JP4079978B2 - Lipids containing n-4 and / or n-7 polyunsaturated fatty acids and method for producing the same - Google Patents
Lipids containing n-4 and / or n-7 polyunsaturated fatty acids and method for producing the same Download PDFInfo
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- JP4079978B2 JP4079978B2 JP2006253323A JP2006253323A JP4079978B2 JP 4079978 B2 JP4079978 B2 JP 4079978B2 JP 2006253323 A JP2006253323 A JP 2006253323A JP 2006253323 A JP2006253323 A JP 2006253323A JP 4079978 B2 JP4079978 B2 JP 4079978B2
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- 238000004519 manufacturing process Methods 0.000 title claims description 14
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- CTMZJQAVRYEWHS-UHFFFAOYSA-N octadeca-8,11,14-trienoic acid Chemical compound CCCC=CCC=CCC=CCCCCCCC(O)=O CTMZJQAVRYEWHS-UHFFFAOYSA-N 0.000 claims description 12
- UCHYLNQKIYPHBX-ARQYDCTJSA-N 5,8,11,14-octadecatetraenoic acid Chemical compound CCC\C=C\C\C=C\C\C=C\C\C=C\CCCC(O)=O UCHYLNQKIYPHBX-ARQYDCTJSA-N 0.000 claims description 11
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、アラキドン酸生産能を有する微生物から得られた、パルミチン酸からステアリン酸への脂肪酸鎖長延長活性が低下又は欠損した微生物を培養することにより、もしくは、アラキドン酸生産能を有する微生物を、パルミトレイン酸もしくはその誘導体、又はこれらを構成成分として含有する油脂を添加した培地で培養することにより、培養物からｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質を採取すること、あるいは該脂質からｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有するトリグリセリド、又はｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を分離精製することを特徴とするｎ−４系又はｎ−７系高度不飽和脂肪酸、又はこれらを含有する脂質の製造方法、並びにｎ−４系又はｎ−７系高度不飽和脂肪酸を含有する脂質、及びこれらを含有する組成物に関する。 The present invention provides a microorganism having an ability to produce arachidonic acid, by culturing a microorganism obtained by reducing or lacking the fatty acid chain length-extending activity from palmitic acid to stearic acid obtained from a microorganism having an ability to produce arachidonic acid. , Palmitoleic acid or its derivatives, or lipids containing n-4 and / or n-7 polyunsaturated fatty acids are collected from the culture by culturing in a medium supplemented with fats and oils containing these as constituents Or triglyceride containing n-4 and / or n-7 polyunsaturated fatty acid, or n-4 and / or n-7 polyunsaturated fatty acid is separated and purified from the lipid. N-4 series or n-7 series highly unsaturated fatty acids, or a method for producing lipids containing these, and n-4 series or n-7 series highly unsaturated fats Lipids containing, and compositions containing them.
一般にｎ−４系又はｎ−７系高度不飽和脂肪酸とは、二重結合を２つ以上持ち、かつメチル基末端から数えて４番目又は７番目の炭素にメチル基側最初の二重結合を持つ脂肪酸である。ｎ−４系又はｎ−７系高度不飽和脂肪酸の二重結合は、通常生体内に存在するリノール酸やα−リノレン酸、アラキドン酸などのｎ−３系、ｎ−６系高度不飽和脂肪酸の二重結合とは異なった配置をとっており、これら通常の脂肪酸の作用と拮抗すると考えられるため、その生理作用が期待され、また、食品、化粧品、医薬品、動物飼料などへの応用が期待されている。 In general, n-4 series or n-7 series polyunsaturated fatty acids have two or more double bonds and the first double bond on the methyl group side at the 4th or 7th carbon from the end of the methyl group. It has fatty acids. Double bonds of n-4 or n-7 highly unsaturated fatty acids are usually n-3 series, n-6 series highly unsaturated fatty acids such as linoleic acid, α-linolenic acid, arachidonic acid and the like present in the living body. It is considered to antagonize the action of these normal fatty acids, and its physiological action is expected, and it is expected to be applied to foods, cosmetics, pharmaceuticals, animal feeds, etc. Has been.
実際、炭素数18以上のｎ−７系高度不飽和脂肪酸は必須脂肪酸の欠乏した動物体内に存在することが知られており、動物体内で様々な作用を持つと考えられている（Wolff RL "New trends in lipid and lipoprotein analyses" American Oil Chemists' Society, 147-180, 1995 ）。炭素数が16のｎ−４系又はｎ−７系高度不飽和脂肪酸は微生物や藻類に存在することが知られているが、炭素数が18以上のｎ−４系又はｎ−７系高度不飽和脂肪酸の供給源は上記動物が知られているのみで、上記動物の体内からの抽出は、供給の安定性や含量が低い点に問題があり、実用性は低かった。 In fact, n-7 polyunsaturated fatty acids having 18 or more carbon atoms are known to exist in animals lacking essential fatty acids and are considered to have various actions in animals (Wolff RL " New trends in lipid and lipoprotein analyzes "American Oil Chemists' Society, 147-180, 1995). It is known that n-4 or n-7 highly unsaturated fatty acids having 16 carbon atoms are present in microorganisms and algae, but n-4 or n-7 highly unsaturated fatty acids having 18 or more carbon atoms. The above animal is known only as the source of saturated fatty acid, and extraction from the body of the animal has a problem in that the supply stability and content are low, and its practicality is low.
このため、特に炭素数が18以上のｎ−４系又はｎ−７系高度不飽和脂肪酸、又はこれらを含有する脂質の製造方法、並びに炭素数が18以上のｎ−４系又はｎ−７系高度不飽和脂肪酸を含有する脂質又はトリグリセリド、及びこれらを含有する組成物の開発が強く望まれている。 Therefore, in particular, an n-4 series or n-7 series highly unsaturated fatty acid having 18 or more carbon atoms, or a method for producing a lipid containing these, and an n-4 or n-7 series having 18 or more carbon atoms Development of lipids or triglycerides containing polyunsaturated fatty acids and compositions containing them is strongly desired.
従って本発明は、炭素数が18以上のｎ−４系又はｎ−７系高度不飽和脂肪酸、もしくはこれらを含有する脂質又はトリグリセリドの製造方法、並びに炭素数が18以上のｎ−４系又はｎ−７系高度不飽和脂肪酸を含有する脂質又はトリグリセリド、及びこれらを含有する組成物を提供しようとするものである。 Therefore, the present invention provides a method for producing n-4 or n-7 polyunsaturated fatty acids having 18 or more carbon atoms, or lipids or triglycerides containing these, and n-4 or n-carbon having 18 or more carbon atoms. It is intended to provide a lipid or triglyceride containing a -7 series polyunsaturated fatty acid, and a composition containing these.
本発明者等は、上記の目的を達成するために種々研究した結果、アラキドン酸生産能を有する微生物を、パルミトレイン酸もしくはその誘導体、又はこれらを構成成分として含有する油脂を添加した培地で培養することにより、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質を生成することを見出した。さらに、アラキドン酸生産能を有する微生物からパルミチン酸からステアリン酸への脂肪酸鎖長延長活性が低下又は欠損した微生物を取得し、これを培養することにより、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質を生成することをも見出し、本発明を完成した。 As a result of various studies to achieve the above object, the inventors of the present invention cultivate a microorganism having an ability to produce arachidonic acid in a medium to which palmitoleic acid or a derivative thereof, or an oil containing these as a constituent component is added. By this, it discovered that the lipid containing an n-4 type | system | group and / or an n-7 type | system | group highly unsaturated fatty acid was produced | generated. Furthermore, by obtaining a microorganism in which the activity of extending the fatty acid chain length from palmitic acid to stearic acid is reduced or absent from a microorganism having the ability to produce arachidonic acid, and culturing this microorganism, the n-4 system and / or the n-7 system The inventors have also found that lipids containing polyunsaturated fatty acids can be produced, thereby completing the present invention.
