JP2017536822A - Pufaを含有する植物性脂質の改質 - Google Patents
Pufaを含有する植物性脂質の改質 Download PDFInfo
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- 238000004817 gas chromatography Methods 0.000 description 1
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- 230000007407 health benefit Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 238000011005 laboratory method Methods 0.000 description 1
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- 229960004488 linolenic acid Drugs 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
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- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
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- 230000001681 protective effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- JIWBIWFOSCKQMA-UHFFFAOYSA-N stearidonic acid Natural products CCC=CCC=CCC=CCC=CCCCCC(O)=O JIWBIWFOSCKQMA-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
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- A—HUMAN NECESSITIES
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- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
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Abstract
Description
エイコサペンタエン酸(EPA)及びドコサヘキサエン酸(DHA)、並びに任意選択でアラキドン酸(ARA)を含む、抽出した植物性脂質の組成物であって、
a)EPAの含有率がARAの含有率より少なくとも5%高く、及び/又は
b)EPA+DHAの合計含有率がARAより少なくとも7%高く、及び/又は
c)ARAの含有率が4%未満であり、EPAの含有率が7%を超え、DHAの含有率が2%を超える、組成物を提供する。
エイコサペンタエン酸(EPA)及びドコサヘキサエン酸(DHA)、並びに任意選択でアラキドン酸(ARA)を含む脂質を含む、植物又はその部分であって、
a)EPAの含有率がARAの含有率より少なくとも5%高く、及び/又は
b)EPA+DHAの合計含有率がARAより少なくとも7%高く、及び/又は
c)ARAの含有率が4%未満であり、EPAの含有率が7%を超え、DHAの含有率が2%を超える、植物又はその部分も提供する。
a)デルタ-5エロンガーゼ遺伝子の発現が後期種子発生段階中に維持されるか又は増大するようなプロモーターの制御下のデルタ-5エロンガーゼ遺伝子、及び/又は
b)デルタ-5デサチュラーゼ遺伝子の発現が後期種子発生段階中に低減するか又は抑制されるようなプロモーターの制御下のデルタ-5デサチュラーゼ遺伝子
を含む核酸を含む植物も提供する。
a)少なくとも脂質のARA含有率が低下し、好ましくは脂質のEPA及び/又はDHA含有率が増大するまで、本発明の植物を成長させるステップ、及び
b)植物又はその部分を採取するステップ、及び
c)採取した材料から脂質組成物を抽出して、前記脂質組成物を入手するステップ
を含む方法によって入手可能な又は入手された植物性脂質の組成物を提供する。
a)本発明の植物を成長させるステップ、
b)脂質のARA含有率が低下し、好ましくは脂質のEPA及び/又はDHA含有率が増大したとき、植物又はその部分を採取するステップ
を含む、方法を提供する。
a)デルタ-5エロンガーゼ遺伝子の発現が後期種子発生段階中に維持されるか又は増大するようなプロモーターの制御下のデルタ-5エロンガーゼ遺伝子、及び/又は
b)デルタ-5デサチュラーゼ遺伝子の発現が後期種子発生段階中に低減するか又は抑制されるようなプロモーターの制御下のデルタ-5デサチュラーゼ遺伝子
を含む核酸を含むことが好ましい。
a)少なくとも脂質のARA含有率が低下し、好ましくは脂質のEPA及び/又はDHA含有率が増大するまで、本発明の植物を成長させるステップ、及び
