JP5491697B2 - 高レベルオメガ−3および低レベル飽和脂肪酸を含む組成物 - Google Patents
高レベルオメガ−3および低レベル飽和脂肪酸を含む組成物 Download PDFInfo
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Description
定義
「脂肪酸(FA)」とは、炭素原子の骨格を有する化合物であって、炭素原子の幾つかの結合は「不飽和」であってもよく、「遊離」酸の場合には骨格のアルファ端に酸(COOH)部分を有する化合物を指す。他端はオメガ(ω)端である。本明細書の目的上、この用語はこれらの塩およびエステル(エチルもしくはコレステロールエステル、アミド、リン脂質、またはモノ−、ジ−もしくはトリ−グリセリドを含むがこれらに限定されるものではない)を排除するものではない。
組成物
ここで提供される特定の組成物は、約10重量%から約50重量%のn−3 HUFA脂肪酸、約15%未満のミリスチン酸、および約20%未満のパルミチン酸を含む。これらの実施形態におけるn−3 HUFA脂肪酸はEPAを含むことができ、例えば、組成物中におけるEPAの他のn−3 HUFA脂肪酸に対する比は少なくとも6:1である。
製造方法
微細藻類培養物由来のDHAオイルは、ヒトもしくは食料産生動物に適度の最終DHA摂取をもたらすことを目的とする食事補助食品として提供されるとき、総C14:0および/もしくはC16:0食事負荷への有意の寄与を構成することができる。例えば、牛乳に0.4〜0.8g/L DHAを組み込むのに必要な量で乳牛に給餌するとき、シゾキトリウム種(Schizochytrium sp.)由来の組成物を含む食事補助食品はミルク中のC14:0およびC16:0の有意の同時増加をもたらした(Franklin et al, J Nutr; 129: 2048-2052 (1999年))。一例として、米国特許第6,568,351号は、これらの組成物を餌に補給することによって、肉におけるC14:0およびC16:0の増加をもたらすウサギの飼育方法を提供する。同様の状況下で、比較的高レベルのC14:0およびC16:0を含有するEPAオイルの補給も総C14:0および/もしくはC16:0食事負荷に対する有意の寄与をもたらすものと予想することができる。本発明以前には、低レベルのC14:0およびC16:0を含むEPAオイルもしくはDHAオイルを目的とする開発を可能とする技術は達成されていなかった。
投与
別の態様において、ここで説明される組成物を、例えば、ヒトもしくは、通常、ヒトが消費するための食品の産生に用いられる動物を含む、動物に与えることができる。
処方物、投与量、治療、および投与方法
動物において望ましいレベルを達成し、もしくは、幾つかの実施形態においては、望ましい効果(1つ以上)を達成するため、ここで提供されるn−3 HUFA脂肪酸および他の生理活性化合物を、単回あるいは、分割投与として投与することができ、例えば、体重当たり少なくとも約1mg/kgから約1g/kg、少なくとも約10mg/kgから約500mg/kg、少なくとも約10mg/kgから約300mg/kgもしくは少なくとも約5mg/kgから約200mg/体重kgもしくは少なくとも約10mg/kgから約200mg/kgで投与することができるが、他の投与量でも有益な効果を提供しえる。投与される量は様々な要素に依存して変化し、この要素は選択される環状組成物およびその臨床効果、動物の疾患、体重、身体状態、健康、年齢、予防もしくは治療のいずれを達成しようとするのか、およびその組成物が化学的に修飾されているかどうかを含むがこれらに限定されるものではない。そのような要素は、臨床試験からの実験データを吟味し、かつ前臨床動物モデルの結果もしくは当該技術分野において利用可能である他の試験システムを吟味する臨床医が容易に決定できる。
この例は、そのn−3 HUFA画分に実質的にEPAのみを含み、かつ比較的低レベルのC14:0およびC16:0を含む微生物バイオマスプロセスを生成するための、微生物の従属栄養性もしくは主として従属栄養性のバッチ培養によって生成される組成物の製造を説明する。用いられる微生物はN.ラエビスである。
培養起源および予備培養条件