すなわち本発明は、アラキドン酸生産能を有する微生物を、パルミトレイン酸もしくはその誘導体、又はこれらを構成成分として含有する油脂を添加した培地で培養し、そして培養物から脂質を採取することを特徴とするｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質の製造方法；並びに
アラキドン酸生産能を有する微生物から得られた、パルミチン酸からステアリン酸への脂肪酸鎖長延長活性が低下又は欠損した微生物を培養し、そして培養物から脂質を採取することを特徴とするｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質の製造方法を提供する。
That is, the present invention is characterized in that a microorganism having an ability to produce arachidonic acid is cultured in a medium to which palmitoleic acid or a derivative thereof, or an oil containing these as a constituent component is added, and lipids are collected from the culture. Method for producing lipids containing n-4 and / or n-7 polyunsaturated fatty acids; and reduced fatty acid chain extension activity from palmitic acid to stearic acid obtained from microorganisms capable of producing arachidonic acid Alternatively, the present invention provides a method for producing a lipid containing an n-4 series and / or an n-7 series highly unsaturated fatty acid, which comprises culturing a deficient microorganism and collecting a lipid from the culture.
本発明はさらに、脂質中の全脂肪酸に対して7,10,13,16−エイコサテトラエン酸の含量が1.5重量％以上である7,10,13,16−エイコサテトラエン酸を含有する脂質、
脂質中の全脂肪酸に対して5,8,11,14−オクタデカテトラエン酸の含量が0.5重量％以上である5,8,11,14−オクタデカテトラエン酸を含有する脂質、
脂質中の全脂肪酸に対して8,11,14−オクタデカトリエン酸の含量が0.1重量％以上である8 11,14−オクタデカトリエン酸を含有する脂質、
The present invention further comprises 7,10,13,16-eicosatetraenoic acid having a content of 7,10,13,16-eicosatetraenoic acid of 1.5% by weight or more based on the total fatty acids in the lipid. Lipids,
A lipid containing 5,8,11,14-octadecatetraenoic acid having a content of 5,8,11,14-octadecatetraenoic acid of 0.5% by weight or more based on the total fatty acids in the lipid;
A lipid containing 8,11,14-octadecatrienoic acid having a content of 8,11,14-octadecatrienoic acid of 0.1% by weight or more based on the total fatty acids in the lipid;
脂質中の全脂肪酸に対して炭素数が18以上のｎ−４系高度不飽和脂肪酸の含量が2.8重量％以上である炭素数が18以上のｎ−４系高度不飽和脂肪酸を含有する脂質、
脂質中の全脂肪酸に対して7,10,13−エイコサトリエン酸の含量が0.1重量%以上である7,10,13−エイコ7サトリエン酸を含有する脂質、
脂質中の全脂肪酸に対して5,8,11−オクタデカトリエン酸の含量が0.1重量％以上である5, 8,11−オクタデカトリエン酸を含有する脂質、
脂質中の全脂肪酸に対して8,11−オクタデカジエン酸の含量が0.1重量％以上である8, 11−オクタデカジエン酸を含有する脂質、
A lipid containing an n-4 highly unsaturated fatty acid having 18 or more carbon atoms, wherein the content of an n-4 highly unsaturated fatty acid having 18 or more carbon atoms is 2.8% by weight or more based on the total fatty acids in the lipid;
A lipid containing 7,10,13-eicosatrienoic acid having a content of 7,10,13-eicosatrienoic acid of 0.1% by weight or more based on the total fatty acids in the lipid;
A lipid containing 5,8,11-octadecatrienoic acid having a content of 5,8,11-octadecatrienoic acid of 0.1% by weight or more based on the total fatty acids in the lipid;
A lipid containing 8,11-octadecadienoic acid having a content of 8,11-octadecadienoic acid of 0.1% by weight or more based on the total fatty acids in the lipid;
脂質中の全脂肪酸に対して炭素数が18以上のｎ−７系高度不飽和脂肪酸の含量が０．３重量％以上である炭素数が18以上のｎ−７系高度不飽和脂肪酸を含有する脂質、
トリグリセリド中の全脂肪酸に対して7,10,13,16−エイコサテトラエン酸の含量が１．５重量％以上である7,10,13,16−エイコサテトラエン酸を含有するトリグリセリド、
トリグリセリド中の全脂肪酸に対して5,8,11,14−オクタデカテトラエン酸の含量が0.5重量％以上である5,8,11,14−オクタデカテトラエン酸を含有するトリグリセリド、
Contains n-7 polyunsaturated fatty acid having 18 or more carbon atoms and a content of n-7 polyunsaturated fatty acid having 18 or more carbon atoms with respect to the total fatty acids in the lipid. Lipids,
A triglyceride containing 7,10,13,16-eicosatetraenoic acid in which the content of 7,10,13,16-eicosatetraenoic acid is 1.5% by weight or more based on the total fatty acids in the triglyceride,
A triglyceride containing 5,8,11,14-octadecatetraenoic acid having a content of 5,8,11,14-octadecatetraenoic acid of 0.5% by weight or more based on the total fatty acids in the triglyceride,
トリグリセリド中の全脂肪酸に対して8,11,14−オクタデカトリエン酸の含量が0.1重量％以上である8,11,14−オクタデカトリエン酸を含有するトリグリセリド、
トリグリセリド中の全脂肪酸に対して炭素数が18以上のｎ−４系高度不飽和脂肪酸の含量が3.0重量％以上である炭素数が18以上のｎ−４系高度不飽和脂肪酸を含有するトリグリセリド、
トリグリセリド中の全脂肪酸に対して7,10,13−エイコサトリエン酸の含量が0.1重量％以上である7,10,13−エイコサトリエン酸を含有するトリグリセリド、
A triglyceride containing 8,11,14-octadecatrienoic acid having a content of 8,11,14-octadecatrienoic acid of 0.1% by weight or more based on the total fatty acids in the triglyceride,
A triglyceride containing an n-4 highly unsaturated fatty acid having 18 or more carbon atoms and a content of n-4 highly unsaturated fatty acids having 18 or more carbon atoms with respect to all fatty acids in the triglyceride,
A triglyceride containing 7,10,13-eicosatrienoic acid having a content of 7,10,13-eicosatrienoic acid of 0.1% by weight or more based on the total fatty acids in the triglyceride,
トリグリセリド中の全脂肪酸に対して5,8,11−オクタデカトリエン酸の含量が0.1重量％以上である5,8,11−オクタデカトリエン酸を含有するトリグリセリド、
トリグリセリド中の全脂肪酸に対して8,11−オクタデカジエン酸の含量が0.1重量％以上である8,11−オクタデカジエン酸を含有するトリグリセリド、及び
トリグリセリド中の全脂肪酸に対して炭素数が18以上のｎ−７系高度不飽和脂肪酸の含量が0.3重量％以上である炭素数が18以上のｎ−７系高度不飽和脂肪酸を含有するトリグリセリド、
並びにこれらの脂質又はトリグリセリドを含有する組成物を提供する。
A triglyceride containing 5,8,11-octadecatrienoic acid having a content of 5,8,11-octadecatrienoic acid of 0.1% by weight or more based on the total fatty acids in the triglyceride,
The triglyceride containing 8,11-octadecadienoic acid having a content of 8,11-octadecadienoic acid of 0.1% by weight or more based on the total fatty acids in the triglyceride, and the carbon number of all fatty acids in the triglyceride A triglyceride containing an n-7 polyunsaturated fatty acid having 18 or more carbon atoms and an n-7 polyunsaturated fatty acid content of 0.3% by weight or more,
As well as compositions containing these lipids or triglycerides.
