JP2005176851A - ドコサヘキサエン酸およびドコサヘキサエン酸を含む化合物 - Google Patents
ドコサヘキサエン酸およびドコサヘキサエン酸を含む化合物 Download PDFInfo
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- JP2005176851A JP2005176851A JP2005009518A JP2005009518A JP2005176851A JP 2005176851 A JP2005176851 A JP 2005176851A JP 2005009518 A JP2005009518 A JP 2005009518A JP 2005009518 A JP2005009518 A JP 2005009518A JP 2005176851 A JP2005176851 A JP 2005176851A
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- oil
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- glucose
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- Y10S435/946—Microorganisms using algae
Abstract
【解決手段】 (a)約106細胞/ml(0.5−1.0g乾燥重量/リットル)のC.cohniiを、約1/4から1/2の塩度の人工海水、1−8%グルコースおよび0.4−0.
8%酵母エキスを含む栄養溶液を初期に含む発酵槽に添加し;(b)C.cohniiを15℃
から34℃の温度および5.0から9.0のpHで培養し;(c)グルコースと酵母エキスを約56時間かけて漸次栄養溶液に添加し;(d)さらにグルコースを約16時間かけて栄養溶液に添加して、C.cohniiがオイルを生産するのを誘発し;(e)培養中を通して
溶解酸素含有量を空気飽和レベルの少なくとも約20%に維持するように、発酵槽を撹拌および通気し;そして(f)約60時間から約90時間後にC.cohniiを回収することか
らなる。
【選択図】 図1
Description
rine Dinoflagellates(海中渦鞭毛虫類の多不飽和脂肪酸)、J.
Protozoal,17:213-219(1970))。
も多量のC20オメガ−3−PUFAである。しかし、一般にDHAは乳幼児用調合乳には含まれていない。合衆国特許第4,670,285号はオメガ−3−脂肪酸含有の乳幼児用調合乳を開示している。しかし、そこで利用されている脂肪酸は卵あるいは魚(Talapia)油から得たもので、前述した好ましくない特性がある。さらに、魚油は一般に他のオメガ−3−脂肪酸であるエイコサペンタエン酸(EPA)を含み、これは持続性抗凝血作用と乳幼児のアラキドン酸濃度抑制のため乳幼児用調合乳に望ましくない成分である。これは乳幼児の体重増加率の減少と関連がある(Carlesonら、INFORM
1:306)。実際、母乳中EPA濃度は非常に低い(DHAの4分の1以下)。
本発明による方法で産生した食用オイルにはいやな味や魚臭がなく従来の供給源から得たDHA含有オイルによくみられる環境汚染物質も含まない。従って、本発明はさらに本発明のオイルを含有する食品も含む。
ecodiniumcohniiであり、これは増殖に還元炭素源を要する偏性有機栄養生物である。C.cohniiが好ましい理由は、十分量(全PUFA量の約1%以上)存在するPUFAがDHAのみであるという脂肪酸プロフィールを有するからである。この有機体の標本は、MK8840と称し、Rockville,Marylandの A
merican Type Culture Collection に寄託されており、受け入れ番号40750号である。ここで用いられる微生物、あるいは微生物の特異型には、野生株、変異株あるいは組換え型を含む。DHA含有オイルを高水準で産生する微生物
はすべて本発明の範囲内と考えられる。本発明の特徴の1つは渦鞭毛虫類等の微生物の食用オイル産生能力の認識と、それに伴うこのようなオイルの信頼できる経済的な供給源の維持の問題の解決である。従って、野性型微生物とDHA含有単細胞オイルを産生するよう設計された組換え微生物は本発明の一面である。このような組換え微生物には、同じ基質で、同じ野性型微生物が産生する量と比べて、単細胞オイル中のDHA量が多くなるように、あるいは総オイル量が多くなるように、あるいはその両方を意図して設計されたものが含まれる。また、さらに費用効果のすぐれた基質を効率よく用いて匹適する野性型と同量のDHA含有単細胞オイルを産生するよう設計された微生物も含まれる。
あると考えようとしなかった。従前の研究者は C.cohniiを首尾よく培養するに
は極めて複雑な栄養混合物が必要であると説明してきた。Goldら、Protozoal,13:255−257(1966);Guillardら、in“Dinoflagellates”,Academic Press(1984);Hendersonら、Phytochemistry27:1679−1683(1988)。これに対して、本発明はグルコースと酵母エキスを含む簡単な培地でDHA産生微生物の培養をなしとげる。これらの成分を海水等の溶液に用いて経済的に有意な増殖速度と細胞濃度が得られる。たとえば、3〜5日の発酵期間で、C.cohnii細胞濃度は少なくとも溶液1lあたりバイオマス10gで、一般には20から約40g/lに達しうる。このような濃度はこれまで到達されえなかった。