珪藻N.ラエビス(UTEX2047)をUniversity of Texas Culture Collection of Algaeから得た。純培養予備培養物をバッチ培養物として500ml三角フラスコ内で、1リットルあたり15mgのNa2SiO39H2Oで改変され、かつ1リットルあたり合計で600mgの硝酸ナトリウムを含有するように調整されたリューウィン珪藻培地(Lewin's Diatom Medium)を含む基本培地において従属栄養的に成長させた。オートクレーブ処理に先立ち、デキストロース(100%グルコース)をこの培地に添加してグルコースレベルを1リットルあたり5グラムとした。実験に先立ち、予備培養物を入れたフラスコを、暗所において25℃で1週間、150rpmのオービタル・シェーカーに置いた。
培養培地
改変リューウィン珪藻培地(mLDM)を現地で入手可能な成分からUTEXウェブサイト(現時点で、www.bio.utexas.edu/research/utex)の処方に従って調製する。15mg/Lのメタ珪酸ナトリウム9H2Oを、1リットルあたり600mgの硝酸ナトリウムの総レベルを達成するのに十分な硝酸ナトリウムと共に培地に添加する。希塩基溶液の滴定、例えば、0.1モル強度の水酸化カリウムの滴定によりmLDMをpH8.5に調整し、121℃で15分間オートクレーブ処理した後、急速に冷却して沈殿の形成を防止した。4種類の純粋な糖、グルコース(G)、スクロース(S)、D(−)フルクトース(F)およびラクトース(L)に加えて、等量で対になった糖の6種類の組み合わせ(GS、GF、GL、SF、SL、FL)を培地に添加し、5g1−1の各糖もしくは糖対組み合わせの最終レベルを有するバッチを生成した。全ての実験は3回ずつ行った。フラスコを暗所に25℃で置き、0.2ミクロンろ過空気を泡立てて培養中の循環を11〜12日維持した。第12日に、2500gで反復遠心分離した後、培地を回収して蒸留水を補充することにより、すべての残留フラスコからバイオマスを採取した。試料を窒素流の下で乾燥させ、トルエン、メタノールおよび硫酸の混合液を用いる1工程インキュベーション手順において脂質を抽出してメチル化した。この混合物を中和し、抽出物を木炭および無水硫酸ナトリウムで精製および乾燥させた。脂肪酸は、抽出前に試料に注入した標準と比較することにより決定する。次に、ヘキサンに溶解した試料からの溶質のアリコートを採取し、105メートル 0.25mm 90% ビス−シアノプロフィールカラム(RTX2330、Restek)および0.25ミクロン膜厚を有するガスクロマトグラフィー(GCFID6890、Agilent Corp)によって分析した。脂肪酸は37成分混合物(Suplco)との比較で保持時間によって同定した。次に、理論的応答因子(AOCS)を用いてピーク面積をピーク応答に合わせて修正し、結果を曲線下の面積として報告した(Sukhija, P.S. & Palmquist, D.L. (1988年) Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. J. Agric. Food Chem. 36: 1202-1206およびWu, J., James, Jr. D.W., Dooner, H.K. & Browse, J. A mutant of Arabidpsis deficient in the elongation of palmitic acid. Plant Physiol. 106: 143-150. (1994年))。
バイオマスの総脂肪酸におけるEPAパーセンテージ
フルクトース: 14.06 S.D.=2.17
ラクトース: 13.71 S.D.=1.17
スクロース: 13.62 S.D.=1.42
グルコース: 12.27 S.D.=1.35
バイオマスの総脂肪酸におけるC16;0パーセンテージ
フルクトース: 21.62 S.D.0.54
ラクトース: 22.52 S.D.1.33
スクロース: 22.90 S.D.1.36
グルコース: 23.50 S.D.0.88
ミリグラムでのフラスコあたりの総脂肪酸生成
フルクトース: 0.77, S.D.=0.42
ラクトース: 0.67, S.D.=0.27
スクロース: 0.65, S.D.=0.32
グルコース: 0.44, S.D.=0.25
この例において報告される研究の結果(上記)は、微生物種ニッチア・ラエビスから比較的高収率の総脂肪酸を生成することが可能な糖の部分を少なくとも含有する培養培地が、同時に、比較的低収率の総脂肪酸を生成する糖の部分を少なくとも含有する培養培地よりも、より多くのEPAおよびより少ないC16:0を含む組成物を生成することが可能であったことを示唆する。
この例は、そのn−3 HUFA画分に実質的にEPAのみを含み、かつ比較的低レベルのC14:0およびC16:0を含む微生物バイオマスプロセスを生成するための、微生物の従属栄養性もしくは主として従属栄養性のバッチ培養によって生成される組成物の製造を説明する。用いられる微生物はN.ラエビスである。