本発明においてｎ−４系又はｎ−７系高度不飽和脂肪酸とは、炭素数が18以上で二重結合を２つ以上持ち、かつメチル基末端から数えて４番目又は７番目の炭素にメチル基側最初の二重結合を持つ脂肪酸であり、具体的には、ｎ−４系高度不飽和脂肪酸としては7,10,13,16−エイコサテトラエン酸や、5,8,11,14−オクタデカテトラエン酸、8,11,14−オクタデカトリエン酸が、ｎ−７系高度不飽和脂肪酸としては7,10,13−エイコサトリエン酸や、5,8,11−オクタデカトリエン酸、8,11−オクタデカジエン酸が挙げられる。 In the present invention, n-4 series or n-7 series polyunsaturated fatty acid means 18 or more carbon atoms, two or more double bonds, and methyl at the 4th or 7th carbon from the methyl group end. It is a fatty acid having the first double bond on the base side. Specifically, as an n-4 polyunsaturated fatty acid, 7,10,13,16-eicosatetraenoic acid, 5,8,11,14 -Octadecatetraenoic acid and 8,11,14-octadecatrienoic acid are 7,10,13-eicosatrienoic acid and 5,8,11-octadecateriene as n-7 polyunsaturated fatty acids Acid, and 8,11-octadecadienoic acid.
また本発明においては、アラキドン酸生産能を有する微生物であれば、すべて使用することができる。アラキドン酸生産能を有する微生物としては、モルティエレラ（Mortierella ）属、コニディオボラス（Conidiobolus）属、フィチウム（Pythium ）属、フィトフトラ（Phytophthora）属、ペニシリューム（Penicillium ）属、クラドスポリューム（Cladosporium）属、ムコール（Mucor ）属、フザリューム（Fusarium）属、アスペルギルス（Aspergillus ）属、ロードトルラ（Rhodotorula ）属、エントモフトラ（Entomophthora ）属、エキノスポランジウム（Echinosporangium）属、サプロレグニア（Saprolegnia ）属に属する微生物を挙げることができる。モルティエレラ（Mortierella ）属モルティエレラ（Mortierella ）亜属に属する微生物では、例えばモルティエレラ・エロンガタ（Mortierella elongata）、モルティエレラ・エキシグア（Mortierella exigua）、モルティエレラ・フィグロフィラ（Mortierella hygrophila）、モルティエレラ・アルピナ（Mortierella alpina）等を挙げることができる。 In the present invention, any microorganism having an ability to produce arachidonic acid can be used. The microorganisms capable of producing arachidonic acid include the genus Mortierella, Conidiobolus, Pythium, Phytophthora, Penicillium, and Cladosporium. , Belonging to the genus Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporangium, Saprolegnia Can do. For microorganisms belonging to the genus Mortierella (Mortierella), for example, Mortierella elongata, Mortierella exigua, Mortierella hygrophila, Mortierella alpina (Mortierella alpina).
具体的にはモルティエレラ・エロンガタ（Mortierella elongata）IFO8570 、モルティエレラ・エキシグア（Mortierella exigua）IFO8571 、モルティエレラ・フィグロフィラ（Mortierella hygrophila）IFO5941 、モルティエレラ・アルピナ（Mortierella alpina）IFO8568 、ATCC16266 、ATCC32221 、ATCC42430 、CBS219.35 、CBS224.37 、CBS250.53 、CBS343.66 、CBS527.72 、CBS529.72 、CBS608.70 、CBS754.68 等の菌株を挙げることができる。 More specifically, Mortierella elongata IFO8570, Mortierella exigua IFO8571, Mortierella hygrophila IFO5941, Mortierella alpina ATCC30 266, ATCC16 CBS219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS529.72, CBS608.70, CBS754.68 and the like can be mentioned.
これらの菌株はいずれも、大阪市の財団法人醗酵研究所（IFO ）、米国のアメリカン・タイプ・カルチャー・コレクション（American Type Culture Collection, ATCC）、又はCentrralbureau voor Schimmelcultures（CBS ）からなんら制限なく入手することができる。また本発明の研究グループが土壌から分離した菌株モルティエレラ・エロンガタSAM0219 （微工研菌寄第8703号）（微工研条寄第1239号）を使用することもできるが、これらの菌株に限定しているわけではない。これらのタイプカルチャーに属する菌株、あるいは自然界から分離した菌株をそのまま用いることができるが、増殖及び／又は単離を１回以上行うことによって得られる元の菌株とは性質の異なる自然突然変異株を用いることもできる。 All of these strains can be obtained without limitation from Osaka Institute of Fermentation (IFO), American Type Culture Collection (ATCC), or Centrralbureau voor Schimmelcultures (CBS). be able to. In addition, the strain Mortierella elongata SAM0219 (Mikken Kenyoku No. 8703) (Mikken Kenjyo No. 1239) isolated from soil by the research group of the present invention can also be used, but is limited to these strains. I'm not doing it. Although strains belonging to these type cultures or strains isolated from nature can be used as they are, natural mutant strains having different properties from the original strains obtained by performing growth and / or isolation once or more can be used. It can also be used.
さらに本発明において、アラキドン酸生産能を有する微生物から得られたパルミチン酸からステアリン酸への脂肪酸鎖長延長反応が低下又は欠損した微生物は、上記アラキドン酸生産能を有する微生物を用いて、例えば以下に記載する変異処理によって得ることができる。また、アラキドン酸生産能を有する微生物のパルミチン酸からステアリン酸への脂肪酸鎖長延長反応に関与する酵素の活性を低下または欠失させる遺伝子操作によっても得ることができる。その例として、該酵素をコードする遺伝子のノックアウトや、アンチセンスによる不活化などが挙げられるが、これらの方法に限定しているわけではない。 Furthermore, in the present invention, a microorganism in which the fatty acid chain length extension reaction from palmitic acid to stearic acid obtained from a microorganism having an arachidonic acid-producing ability is reduced or lost can be obtained by using, for example, the following microorganism having an arachidonic acid-producing ability. It can be obtained by the mutation treatment described in 1. It can also be obtained by genetic manipulation that reduces or eliminates the activity of the enzyme involved in the fatty acid chain lengthening reaction from palmitic acid to stearic acid in microorganisms having the ability to produce arachidonic acid. Examples thereof include knockout of a gene encoding the enzyme and inactivation by antisense, but are not limited to these methods.