b)植物又はその部分を採取するステップ、及び
c)採取した材料から脂質組成物を抽出して、前記脂質組成物を入手するステップ
を含む方法によって入手可能な又は入手された植物性脂質の組成物も提供する。
a)本発明の植物を成長させるステップ、
b)脂質のARA含有率が低下し、好ましくは脂質のEPA及び/又はDHA含有率が増大したとき、植物又はその部分を採取するステップ
を含む、方法も提供する。
a)本発明の植物を成長させるステップ、及び
b)脂質のARA含有率が低下し、好ましくは脂質のEPA及び/又はDHA含有率が増大したとき、植物の種子を採取するステップ
を含む、方法を提供する。
植物の成長及びサンプリング
(VC-LTM593-1qcz rcの2コピーを含有する)LBFLFK品目の同型接合体T3植物、(VC-LTM593-1qcz rcの1コピーを含有する)LBFGKN品目の同型接合体T3植物、(VC-LJB2197-1qczとVC-LLM337-1qcz rcのそれぞれ1コピーを含有する)LANPMZ品目の同型接合体T4植物、及び(VC-LJB2755-2qcz rcとVC-LLM391-2qcz rcのそれぞれ1コピーを含有する)LAODDN品目の同型接合体T4植物を農地に植え付けた。これらの品目の植物は、参照により本明細書に組み込まれる優先権書類の実施例中に記載されたように入手し繁殖させた。全ての品目は、オストレオコッカス・タウリ(Ostreococcus tauri)から得たそれをベースとするデルタ-5エロンガーゼをコードする1遺伝子(「d5EloOT_GA3」)を含む。全ての品目は、ヤブレツボカビ属の種(Thraustochytrium sp.)から得たそれをベースとするデルタ-5デサチュラーゼをコードする1遺伝子(「d5DesTc_GA2」)をさらに含有する。品目LBFLFK及びLBFGKNは、別のプロモーター(Conlininの代わりにSETL)の制御下のデルタ-5デサチュラーゼ遺伝子のさらなるコピーを含有する。最初の開花日後の週に、個々の総状花序に、最も新しく開いた花の真上の茎に眼で見える印を付けた。それぞれの総状花序に関して、印の真下の3個の莢は同齢である(即ち、同日に開花した又は授粉された)と考えた。マーキング後14日で開始しマーキング後46日まで、それぞれの総状花序における印の下の3個の莢を様々な時間地点で回収した。それぞれの時間地点で、50の個々の植物から約150の莢をサンプリングした。それぞれ個々の植物は、その寿命中に一度だけサンプリングした。未成熟種子は総状花序からの除去直後に莢から切開し、ドライアイスで即座に凍結させた。種子の齢は総状花序における印の齢によって決定し、15日齢の印の真下から得た3個の莢(及び内部の種子)は開花後15日であると考えたことを意味する。それぞれの品目に関して、それぞれの時間地点で、約150の莢由来の種子を1サンプルにプールした。分析用に、それぞれの種子サンプルを凍結状態で粉末状にし、粉末は等量に分配して、脂質分析及び定量リアルタイムPCRによる遺伝子発現分析の技術用レプリカとして使用した。
植物油の脂質抽出及び脂質分析
Ullman,Encyclopedia of Industrial Chemistry,Bd.A2,S.89-90 und S.443-613,VCH:Weinheim(1985);Fallon,A.,et al.,(1987)「Applications of HPLC in Biochemistry」in:Laboratory Techniques in Biochemistry and Molecular Biology,Bd.17;Rehm et al.(1993) Biotechnology,Bd.3,Kapitel III:「Product recovery and purification」,S.469-714,VCH:Weinheim;Belter,P.A.,et al.(1988) Bioseparations:downstream processing for Biotechnology,John Wiley and Sons;Kennedy,J.F.,und Cabral,J.M.S.(1992) Recovery processes for biological Materials,John Wiley and Sons;Shaeiwitz,J.A.,und Henry,J.D.(1988) Biochemical Separations,in:Ullmann's Encyclopedia of Industrial Chemistry,Bd.B3;Kapitel11,S.1-27,VCH:Weinheim、及びDechow,F.J.(1989) Separation and purification techniques in biotechnology,Noyes Publicationsを含む標準的参考文献中に記載されたように脂質を抽出した。
定量リアルタイムPCRのプロトコール