C16:0=19.3
C14:0=7.75
C22:6=2.97
この培養条件は1.254の脂肪酸収率も生じており、これは赤色光下のラクトースに次ぐほぼ最高記録であった。
この例は、まず第一にヒトもしくは動物の摂取に適するような十分な容量で生成することができる、EPAに富む組成物を含有する様々な製品を提供する。生成は微細藻類の培養によるものが最も実現可能である。EPAの合成をまかなう遺伝子の組がそれを有する生物から採取されて別の生物、例えば、植物もしくは動物に、該遺伝子の発現をまかなう遺伝子制御機構と共に合理的なレベルで移される場合、微細藻類をEPAの本来の源として頼る必要はもはやない。
この例は、実質的にEPAのみをそれらのn−3 HUFA成分として含有し、かつ比較的低いか、もしくは検出不能のレベルのC14:0およびC16:0を含有する補助食品を非水産養殖食料生成動物に給餌することによる、EPA食品の生成を説明する。実質的にEPAのみをそれらのn−3 HUFA成分として含有し、かつ比較的低いか、もしくは検出不能のレベルのC14:0およびC16:0を含有する組成物の非水産養殖食料生成動物への投与およびその結果生じる、望ましい改変脂肪酸プロフィールを有する新規食品の生成について説明する。
材料および方法
パイロット補足研究を着手するのにオークランド大学動物倫理委員会から倫理的許可を得た。この研究は、4頭のフリージアン乳牛の餌に1日量40から160のエチル−エイコサペンタエン酸(純度>98%)を9日間補給することを含んでいた。乳牛を、第1日目に160グラムのEPAを補給し、毎日15グラムずつ用量を減少させて第9日目に40グラムに到達する動物を含む高−低用量(HTLD)群(n=2)および第1日目に40グラムを補給し、毎日15グラムずつ用量を増加させて第9日目に160グラムに到達する動物を含む低−高用量(LTHD)群(n=2)に分割した。給餌およびミルクのサンプリングは、2004年8月19日から29日にウエストオークランドの酪農場で、その群れを世話した経験を有する監督獣医師の監督の下で行なった。補助食品はドレンチングガンにより、1日1回午前中に、搾乳に続いて与えた。毎日、補給に先立ち、ミルクの50ml試料を各動物から2本収集し、収集して1時間以内に100mlポリエチレン容器内で温度制御−20℃冷蔵庫において保存した。ミルク試料を解凍して脂肪酸を抽出し、分析用に用意した。乳脂肪を、ヘキサン中に溶解し、2モルレベルKOHメタノールと反応させることによってメチル化した。2mlのN−ヘキサンに溶解し、次いで20マイクロリットルの2モルレベルKOHメタノールを添加した20ミリグラムの脂肪を3秒間静置し、30秒間激しくボルテックスした。次に、その混合物を6分間静置した後、水中に2モルレベルのHClでメチルレッドに中和し、次いで再度ボルテックスして遠心分離した(Terry Knight publications) Knight T.W., et al., New Zealand Journal Of Agricultural Research 47:3: 287-297 SEP (2004年)にも報告される、agilentウェブサイトからの改変法)。次に、ヘキサンのアリコートを採取し、105メートル 0.25mm RTX2330 90%ビスシアノプロフィールカラム(Restek)および0.25ミクロン膜厚を有するGCFID6890(Agilent Corp)によって分析した。脂肪酸は37成分混合物(Suplco)との比較で保持時間によって同定した。理論的応答因子(AOCS参照)を用いてピーク面積をピーク応答に合わせて修正した。結果を曲線下面積として報告した。
結果および考察
オメガ−3脂肪酸、本研究において、エイコサペンタエン酸(C20:5 EPA)およびドコサペンタエン酸(C22:5 DPA)は、HTLD群においてはそれぞれ第2日および第3日に、LTHD群においては第5日および7日に基線から有意に増加した(図1および図2を参照)。総脂肪酸のパーセンテージ(%TFA)としてのEPAおよびDPAは、HTLD群においてはそれぞれ第5日(1.55)および6日(0.41)に、LTHD群においては第9日(1.29)および11日(0.38)にピークであった。DHAは基線では検出不能であって、この研究を通して不変のままであり、これはEPAからDHAへの変換が泌乳牛においては有意の程度までは生じない可能性を示唆する。従来の著者は、ヒトにおいて特定の食事条件の下で生じる、DHAをEPAに逆変換する代謝経路が、ウシ科動物においてはより大きな程度で起こり得ることを示唆していた(Barclayら、1998)が、Spainら(1995年)は十二指腸に注入した魚油がDHAよりも高レベルの血漿中EPAを導くことを報告しており、これはDHAが、ヒトと同様に、ミルク以外の幾つかの組織および細胞区画に優先的に捕捉され得ることを示唆する(Spain J.N., et al., J Dairy Sci 78:1142-1153 (1995年)を参照)。DHAがそのような区画から放出され、長期の時間スケールにわたってミルク中に蓄積されることは無視することができる。