さらに本発明には、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸の脂質又はトリグリセリド中の割合を全体的に高めるため、あるいは特定の脂肪酸の割合を高めるために、上記アラキドン酸生産能を有する微生物又は該微生物から得られるパルミチン酸からステアリン酸への脂肪酸鎖長延長反応が低下又は欠損した微生物に対して、変異処理や遺伝子操作を施すことによって得られる微生物の使用も包含される。その一例として、アラキドン酸生産能を有する微生物から得られるΔ１２不飽和化反応が低下又は欠損した微生物を使用することができる。 Furthermore, the present invention provides the above-mentioned arachidonic acid production in order to increase the ratio of n-4 series and / or n-7 series polyunsaturated fatty acids in the lipid or triglyceride as a whole, or to increase the ratio of specific fatty acids. The use of microorganisms obtained by performing mutation treatment or genetic manipulation on microorganisms having the ability to reduce or lack fatty acid chain extension reaction from palmitic acid to stearic acid obtained from the microorganisms is also included. . As an example, a microorganism having a reduced or deficient Δ12 desaturation reaction obtained from a microorganism capable of producing arachidonic acid can be used.
すなわち該微生物をパルミトレイン酸もしくはその誘導体、又はこれらを構成成分として含有する油脂を添加した培地で培養することにより、ｎ−４系高度不飽和脂肪酸の割合が少ないか又は含有しない、ｎ−７系高度不飽和脂肪酸含有脂質を得ることができる。またこのΔ１２不飽和化反応が低下又は欠損した微生物にさらに変異処理や遺伝子操作を施して得られるパルミチン酸からステアリン酸への脂肪酸鎖長延長反応が低下又は欠損した微生物を使用することによっても、ｎ−４系高度不飽和脂肪酸の割合が少ないか又は含有しない、ｎ−７系高度不飽和脂肪酸含有脂質を得ることができる。 That is, by culturing the microorganism in a medium to which palmitoleic acid or a derivative thereof, or an oil containing these as a constituent component is added, the ratio of n-4 highly polyunsaturated fatty acids is small or not contained, n-7 A highly unsaturated fatty acid-containing lipid can be obtained. In addition, by using a microorganism in which the fatty acid chain length extension reaction from palmitic acid to stearic acid obtained by further performing mutation treatment or genetic manipulation on a microorganism in which this Δ12 desaturation reaction is reduced or missing is reduced or absent, It is possible to obtain an n-7 polyunsaturated fatty acid-containing lipid in which the ratio of the n-4 polyunsaturated fatty acid is small or not contained.
また、Δ６不飽和化反応が強化された微生物を用いることにより、ｎ−７系高度不飽和脂肪酸の割合を高めることが可能であり、あるいはΔ１２不飽和化反応が強化された微生物を用いることにより、ｎ−４系高度不飽和脂肪酸の割合を高めることが可能である。
本発明において変異処理は、放射線（Ｘ線、ガンマー線、中性子線）照射や紫外線照射、高熱処理等を行ったり、また微生物を適当なバッファー中などに懸濁し、変異原を加えて一定時間インキュベート後、適当に希釈して寒天培地に植菌し、変異株のコロニーを得るといった一般的な突然変異操作を行うこともできる。
Further, by using a microorganism with enhanced Δ6 desaturation reaction, it is possible to increase the ratio of n-7 highly unsaturated fatty acid, or by using a microorganism with enhanced Δ12 desaturation reaction. It is possible to increase the ratio of n-4 polyunsaturated fatty acids.
In the present invention, the mutation treatment is performed by irradiation with radiation (X-ray, gamma ray, neutron beam), ultraviolet irradiation, high heat treatment, etc., or suspending microorganisms in an appropriate buffer, etc., adding the mutagen and incubating for a certain time. Thereafter, a general mutation operation such as appropriately diluting and inoculating the agar medium to obtain a mutant colony can also be performed.
変異原としては、ナイトロジェンマスタード、メチルメタンサルホネートやN-メチル-N'-ニトロ-N- ニトロソグアニジン（NTG ）等にアルキル化剤、5-ブロモウラシル等の塩基類似体、マイトマイシンC 等の抗生物質、6-メルカプトプリン等の塩基合成阻害剤、プロフラビン等の色素類、4-ニトロキノリン-N- オキシド等のある種の発がん剤、塩化マンガン、ホルムアルデヒド等の化合物を挙げることができる。また、使用する微生物は、生育菌体（菌糸）でもよいし、胞子でもよい。 Mutagens include nitrogen mustard, methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), alkylating agents, base analogs such as 5-bromouracil, mitomycin C, etc. Examples include antibiotics, base synthesis inhibitors such as 6-mercaptopurine, pigments such as proflavine, certain carcinogens such as 4-nitroquinoline-N-oxide, and compounds such as manganese chloride and formaldehyde. The microorganism used may be a growing cell (mycelium) or a spore.
例えば本発明の変異株として、アラキドン酸生産能を有するモルティエレラ・アルピナIFO8568 から本発明者らが誘導した，脂肪酸鎖長延長活性が低下したモルティエレラ・アルピナSAM 2268（FERM P-17762）を使用することができるが、この菌株に限定しているわけでなく、アラキドン酸生産能を有し脂肪酸鎖長延長活性が低下又は欠損した菌株をすべて使用することができる。 For example, as a mutant strain of the present invention, Mortierella alpina SAM 2268 (FERM P-17762) having a reduced fatty acid chain length-inducing activity derived from Mortierella alpina IFO8568 capable of producing arachidonic acid was used. However, the present invention is not limited to this strain, and all strains that have the ability to produce arachidonic acid and have reduced or lacked fatty acid chain length extension activity can be used.
本発明に使用される菌株を培養する為には、その菌株の胞子、菌糸、又は予め培養して得られた前培養液を、液体培地又は固体培地に接種し培養する。液体培地の場合に、炭素源としてはグルコース、フラクトース、キシロース、サッカロース、マルトース、可溶性デンプン、糖蜜、グリセロール、マンニトール等の一般的に使用されているものが、いずれも使用できるが、これらに限られるものではない。 In order to culture the strain used in the present invention, the spore, mycelium of the strain or a preculture obtained by culturing in advance is inoculated into a liquid medium or a solid medium and cultured. In the case of a liquid medium, commonly used carbon sources such as glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, etc. can be used, but are not limited to these. It is not a thing.
窒素源としてはペプトン、酵母エキス、麦芽エキス、肉エキス、カザミノ酸、コーンスティープリカー、大豆タンパク、脱脂ダイズ、綿実カス等の天然窒素源の他に、尿素等の有機窒素源、ならびに硝酸ナトリウム、硝酸アンモニウム、硫酸アンモニウム等の無機窒素源を用いることができる。この他必要に応じリン酸塩、硫酸マグネシウム、硫酸鉄、硫酸銅等の無機塩及びビタミン等も微量栄養源として使用できる。 Nitrogen sources include natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor, soy protein, defatted soybean, cottonseed residue, etc., organic nitrogen sources such as urea, and sodium nitrate Inorganic nitrogen sources such as ammonium nitrate and ammonium sulfate can be used. In addition, inorganic salts such as phosphate, magnesium sulfate, iron sulfate, and copper sulfate, vitamins, and the like can be used as a trace nutrient source as necessary.
これらの培地成分は微生物の生育を害しない濃度であれば特に制限はない。実用上一般に、炭素源は0.1〜40重量% 、好ましくは１〜２５重量% の濃度とするのが良い。炭素源は培養途中に逐次流加しても構わない。又、初発の窒素源添加量は0.1〜10重量% 、好ましくは0.1〜６重量% とし、培養途中に窒素源を流加しても構わない。 These medium components are not particularly limited as long as they do not impair the growth of microorganisms. In practice, the carbon source is generally 0.1 to 40% by weight, preferably 1 to 25% by weight. The carbon source may be fed sequentially during the culture. The initial nitrogen source addition amount is 0.1 to 10% by weight, preferably 0.1 to 6% by weight, and the nitrogen source may be fed during the cultivation.