Spectrum Plant Total RNA-KITパーツ番号STRN50(SIGMA-ALDRICH GmbH、ミュンヘン、ドイツ)を使用して、プロトコール「SG-MA_0007-2009RNA isolation」に従いRNAを抽出した。平均して全体RNAの濃度は約450ng/μlであった。260/280比は2.2であり260/230比は2.3であった。
増幅 95℃で15秒間/60℃で60秒間 40サイクル反復
実施例3中の手順に従い、開花後の様々な時間で各品目に関して種子中のmRNA濃度を決定した。表QPCR1及び表QPCR2は、それぞれデルタ-5エロンガーゼ遺伝子とデルタ-5デサチュラーゼ遺伝子をコードするmRNAの量を記載する。値のない箇所は、各品目の植物に関して各日に測定値を得られなかったことを示した。mRNA濃度は任意単位で与え、したがって各々表QPCR1及び表QPCR2内で値は相応値であり、絶対値を表内で比較することはできないが、傾向及び動向に関して比較を妥当に行うことができる。
脂質組成データ
品目の種子脂質の組成を実施例2中で前に記載したように分析した。表FA1において見ることができるように、品目LANPMZ及びLAODDNの全抽出種子脂質におけるARAの含有率は経時的に有意に低下しなかったが、一方で品目LBFGKN及びLBFLFKの全抽出種子脂質におけるARAの含有率はそれぞれ0.53%及び0.72%低下した。表FA2は、全ての品目に関して全抽出種子脂質におけるEPA含有率が増大し続けたことを示す。表FA3は、全ての品目に関して全抽出種子脂質におけるDHA含有率も増大したことを示す。
ATCC PTA-122340
Claims (12)
- エイコサペンタエン酸(EPA)及びドコサヘキサエン酸(DHA)、並びに任意選択でアラキドン酸(ARA)を含む、抽出した植物性脂質組成物であって、
a)EPAの含有率がARAの含有率より少なくとも5%高く、及び/又は
b)EPA+DHAの合計含有率がARAより少なくとも7%高く、及び/又は
c)ARAの含有率が4%未満であり、EPAの含有率が7%を超え、且つDHAの含有率が2%を超える、
前記組成物。 - エイコサペンタエン酸(EPA)及びドコサヘキサエン酸(DHA)、並びに任意選択でアラキドン酸(ARA)を含む脂質を含む、植物又はその部分であって、
a)EPAの含有率がARAの含有率より少なくとも5%高く、及び/又は
b)EPA+DHAの合計含有率がARAより少なくとも7%高く、及び/又は
c)ARAの含有率が4%未満であり、EPAの含有率が7%を超え、且つDHAの含有率が2%を超える、
前記植物又はその部分。 - エイコサペンタエン酸(EPA)及びドコサヘキサエン酸(DHA)及びアラキドン酸(ARA)を含む脂質を含む、植物又はその部分であって、前記植物又はその部分の成長時に、好ましくはEPA及び/又はDHAの含有率が増大しながらARAの含有率が低下する、前記植物又はその部分。
- a)デルタ-5エロンガーゼ遺伝子の発現が後期種子発生段階中に維持されるか又は増大するようなプロモーターの制御下のデルタ-5エロンガーゼ遺伝子、及び/又は
b)デルタ-5デサチュラーゼ遺伝子の発現が後期種子発生段階中に低減するか又は抑制されるようなプロモーターの制御下のデルタ-5デサチュラーゼ遺伝子、
を含む核酸を含む、請求項2又は3記載の植物。 - アマナズナ属又はアブラナ属に属する、請求項2〜4のいずれか1項記載の植物。
- 請求項2〜5のいずれか1項記載の植物の種子。
- a)少なくとも脂質のARA含有率が低下し、且つ好ましくは脂質のEPA及び/又はDHA含有率が増大するまで、請求項2〜5のいずれか1項記載の植物を成長させるステップ、及び
b)前記植物又はその部分を採取するステップ、及び
c)採取した材料から脂質組成物を抽出して、前記脂質組成物を入手するステップ、
を含む方法によって入手可能な又は入手された植物性脂質組成物。 - 前記方法が前記脂質組成物を入手するために、抽出した脂質を、ゴム質除去、脱臭、漂白、脱色、乾燥、不凍処理及び/又は分別することをさらに含む、請求項7記載の植物性脂質組成物。
- 請求項1又は8記載の脂質組成物を含む食料品又は飼料。
- 植物性脂質組成物を改変する方法であって、請求項2〜5のいずれか1項記載の植物を成長させ、エイコサペンタエン酸(EPA)及びドコサヘキサエン酸(DHA)及びアラキドン酸(ARA)を含む脂質を生成するステップを含み、前記成長及び脂質生成のステップは、好ましくはEPA及び/又はDHAの含有率が増大しながら、ARAの含有率が低下するまで続けられる、前記方法。
- 植物性脂質組成物を生成する方法であって、
a)請求項2〜5のいずれか1項記載の植物を成長させるステップ、
b)脂質のARA含有率が低下し、且つ好ましくは脂質のEPA及び/又はDHA含有率が増大したとき、前記植物又はその部分を採取するステップ
を含む、前記方法。 - ARAが少なくとも0.5重量%、好ましくは最大で4重量%〜最大で3.5重量%減少し、一方好ましくはEPAが少なくとも1重量%、好ましくは少なくとも6重量%〜少なくとも7重量%増大する、請求項10又は11記載の方法。
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