fermentor;STF)かエアリフト発酵槽(ALF)で有機体と増殖することが好ましく、いずれのタイプも当該技術分野専門家既知である。STFを選んだ場合は、Rushton型高効率タービンかピッチドブレードあるいはマリンインペラーを用いて撹拌する。撹拌と散布が微生物への酸素供給をくり返す。バイオマスの増加につれて、酸素要求量が増えるので、通常撹拌速度を上げる。翅端速度は約500cm/秒以下に、好ましくは約300cm/秒以下に保つことが望ましい。大きな翅端速度にも剪断をうけることなく耐えうる微生物株の選択は当該技術分野の範囲内である。このような株の使用は本発明に明らかに含まれる。
lである。従って30lの発酵槽では、生存能力のある細胞を1lあたり20g乾燥重量の濃度で含む種付け培養基1〜3lを添加する。
HA生合成には酸素が必要であるので、DHAの収率を上げるには空気飽和濃度の約10〜50%のD.0.濃度が必要である。翅端撹拌速度150〜200cm/秒と給気速度1VVM(1分あたりの空気体積/発酵槽体積)の組み合わせによりバイオマス濃度約25g乾燥重量/培養物1lで約20〜30%のD.0.濃度が提供できる。細胞濃度が高ければより高いD.0.濃度が必要となるが、これは02散布による給気速度の増加が発酵槽の
空気圧の上昇で達成できる。
6.0から約7.0のpH範囲を用いることが好ましい。接種前にはKOHやNaOH等の塩基を用いて培養基pHを調整する。発酵の後期には、培養基はアルカリ性になる傾向がある。必要であれば、増殖期中のアルカリ性を修正するために無機酸のpH調節剤を用いてもよい。渦鞭毛虫での単細胞オイルの産生は定常期の賦課(たとえば窒素枯渇やpH上昇)によって誘発される。YE欠乏は、利用できるグルコースが残存しているのに培養基がYEを使い果すようにYE供給量を制限することに起因する。本発明は、単細胞オイルの効率のよい産生を促進するものが、炭素源の窒素源に対する比であることを認めている。例としてグルコースとYEを用いると、好ましい炭素源と窒素源の比は、YE1に対してグルコース約10〜15である。他の炭素源と窒素源における同様の比は当該技術専門家により算出できる。
抽出可能なオイルから成る。株の選択によりこの比率を上げることができ、このような選択は本発明の範囲内である。本オイルは、一般に次の脂肪酸組成を有するトリグリセリドを約70%以上含むことが好ましい。
20〜25%パルミチン酸(C16:0)
10〜15%オレイン酸(C18:0)
30〜40%DHA(C22:6)
0〜10%その他(ホスファチジルコリン等の極性脂質を含む他のオイル成分もDHAに含まれることがある)
粗オイルの特性は黄澄色で室温で液体である。本オイルはDHAを少なくとも約20重量%含むことが望ましく、約35重量%以上含むことが最も望ましい。
ercritical F1uid Extraction,Butterworth,1986.に記載されている。抽出溶媒がへキサンである場合は、乾燥バイオマスに対するへキサンの適当な割合は、乾燥バイオマス1kgあたりへキサン約4lである。へキサンとバイオマスを撹拌反応槽に入れ約20〜50℃の温度で約2時間混合することが好ましい。混合後、バイオマスを濾過しオイルを含むへキサンから分離する。あるいは、湿性バイオマスペースト(固形分30〜35%)をエタノール、イソプロパノールあるいはへキサン/イソプロパノール混合物等のより極性の高い溶媒で直接抽出できる。残澄バイオマス、すなわち C.cohnii等の微生物の単細胞食用オイル抽出後のバイオマスは動物の飼料として使用できるがこれは約35〜40%のタンパク質、8〜10%の灰分、45〜50%の炭水化物を含む。タンパク質含有率が高くDHA濃度も高いことから、バイオマスペースト全体を水産養殖(例、エビ,カキ,魚)飼料として使用できる。
食品には魚油の持つ不快な特殊感覚器に印象を与える特性がない。従って食品は乳幼児にも成人にもより容易に認容される。本発明の乳幼児用調合乳は0.05重量%のDHA含有単細胞オイルを含むことが好ましい。さらに固形分の多い本発明のベビーフードは、約0.5重量%のDHA含有単細胞オイルを含むことが好ましい。いずれの場合でも、オイルが少なくとも約35%のDHAを含むことが最も好ましい。
作業容量30lのSTFに濃度2分の1の人工海水を入れた。IO6lを水道水18lと化合した。培養基の入った発酵槽を滅菌し28℃まで冷却した。濃縮YE(455g/l)400ml、グルコースシロップ(400g/l)900mlおよび約2×107細
胞/mlあるいはバイオマス20g/l(最終濃度は約7×106細胞/mlあるいはバ