本発明の微生物の培養温度は使用する微生物によりことなるが、５〜40℃、好ましくは10〜30℃とし、培地のpHは4 〜10、好ましくは５〜9 として通気攪拌培養、振盪培養、又は静置培養を行う。培養は通常2〜30日間、好ましくは5〜20日間、より好ましくは5 〜15日間行う。
固体培養で培養する場合は、固形物重量に対して50〜100 重量% の水を加えたふすま、もみがら、米ぬか等を用い、５〜40℃、好ましくは10〜30℃の温度において、3 〜14日間培養を行う。この場合に必要に応じて培地中に窒素源、無機塩類、微量栄養源を加えることができる。
The culture temperature of the microorganism of the present invention varies depending on the microorganism to be used, but it is 5 to 40 ° C., preferably 10 to 30 ° C., and the pH of the medium is 4 to 10, preferably 5 to 9. Alternatively, static culture is performed. The culture is usually performed for 2 to 30 days, preferably 5 to 20 days, more preferably 5 to 15 days.
When cultivating in solid culture, use bran, rice bran, rice bran, etc. to which 50 to 100% by weight of water has been added based on the weight of the solid, and use a temperature of 5 to 40 ° C, preferably 10 to 30 ° C. Incubate for ~ 14 days. In this case, a nitrogen source, inorganic salts, and a trace nutrient source can be added to the medium as necessary.
本発明においては、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸の生成量を高めるために、テトラデカン、ヘキサデカン等の炭化水素、テトラデカン酸、ヘキサデカン酸等の脂肪酸又はその塩（例えばナトリウム塩、カリウム塩等）及びエステル、又は該脂肪酸が構成成分として含まれる油脂（例えば、ヤシ油、パーム核油）等を基質として添加することができる。またアラキドン酸生産能を有する微生物を用いる場合には、前駆体としてパルミトレイン酸もしくはその誘導体、又はこれらを構成成分として含有する油脂を添加する。これら基質又は前駆体それぞれの総添加量は培地に対して0.001 〜10重量% 、好ましくは0.5 〜10重量% である。 In the present invention, a hydrocarbon such as tetradecane and hexadecane, a fatty acid such as tetradecanoic acid and hexadecanoic acid, or a salt thereof (for example, sodium) in order to increase the production amount of n-4 series and / or n-7 series highly unsaturated fatty acid. Salts, potassium salts and the like) and esters, or fats and oils (for example, coconut oil and palm kernel oil) containing the fatty acid as a constituent component can be added as a substrate. When using a microorganism having an ability to produce arachidonic acid, palmitoleic acid or a derivative thereof, or an oil containing these as a constituent component is added as a precursor. The total amount of each of these substrates or precursors is 0.001 to 10% by weight, preferably 0.5 to 10% by weight, based on the medium.
これらの添加物は生産微生物を接種する前又はその直後に加えてもよく、又は培養を開始した後に加えてもよく、あるいは両時点で加えてもよい。培養開始後の添加は1 回でもよく、又は複数回に分けて間欠的に添加してもよい。あるいは、連続的に添加することもできる。又、これらの添加物を唯一の炭素源として培養してもよい。
また、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸含有脂質又はトリグリセリドを商品化が可能な収率で得るには、液体培地を用い、通気撹拌培養が好ましく、通常の撹拌式発酵槽、あるいは気泡塔型培養装置を使用することもできる。通気量としては0.1〜３ vvmが、撹拌速度としては10〜500 rpmが望ましい。
These additives may be added before or immediately after inoculation with the production microorganism, or may be added after the start of culture, or may be added at both time points. The addition after the start of the culture may be performed once, or may be added intermittently divided into a plurality of times. Alternatively, it can be added continuously. Alternatively, these additives may be cultured as the sole carbon source.
In addition, in order to obtain n-4 series and / or n-7 series highly unsaturated fatty acid-containing lipids or triglycerides in a yield that can be commercialized, aeration stirring culture is preferable using a liquid medium, and normal stirring fermentation is used. A tank or a bubble column type culture apparatus can also be used. The aeration rate is preferably 0.1-3 vvm, and the stirring speed is preferably 10-500 rpm.
このように培養して、菌体内にｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質が生成蓄積される。液体培地を使用した場合には、菌体培養によって脂質を製造する途中の培養液もしくはその殺菌した培養液、または培養終了時の培養液もしくはその殺菌した培養液、またはそれぞれから集菌した培養菌体もしくはその乾燥物からｎ−４系及び／又はｎ−７系高度不飽和脂肪酸含有脂質を採取する。例えば培養菌体からは次のようにしてｎ−４系及び／又はｎ−７系高度不飽和脂肪酸含有脂質の採取、および該脂質からｎ−４系及び／又はｎ−７系高度不飽和脂肪酸含有トリグリセリド又はｎ−４系及び／又はｎ−７系高度不飽和脂肪酸の単離を行う。 By culturing in this way, lipids containing n-4 and / or n-7 polyunsaturated fatty acids are produced and accumulated in the cells. When a liquid medium is used, a culture solution in the middle of producing lipids by cell culture or a sterilized culture solution thereof, a culture solution at the end of the culture or a sterilized culture solution thereof, or a culture collected from each The n-4 series and / or n-7 series highly unsaturated fatty acid-containing lipid is collected from the body or its dried product. For example, n-4 and / or n-7 highly unsaturated fatty acid-containing lipids are collected from cultured cells as follows, and n-4 and / or n-7 highly unsaturated fatty acids are collected from the lipid. The contained triglycerides or n-4 and / or n-7 polyunsaturated fatty acids are isolated.
培養終了後、培養液より遠心分離法や濾過等の常用の固液分離手段により培養菌体を得る。菌体は十分水洗し、好ましくは乾燥する。乾燥は凍結乾燥、風乾等によって行うことができる。乾燥菌体は、例えばダイノミルや超音波などにより破砕した後、好ましくは窒素気流下で有機溶媒によって抽出処理する。有機溶媒としてはエーテル、ヘキサン、メタノール、エタノール、クロロホルム、ジクロロメタン、石油エーテル等を用いることができ、又メタノールと石油エーテルの交互抽出やクロロホルム- メタノール- 水の一層系の溶媒を用いた抽出によっても良好な結果を得ることができる。抽出物から減圧下で有機溶媒を留去することにより、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有した脂質が得られる。 After completion of the culture, cultured cells are obtained from the culture solution by conventional solid-liquid separation means such as centrifugation or filtration. The cells are washed thoroughly with water and preferably dried. Drying can be performed by freeze drying, air drying, or the like. The dried cells are crushed by, for example, dynomill or ultrasonic waves, and then extracted with an organic solvent, preferably under a nitrogen stream. As the organic solvent, ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, etc. can be used. Alternatively, alternate extraction of methanol and petroleum ether and extraction using a one-layer solvent of chloroform-methanol-water can also be used. Good results can be obtained. By distilling off the organic solvent from the extract under reduced pressure, a lipid containing an n-4 series and / or an n-7 series highly unsaturated fatty acid is obtained.
