養を行った。培養過程において、毎日、所定時刻にサンプリングしてそのバイオマス(乾燥重量)を測定した。
初期接種量による黄緑色藻成長への影響
混合栄養培養条件において、グルコースを有機炭素源として黄緑色藻を培養した。前記混合栄養培養条件としては、グルコース濃度は10g/L、ペプトン濃度は2g/L、その他の栄養塩の濃度はBG11培地と同様であり、光照射強度は50μmol photons m−2s−1、培養温度は25℃、初期接種量は0.1〜3g/Lであった。1500mL円柱状エアリフト型式反応器を培養装置として、単一の培養体積を1000mLとした。濾過後の空気を、通気速度0.1vvmで前記円柱状エアリフト型式反応器に導入した。培養過程において、毎日、所定時刻にサンプリングしてそのバイオマス(乾燥重量)を測定した。
黄緑色藻の回分発酵培養
混合栄養培養条件において、グルコースを有機炭素源として黄緑色藻に回分発酵培養を行った。前記混合栄養培養条件としては、グルコース濃度は60g/L、ペプトン濃度は12g/L、リン酸塩は0.4g/L、その他の栄養塩の濃度はBG11培地と同様であり、初期接種量は0.3g/Lであった。10L透光型発酵槽を培養装置として、培養体積を8Lとした。外付けLEDランプを光源として、光照射強度を50μmol photons m−2s−1とした。濾過後の空気を、通気速度0.1vvmで前記発酵槽に導入した。培養温度と撹拌速度はそれぞれ25℃と180rpmに設定される。培養過程において、毎日、所定時刻にサンプリングしてそのバイオマス(乾燥重量)と培地中のグルコース残留量を測定した。前記グルコース残留量の測定は生物センサにより行われる。
黄緑色藻の流加発酵培養
混合栄養培養条件において、グルコースを有機炭素源として黄緑色藻に流加発酵培養を行った。前記混合栄養培養条件としては、グルコース濃度は10g/L、ペプトン濃度は2g/L、リン酸塩は0.4g/L、その他の栄養塩の濃度はBG11培地と同様であり、初期接種量は0.3g/Lであった。毎日、培地にグルコース10g/L及びペプトン2g/Lを添加した。10L透光型発酵槽を培養装置、培養体積を8Lとした。外付けLEDランプを光源、光照射強度を50μmol photons m−2s−1とした。濾過後の空気を、通気速度0.1vvmで前記発酵槽に導入した。培養温度と撹拌速度はそれぞれ25℃と180rpmに設定される。培養過程において、毎日、所定時刻にサンプリングしてそのバイオマス(乾燥重量)と培地中のグルコース残留量を測定した。前記グルコース残留量の測定は生物センサにより行われる。なお、毎日、サンプリング過程はグルコースとペプトンを添加する前に行われる。
Claims (1)
- トリボネマ属の黄緑色藻に従属栄養培養を行うステップと、トリボネマ属の黄緑色藻の培養液を収集して、トリボネマ属の黄緑色藻バイオマスを得るステップと、抽出及び/又は圧搾処理を行って、ω−7脂肪酸合成物を得るステップとを備え、
前記トリボネマ属の黄緑色藻バイオマスを得るステップでは、遠心及び/又は濾過及び/又は空気浮上方式により、トリボネマ属の黄緑色藻の培養液を収集して、トリボネマ属の黄緑色藻バイオマスを得ることを特徴とするω−7脂肪酸合成物の生産方法。
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