この例は、EPAのみの乳製品の消費によるn−3 HUFAのヒト組織への組み込みを説明する。好ましい実施形態において、この方法は(i)比較的低レベルのミリスチン酸およびパルミチン酸のEPAのみの補助食品を用いる泌乳牛の補給によりEPAのみのミルクを生成し、(ii)該乳牛からミルクを採取し、(iii)該ミルクをヒトによる消費に適する乳製品(クリーム状ミルクおよび強化クリームを含む)に処理し、(iV)次いで、これらを、該乳製品の十分なn−3 HUFA成分がヒトによって吸収されて組織に組み込まれるように、ヒトが十分に長い期間にわたって消費することを含む。
強化食品の調製
1種類のクリーム状ミルク(クリーム状ミルク)および2種類の強化クリーム(強化クリームAおよび強化クリームB)を調製した。クリーム状ミルクは、ミルクを3つの20リットル容器内で一晩、4℃で静置した後、クリーム状ミルクを容器の頂部から抜き取り、合わせたクリーム状ミルクをホモジナイズすることによって調製した。該クリーム状ミルクを600ml遠心ボトル内で5分間、2000gで遠心分離し、強化クリームをボトルの頂部からすくい取り、該強化クリームをホモジナイズすることによって2種類の強化クリームをさらに調製した。次に、クリーム状ミルクおよび強化クリームを様々なn−3食品に組み込んだが、これにはバナナおよびイチゴをクリーム状ミルクおよび強化クリームと共に液状化することによって製造されたフルーツスムージー、強化クリームを既存のビーフシチューに混合することによって製造されたクリーム状ビーフシチュー、強化クリームをホイップし、既存のチョコレート・ログ・ケーキの頂部に乗せることによって調製されたホイップ強化クリームを有するチョコレート・ログ・ケーキが含まれる。その後、これらのn−3 HUFA強化食品を消費に利用可能なものとした。
乳脂肪分析
クリーム状ミルクおよび強化クリームからの試料を20℃で2〜3日間凍結保存した後、脂肪酸抽出のために解凍し、ガスクロマトグラフィーによって分析した。約30mg(20μL)の溶融脂肪(60℃)を15mL kimmax管に加え、2mlのヘプタンに溶解し、20μLの2M KOHメタノール溶液を添加することによって脂肪酸試料を分析用に調製した。その後、これらの試料を濁るまで30秒間ボルテックスし、5分間静置した。次に、それらの混合物を、25μLの2M HCLを添加することによって中和して赤色を生じさせ、十分に混合されるまで再度ボルテックスした後、3,000rpmで2分間遠心分離した。その後、試料の上部ヘプタン層をパスツールピペットで除去し、ガスクロマトグラフィー(GC)分析用にオートサンプラー・バイアルに入れた。GC分析は、炎イオン化検出器(GCFID6890、Agilent Corp)が装着され、105メートル 0.25mm 90%ビスシアノプロフィールカラム(RTX2330、Restek)を備え、0.25ミクロン膜厚を有するガスクロマトグラフィーにおいて行った。脂肪酸は37成分混合物(Suplco)との比較で保持時間によって同定した。理論的応答因子(AOCS参照)を用いてピーク面積をピーク応答に合わせて修正し、結果を曲線下面積として報告した。
血液分析
基準となる食後(通常の昼食の4時間後)および空腹時(絶食から10時間後)の血液試料を、補給開始の前、並びに補給開始後、それぞれ、24時間および39時間に採取した。血清試料は、血液を1時間凝固させた後、血液を1000gで15分間遠心分離し、血清を遠心管の頂部から抜き取ることによって調製した。赤血球(RBC)は、1000gで2分間遠心分離し、血清を抜き取った後、以下の方法、すなわち、細胞をpH7.4に調整したリン酸緩衝生理食塩水で洗浄した後、再度遠心分離して流体を抜き取ることをを2回繰り返して調製した。血清およびRBC試料を20℃で2〜3日間凍結させた後、脂肪酸抽出のために解凍し、ガスクロマトグラフィーによって分析した。
乳脂肪分析