また上記の方法に代えて湿菌体を用いて抽出をおこなうことができる。この場合にはメタノール、エタノール等の水に対して相溶性の溶媒、又はこれらと水及び／又は他の溶媒とからなる水に対して相溶性の混合溶媒を使用する。その他の手順は上記と同様である。
上記のようにして得られた脂質中には、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸が、トリグリセリド、ジグリセリド、モノグリセリド、ステロールエステルなどの中性脂質や、フォスファチジルコリン、リゾフォスファチジルコリン、フォスファチジルエタノールアミン、リゾフォスファチジルエタノールアミン、フォスファチジルイノシトール、リゾフォスファチジルイノシトール、フォスファチジルセリン、リゾフォスファチジルセリン、フォスファチジン酸、リゾフォスファチジン酸などの極性脂質の構成成分として、あるいは遊離脂肪酸として存在している。
Moreover, it can replace with said method and can extract using a wet cell. In this case, a solvent compatible with water such as methanol and ethanol, or a mixed solvent compatible with water composed of these and water and / or other solvents is used. Other procedures are the same as described above.
Among the lipids obtained as described above, n-4 and / or n-7 polyunsaturated fatty acids are neutral lipids such as triglycerides, diglycerides, monoglycerides, sterol esters, phosphatidylcholines, Lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, lysophosphatidylinositol, phosphatidylserine, lysophosphatidylserine, phosphatidic acid, lysophosphatidine It exists as a constituent of polar lipids such as acids or as free fatty acids.
例えばモルティエレラ亜属に属する微生物を用いて製造されるｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質の油脂組成としては、中性脂質が70〜100重量％、極性脂質が０〜30重量％であり、中性脂質の主な成分であるトリグリセリドは70〜99重量％である。また該脂質の脂肪酸組成としては、脂質中の全脂肪酸に対して、
7,10,13,16−エイコサテトラエン酸が1.5〜70重量％、好ましくは2.0〜70重量％、より好ましくは5.0〜70重量％、
For example, the fat and oil composition of lipids containing n-4 series and / or n-7 series highly unsaturated fatty acids produced using microorganisms belonging to the genus Mortierella are neutral lipids of 70-100% by weight, polar The lipid is 0 to 30% by weight, and the triglyceride which is the main component of the neutral lipid is 70 to 99% by weight. The fatty acid composition of the lipid is based on the total fatty acids in the lipid.
7,10,13,16-eicosatetraenoic acid is 1.5 to 70% by weight, preferably 2.0 to 70% by weight, more preferably 5.0 to 70% by weight,
5,8,11,14−オクタデカテトラエン酸が0.5〜60重量％、好ましくは0.6〜60重量％、より好ましくは1.0〜60重量％、
8,11,14−オクタデカトリエン酸が0.1〜60重量％、好ましくは0.5〜60重量％、より好ましくは0.7〜60重量％、
7,10,13−エイコサトリエン酸が0.1〜60重量％、好ましくは0.4〜60重量％、より好ましくは0.5〜60重量％、
5,8,11−オクタデカトリエン酸が0.1〜60重量％、好ましくは0.2〜60重量％、より好ましくは0.3〜60重量％、
8,11−オクタデカジエン酸が0.1〜60重量％、好ましくは0.2〜60重量％、より好ましくは0.3〜60重量％、
5,8,11,14-octadecatetraenoic acid is 0.5-60% by weight, preferably 0.6-60% by weight, more preferably 1.0-60% by weight,
8,11,14-octadecatrienoic acid is 0.1 to 60% by weight, preferably 0.5 to 60% by weight, more preferably 0.7 to 60% by weight,
7,10,13-eicosatrienoic acid is 0.1-60% by weight, preferably 0.4-60% by weight, more preferably 0.5-60% by weight,
5,8,11-octadecatrienoic acid is 0.1 to 60% by weight, preferably 0.2 to 60% by weight, more preferably 0.3 to 60% by weight,
8,11-octadecadienoic acid is 0.1-60 wt%, preferably 0.2-60 wt%, more preferably 0.3-60 wt%,
また脂質中のトリグリセリド中の全脂肪酸に対して、
7,10,13 16−エイコサテトラエン酸が1.5〜70重量％、好ましくは4.8〜70重量％、より好ましくは7.8〜70重量％、
5,8,11,14−オクタデカテトラエン酸が0.5〜60重量％、好ましくは1.0〜60重量％、より好ましくは2.1〜60重量％、
Also, for all fatty acids in triglycerides in lipids,
7,10,13 16-eicosatetraenoic acid is 1.5 to 70% by weight, preferably 4.8 to 70% by weight, more preferably 7.8 to 70% by weight,
5,8,11,14-octadecatetraenoic acid is 0.5-60% by weight, preferably 1.0-60% by weight, more preferably 2.1-60% by weight,
8,11,14−オクタデカトリエン酸が0.1〜60重量％、好ましくは0.5〜60重量％、より好ましくは0.8〜60重量％、
7, 10, 13−エイコサトリエン酸が0.1〜60重量％、好ましくは0.5〜60重量％、より好ましくは1.1〜60重量％、
5,8,11−オクタデカトリエン酸が0.1〜60重量％、好ましくは0.2〜60重量％、
8,11−オクタデカジエン酸が0.1〜60重量％、好ましくは0.2〜60重量％である。
8,11,14-octadecatrienoic acid is 0.1 to 60% by weight, preferably 0.5 to 60% by weight, more preferably 0.8 to 60% by weight,
7, 10, 13-eicosatrienoic acid is 0.1-60 wt%, preferably 0.5-60 wt%, more preferably 1.1-60 wt%,
5,8,11-octadecatrienoic acid is 0.1 to 60% by weight, preferably 0.2 to 60% by weight,
8,11-octadecadienoic acid is 0.1 to 60% by weight, preferably 0.2 to 60% by weight.