イオマス約700mg/lになる)含有する種発酵槽からの接種物1lを培養基に加えた。撹拌は翅端速度120cm/秒に設定し給気は1VVM(30l/分)に設定した。30時間後にグルコースシロップ(900ml)を追加し、さらに次の42時間の間に4.2lを追加した。従って計6lのグルコースシロップを加えた。6時間後に濃縮YE溶液(400ml)を加え次の48時間の間にさらに1.2l追加し、計2.0l加えた。D.0.20%以上に維持するため24時間後に翅端速度を150cm/秒に、48時間後には160cm/秒に上げた。72時間後には翅端速度を200cm/秒に上げ、最後投入グルコースが細胞オイルに転化するに十分な時間をおいて培養物を増殖させた。培養条件を図1にグラフで示す。その後細胞球粒を保持して遠心分離により培養物を採収した。採収した細胞球粒を凍結乾燥して水分含有量約4%とした。へキサン(2.8l)を乾燥バイオマスに加えガラスケトルに入れ50℃で1.5時間撹拌した。回転蒸発装置を用いてへキサンを除去し、粗DHA含有オイル約175gを産生した。
作業容量350lのSTFにI.O.(登録商標)4.3kgと水道水230lを化合して作った濃度2分の1の人工海水培養基を入れた。培養基を入れた発酵槽を滅菌し28℃まで冷却した。濃縮YE(400g/l)6.8l、グルコースシロップ(400g/l)12.5lおよび種発酵槽(106 細胞/mlあるいはバイオマス濃度約1.3g/l)からの C.cohnii接種物30lを培養基に加えた。撹拌は翅端速度73cm/
秒に設定し給気は1VVM(280l/分)に設定した。約44時間後にグルコースシロップ(12l)を追加し次の32時間の間にさらに43lを加えた。従って、計67.5lのグルコースシロップを加えた。グルコース添加と細胞増殖を図2にグラフで表す。
げ、55時間後には225cm/秒に上げた。76時間後には翅端速度を150cm/秒に下げて、最終投入グルコースが細胞オイルに転化するに十分な時間をおいて培養物を増殖させた。その後培養物を採収した。採収した細胞を乾燥し水分含有量約4%とした。へキサンを乾燥バイオマスに加えてガラスケトルに入れ25℃で2時間撹拌した。回転蒸発装置を用いてへキサンを除去し、粗DHA含有オイル約700gを産生した。
作業容量30lのSTFにI.O.(登録商標)565gと水道水15lを化合して作った正規濃度の人工海水を入れた。培養基を入れた発酵槽を滅菌し28℃に冷却した。濃縮YE(400g/l)400ml、グルコースシロップ(400g/l)1.9l、および種発酵槽(106細胞/lあるいはバイオマス約20g/l)からのC.cohnii接種物1lを培養基に加えた。撹拌は翅端速度80cm/秒に設定し給気は1VVM(20l/分)に設定した。94時間後にグルコースシロップ(1.5l)を追加し116時間後にさらに1.1l追加した。従って、計4.5lのグルコースシロップを加えた。
上げた。66時間後に、定常期を導入したがこれを成しとげるために4NKOHでPHを7.0にスパイクし撹拌翅端速度は行程中さらには上げなかった。図3に示すように、最終投入グルコースが細胞オイルに転化するに十分な時間をおいて培養物を増殖させた。その後培養物を採収した。採収した細胞を乾燥し水分含有量約4%とした。乾燥バイオマスにへキサンを加えガラスケトルに入れ50℃で1.5時間撹拌した。回転蒸発装置を用いてへキサンを除去し、粗DHA含有オイル約65g産生した。
Claims (11)
- C.cohniiバイオマスから抽出された食用単細胞粗オイルであって、ドコサヘキサエン酸
(DHA)がオイルの少なくとも35%をなし、そしてオイルが70%を超えるトリグリセリドを含む、上記食用単細胞粗オイル。 - ヘキサン抽出によりC.cohniiバイオマスから得られた、請求項1記載の粗オイル。
- オイルが栄養溶液1リットルあたり少なくとも1.5グラムの濃度で存在する、請求項1記載の粗オイルを含む栄養溶液。
- 有意な量のエイコサペンタエン酸(EPA)を含まない、請求項1又は2記載の粗オイル。
- 請求項1又は2記載の粗オイルを追加の加工工程に供することにより得られる食用単細胞オイルを含む乳幼児用調合乳。
- 請求項1又は2記載の粗オイルを追加の加工工程に供することにより得られる食用単細胞オイルを含むベビーフード。
- 請求項1又は2記載の粗オイルを追加の加工工程に供することにより得られる食用単細胞オイルを含む食品。
- 請求項1又は2記載の粗オイルを追加の加工工程に供することにより得られる食用単細胞オイルを含む医薬品。
- 完全非経口栄養(TPN)の供給に適した,請求項8記載の医薬品。
- 請求項1又は2記載の粗オイルを追加の加工工程に供することにより得られる食用単細胞オイルを含む栄養補助食品。
- 栄養補助食品が上記オイルを封入するゼラチンカプセルの形態の中にある、請求項10記載の栄養補助食品。
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Cited By (3)
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WO2011013707A1 (ja) * | 2009-07-29 | 2011-02-03 | 味の素株式会社 | L-アミノ酸の製造法 |
JPWO2011013707A1 (ja) * | 2009-07-29 | 2013-01-10 | 味の素株式会社 | L−アミノ酸の製造法 |
US8771981B2 (en) | 2009-07-29 | 2014-07-08 | Ajinomoto Co., Inc. | Method for producing an L-amino acid |
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