脂質分析は、クリーム状ミルクが6.70重量%の総脂質を含み、強化クリーム1および2が、それぞれ、53.74重量%および49.85重量%の総脂質を含むことを示した。総乳脂肪酸のパーセンテージとしてのN−3 HUFA含有率は、クリーム状ミルク、強化クリーム1および2について、それぞれ、EPAが0.49、0.57および0.41%、DPAが0.12、0.12、および0.11%、並びにDHAが0.04、0.04および0.03%であった。クリーム状ミルク、並びに強化クリーム1および2において、それぞれ、C16:0は総脂肪酸の21.84、21.80および22.04%を構成し、かつCLAは2.06、2.21および2.17%を構成していた。当該技術分野において公知のように、脂質の80%が脂肪酸を含んでなるものと仮定した。
n−3 HUFA強化食品の消費
38歳の被験者は精製魚油カプセルの常用者であったが、この実験の開始の2週間前に補助食品の摂取を止めていた。この実験の過程で、被験者が魚および魚油もしくはオメガ−3補助食品を消費することはなかった。クリーム状ミルクおよび強化クリームを除いて、補給された食事で消費した残りのものも、葉の多い緑色野菜、亜麻仁油もしくは他のオメガ−3脂肪酸の豊富源を含まない、アルファ−リノレン酸(C18:3n−3)が比較的少ないものであった。補給の最初の24時間において、3キログラムのクリーム状ミルク、250グラムの強化クリーム1および400グラムの強化クリーム2を含む強化食品を消費した。24時間から30時間までの間に、さらに2.0キログラムのクリーム状ミルクおよびさらに400グラムの強化クリーム2を強化食品において消費した。
血液試料中の脂肪酸
基準時、絶食血清試料中の総脂肪酸のパーセンテージとしてのEPAおよびDPAおよびDHAは、補給後39時間に採取された血液からの血清における3.74、0.97および2.5と比較して、それぞれ、1.36、0.84および3.0であった。食後血清試料においては、EPAおよびDPAおよびDHAは基準時、それぞれ、総脂肪酸の1.44、0.90、3.50パーセントと測定され、それに対して補給後24時間に採取された試料においては2.23、0.72、および1.89パーセントであった。食事後赤血球においては、EPAおよびDPAおよびDHAは基準時、それぞれ、総脂肪酸の0.92、1.98および6.9パーセンテージと測定され、それに対して補給後24時間に採取された試料においては、それぞれ、1.55、3.01および8.39であった。血液中のCLAレベルは測定しなかった。
Claims (11)
- 脂肪酸全量のうち、
10%から50%のn−3 HUFA脂肪酸、
15%未満のミリスチン酸(C14:0)、
20%未満のパルミチン酸(C16:0)、
を含む組成物の製造方法であって、
前記組成物中のEPAのDHAに対する比が少なくとも6:1であり、前記組成物が、珪藻の、白色光照射工程を含む従属栄養性培養によって製造される、組成物の製造方法。 - 前記組成物が、8%未満のミリスチン酸を含む、請求項1に記載の製造方法。
- 前記珪藻の培養がニッチア・ラビエスの培養を含む、請求項1または請求項2に記載の製造方法。
- 前記特定量のn−3 HUFA脂肪酸、ミリスチン酸、およびパルミチン酸が、生物の成長が終止した後にさらに精製もしくは希釈を行うことなく得られる、請求項1〜請求項3のいずれか1項に記載の製造方法。
- ヒトまたは動物消費用の栄養補助食品として前記組成物を処方することを含む、請求項1〜請求項4のいずれか1項に記載の製造方法。
- 経口投与用の形態に前記組成物を処方することを含む、請求項1〜請求項4のいずれか1項に記載の製造方法。
- 動物性食品の製造方法であって、請求項1〜請求項4のいずれか1項に記載の製造方法により得られた組成物を、動物に対し、前記動物の選択された組織もしくは液体における脂質組成を変更するのに十分な量および時間で投与、もしくは供給する工程、並びに前記選択された動物組織または液体を食品として採取する工程を含む方法。
- 前記動物性食品がミルクベース製品である、請求項7に記載の方法。
- 前記ミルクベース食品が、バター、チーズ、チョコレート、カッテージチーズ、クリーム、ミルク、粉末化ミルク、コンデンスミルク、スキムミルク、アイスクリーム、ヨーグルトおよび幼児用処方物からなる群より選択されるものである、請求項8に記載の方法。
- 前記白色光が、毎秒1平方メートルあたり1〜10マイクロモル光子である、請求項1〜4のいずれか1項に記載の製造方法。
- 前記珪藻の培養がニッチアの培養を含む、請求項1または請求項2に記載の製造方法。
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