なお本発明においては、
脂質中の全脂肪酸に対して7,10,13,16−エイコサテトラエン酸の含量が1.5重量％以上、好ましくは2.0重量％以上、より好ましくは5.0重量％以上である7,10,13,16−エイコサテトラエン酸を含有する脂質、
脂質中の全脂肪酸に対して5,8,11,14−オクタデカテトラエン酸の含量が0.5重量％以上、好ましくは0.6重量％以上、より好ましくは1.0重量％以上である5,8,11,14−オクタデカテトラエン酸を含有する脂質、
In the present invention,
The content of 7,10,13,16-eicosatetraenoic acid is 1.5% by weight or more, preferably 2.0% by weight or more, more preferably 5.0% by weight or more based on the total fatty acids in the lipid. , 16-lipid containing eicosatetraenoic acid,
The content of 5,8,11,14-octadecatetraenoic acid is 0.5% by weight or more, preferably 0.6% by weight or more, more preferably 1.0% by weight or more based on the total fatty acids in the lipid. , 14-octadecatetraenoic acid containing lipids,
脂質中の全脂肪酸に対して8,11,14−オクタデカトリエン酸の含量が0.1重量％以上、好ましくは0.5重量％以上、より好ましくは0.7重量％以上である8,11,14−オクタデカトリエン酸を含有する脂質、
脂質中の全脂肪酸に対して炭素数が18以上のｎ−４系高度不飽和脂肪酸の含量が2.8重量％以上、好ましくは3.5重量％以上、より好ましくは6.8重量％以上である、炭素数が18以上のｎ−４系高度不飽和脂肪酸を含有する脂質、
脂質中の全脂肪酸に対して7,10,13−エイコサトリエン酸の含量が0.1重量％以上、好ましくは0.4重量％以上、より好ましくは0.5重量％以上である、7,10,13−エイコサトリエン酸を含有する脂質、
8,11,14-octadecataenoic acid having a content of 8,11,14-octadecatrienoic acid of 0.1% by weight or more, preferably 0.5% by weight or more, more preferably 0.7% by weight or more based on the total fatty acids in the lipid Lipids containing trienoic acid,
The content of n-4 highly unsaturated fatty acids having 18 or more carbon atoms with respect to all fatty acids in the lipid is 2.8% by weight or more, preferably 3.5% by weight or more, more preferably 6.8% by weight or more, A lipid containing 18 or more n-4 polyunsaturated fatty acids,
The 7,10,13-eicosatrienoic acid content with respect to the total fatty acids in the lipid is 0.1% by weight or more, preferably 0.4% by weight or more, more preferably 0.5% by weight or more. Lipids containing satrienoic acid,
脂質中の全脂肪酸に対して5,8,11−オクタデカトリエン酸の含量が0.1重量％以上、好ましくは0.2重量％以上、より好ましくは0.3重量％以上である5,8,11−オクタデカトリエン酸を含有する脂質、
脂質中の全脂肪酸に対して8,11−オクタデカジエン酸の含量が0.1重量％以上、好ましくは0.2重量％以上、より好ましくは0.3重量％以上である8,11−オクタデカジエン酸を含有する脂質、あるいは
脂質中の全脂肪酸に対して炭素数が18以上のｎ−７系高度不飽和脂肪酸の含量が0.3重量％以上、好ましくは0.9重量％以上、より好ましくは1.2重量％以上である炭素数が18以上のｎ−７系高度不飽和脂肪酸を含有する脂質、
を得ることができる。
5,8,11-octadecataic acid having a content of 5,8,11-octadecatrienoic acid of 0.1% by weight or more, preferably 0.2% by weight or more, more preferably 0.3% by weight or more based on the total fatty acids in the lipid Lipids containing trienoic acid,
Contains 8,11-octadecadienoic acid with a content of 8,11-octadecadienoic acid of 0.1% by weight or more, preferably 0.2% by weight or more, more preferably 0.3% by weight or more based on the total fatty acids in the lipid The content of the n-7 polyunsaturated fatty acid having 18 or more carbon atoms with respect to the total fatty acid in the lipid or the lipid is 0.3 wt% or more, preferably 0.9 wt% or more, more preferably 1.2 wt% or more A lipid containing an n-7 polyunsaturated fatty acid having 18 or more carbon atoms,
Can be obtained.
なお、上記の脂質とは、その分子内で高級脂肪酸が何らかの化学結合、代表的にはエステル結合を形成した、水に不溶でかつアルコール、クロロホルム、ベンゼンなどの有機溶媒に可溶な物質を言う。ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有した脂質の例としては、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸の低級アルキルエステル、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を構成成分として含むグリセリンエステル又はステロールエステル、及びこれらのうちの任意の２種以上の混合物などが挙げられる。 The above-mentioned lipid means a substance in which a higher fatty acid forms some chemical bond in the molecule, typically an ester bond, and is insoluble in water and soluble in organic solvents such as alcohol, chloroform and benzene. . Examples of lipids containing n-4 and / or n-7 highly unsaturated fatty acids include n-4 and / or n-7 highly unsaturated fatty acids, n-4 and / or n-7. Lower alkyl esters of highly polyunsaturated fatty acids, glycerol esters or sterol esters containing n-4 and / or n-7 highly unsaturated fatty acids as components, and mixtures of any two or more of these Can be mentioned.
上記ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸の低級アルキルエステルとは、炭素数１〜６個、好ましくは１〜４個、より好ましくは１〜３個の低級アルコールとｎ−４系及び／又はｎ−７系高度不飽和脂肪酸のエステルを言う。また、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を構成成分として含むグリセリンエステルとは、グリセリン１分子に対して、少なくとも１分子のｎ−４系及び／又はｎ−７系高度不飽和脂肪酸がエステル結合した物質を言う。 The lower alkyl ester of the n-4 series and / or n-7 series highly unsaturated fatty acid is a C1-C6, preferably 1-4, more preferably 1-3 lower alcohol and n-. Refers to esters of 4- and / or n-7 polyunsaturated fatty acids. Moreover, the glycerol ester which contains n-4 type | system | group and / or n-7 type | system | group highly unsaturated fatty acid as a structural component is at least 1 molecule | numerator n-4 type | system | group and / or n-7 type | system | group high degree with respect to 1 molecule of glycerol. A substance in which unsaturated fatty acids are ester-bonded.
その例として、トリグリセリド、ジグリセリド、モノグリセリド、フォスファチジルコリン、リゾフォスファチジルコリン、フォスファチジルエタノールアミン、リゾフォスファチジルエタノールアミン、フォスファチジルイノシトール、リゾフォスファチジルイノシトール、フォスファチジルセリン、リゾフォスファチジルセリン、フォスファチジン酸、リゾフォスファチジン酸、グリセロ糖脂質などが挙げられる。 Examples include triglycerides, diglycerides, monoglycerides, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, lysophosphatidylinositol, phosphatidylserine, Examples include lysophosphatidylserine, phosphatidic acid, lysophosphatidic acid, and glyceroglycolipid.
また、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を構成成分として含むステロールエステルとは、ステロールとｎ−４系及び／又はｎ−７系高度不飽和脂肪酸がエステル結合した物質を言う。その例として、コレステロールエステル、デスモステロールエステルなどが挙げられる。
ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有した脂質は以上の例に限定されず、スフィンゴリン脂質、他のリン脂質、セラミド、スフィンゴ糖脂質、他の糖脂質など、上記の定義に包含される任意の脂質を含む。
Moreover, the sterol ester which contains n-4 type and / or n-7 type | system | group highly unsaturated fatty acid as a structural component is the substance which the sterol and the n-4 type | system | group and / or n-7 type | system | group highly unsaturated fatty acid ester-bonded. To tell. Examples thereof include cholesterol esters and desmosterol esters.
Lipids containing n-4 and / or n-7 polyunsaturated fatty acids are not limited to the above examples. Sphingophospholipids, other phospholipids, ceramides, glycosphingolipids, other glycolipids, etc. Any lipid included in the definition of
さらに本発明においては、
脂質中のトリグリセリド中の全脂肪酸に対して7,10,13,16−エイコサテトラエン酸の含量が1.5重量％以上、好ましくは4.8重量％以上、より好ましくは7.8重量％以上である、7,10,13,16−エイコサテトラエン酸を含有するトリグリセリド、
トリグリセリド中の全脂肪酸に対して5,8,11,14−オクタデカテトラエン酸の含量が0.5重量％以上、好ましくは1.0重量％以上、より好ましくは2.1重量％以上である、5,8,11,14−オクタデカテトラエン酸を含有するトリグリセリド、
Furthermore, in the present invention,
The content of 7,10,13,16-eicosatetraenoic acid is 1.5% by weight or more, preferably 4.8% by weight or more, more preferably 7.8% by weight or more based on the total fatty acids in the triglycerides in the lipids. , 10,13,16-triglycerides containing eicosatetraenoic acid,
The content of 5,8,11,14-octadecatetraenoic acid is 0.5% by weight or more, preferably 1.0% by weight or more, more preferably 2.1% by weight or more, based on the total fatty acids in the triglyceride, A triglyceride containing 11,14-octadecatetraenoic acid,
トリグリセリド中の全脂肪酸に対して8,11,14−オクタデカトリエン酸の含量が0.1重量％以上、好ましくは0.5重量％以上、より好ましくは0.8重量％以上である、8,11,14−オクタデカトリエン酸を含有するトリグリセリド、
トリグリセリド中の全脂肪酸に対して炭素数が18以上のｎ−４系高度不飽和脂肪酸の含量が3.0重量％以上、好ましくは6.6重量％以上、より好ましくは10.4重量％以上である炭素数が１８以上のｎ−４系高度不飽和脂肪酸を含有するトリグリセリド、
The 8,11,14-octadecatrienoic acid content of the total fatty acid in the triglyceride is 0.1% by weight or more, preferably 0.5% by weight or more, more preferably 0.8% by weight or more. Triglycerides containing decatrienoic acid,
The content of n-4 polyunsaturated fatty acids having 18 or more carbon atoms with respect to all fatty acids in triglycerides is 3.0 wt% or more, preferably 6.6 wt% or more, more preferably 10.4 wt% or more. A triglyceride containing the above n-4 polyunsaturated fatty acid,
トリグリセリド中の全脂肪酸に対して7,10,13−エイコサトリエン酸の含量が0.1重量％以上、好ましくは0.5重量％以上、より好ましくは1.1重量％以上である7,10,13−エイコサトリエン酸を含有するトリグリセリド、
トリグリセリド中の全脂肪酸に対して5,8,11−オクタデカトリエン酸の含量が0.1重量％以上、好ましくは0.2重量％以上である5,8,11−オクタデカトリエン酸を含有するトリグリセリド、
7,10,13-eicosatrie having a content of 7,10,13-eicosatrienoic acid of 0.1% by weight or more, preferably 0.5% by weight or more, more preferably 1.1% by weight or more based on the total fatty acids in the triglyceride Triglycerides containing enoic acid,
A triglyceride containing 5,8,11-octadecatrienoic acid having a content of 5,8,11-octadecatrienoic acid of 0.1% by weight or more, preferably 0.2% by weight or more based on the total fatty acids in the triglyceride,
トリグリセリド中の全脂肪酸に対して8,11−オクタデカジエン酸の含量が0.1重量％以上、好ましくは0.2重量％以上である8,11−オクタデカジエン酸を含有するトリグリセリド、
トリグリセリド中の全脂肪酸に対して炭素数が18以上のｎ−７系高度不飽和脂肪酸の含量が0.3重量％以上、好ましくは0.9重量％以上、より好ましくは1.5重量％以上である炭素数が１８以上のｎ−７系高度不飽和脂肪酸を含有するトリグリセリド、
を得ることができる。
A triglyceride containing 8,11-octadecadienoic acid having a content of 8,11-octadecadienoic acid of 0.1% by weight or more, preferably 0.2% by weight or more, based on the total fatty acids in the triglyceride,
The content of n-7 polyunsaturated fatty acid having 18 or more carbon atoms with respect to all fatty acids in the triglyceride is 0.3 wt% or more, preferably 0.9 wt% or more, more preferably 1.5 wt% or more. A triglyceride containing the above n-7 polyunsaturated fatty acid,
Can be obtained.
培養物から採取したｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質からｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有するトリグリセリドを分離精製するには、常法に従って、例えば脱酸法、脱臭法、脱ガム法、脱水法、水蒸気蒸留法、分子蒸留法、冷却分離法、カラムクロマトグラフィー法などにより行う。例えば前述の操作に従って培養物からヘキサンを用いてｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質を抽出し、この抽出油から例えば脱酸、脱臭、脱ガム等の精製処理によりｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有するトリグリセリドを得ることができる。 To separate and purify triglycerides containing n-4 and / or n-7 highly unsaturated fatty acids from lipids containing n-4 and / or n-7 highly unsaturated fatty acids collected from the culture According to a conventional method, for example, a deoxidation method, a deodorization method, a degumming method, a dehydration method, a steam distillation method, a molecular distillation method, a cooling separation method, a column chromatography method or the like is performed. For example, a lipid containing n-4 and / or n-7 polyunsaturated fatty acids is extracted from the culture using hexane according to the above-described operation, and purification such as deoxidation, deodorization, degumming and the like is performed from the extracted oil. By the treatment, triglycerides containing n-4 and / or n-7 polyunsaturated fatty acids can be obtained.
ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質からｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を分離するには、混合脂肪酸あるいは混合脂肪酸エステルの状態で、常法により、例えば、尿素付加法、冷却分離法、カラムクロマトグラフィー法などにより濃縮分離することにより行う。
より具体的にはｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を直接分離することもできるが、低級アルコールとのエステル、例えばｎ−４系及び／又はｎ−７系高度不飽和脂肪酸のエチルエステルとして分離するのが好ましい。
To separate n-4 series and / or n-7 series highly unsaturated fatty acids from lipids containing n-4 series and / or n-7 series highly unsaturated fatty acids, in the state of mixed fatty acid or mixed fatty acid ester In the usual manner, for example, it is carried out by concentration and separation by the urea addition method, the cooling separation method, the column chromatography method or the like.
More specifically, n-4 series and / or n-7 series highly unsaturated fatty acids can be directly separated, but esters with lower alcohols such as n-4 series and / or n-7 series highly unsaturated. It is preferably separated as the fatty acid ethyl ester.
このようなエステルにすることにより、他の脂質成分から容易に分離することができ、また、培養中に生成する他の脂肪酸、例えばパルミチン酸等（これらも、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸のエステル化に際してエステル化される）から容易に分離することができる。例えば、高度不飽和脂肪酸のエチルエステルを得るには、前記の抽出脂質を無水エタノール- 塩酸5 〜10% 、BF３-エタノール10〜50% 等により、室温にて1 〜24時間処理するのが好ましい。 By making such an ester, it can be easily separated from other lipid components, and other fatty acids produced during cultivation, such as palmitic acid (also these are n-4 series and / or n- Can be easily separated from the 7-series highly unsaturated fatty acid. For example, in order to obtain ethyl ester of highly unsaturated fatty acid, it is preferable to treat the extracted lipid with anhydrous ethanol-hydrochloric acid 5-10%, BF3-ethanol 10-50%, etc. at room temperature for 1-24 hours. .
前記の処理液からｎ−４系及び／又はｎ−７系高度不飽和脂肪酸のエチルエステルを回収するには、ヘキサン、エーテル、酢酸エチル等の有機溶媒で抽出するのが好ましい。次に、この抽出液を無水硫酸ナトリウム等により乾燥し、有機溶媒を好ましくは減圧下で留去することにより脂肪酸エステルを主成分として含む混合物が得られる。この混合物には、目的とするｎ−４系及び／又はｎ−７系高度不飽和脂肪酸のエチルエステルの他に、パルミチン酸エチルエステル等の脂肪酸エチルエステルが含まれている。 In order to recover the ethyl ester of the n-4 series and / or n-7 series highly unsaturated fatty acid from the treatment liquid, it is preferable to extract with an organic solvent such as hexane, ether, ethyl acetate or the like. Next, this extract is dried with anhydrous sodium sulfate or the like, and the organic solvent is preferably distilled off under reduced pressure to obtain a mixture containing a fatty acid ester as a main component. This mixture contains fatty acid ethyl esters such as palmitic acid ethyl ester in addition to the desired n-4 and / or n-7 highly unsaturated fatty acid ethyl ester.
この混合物はそのまま又は本発明の脂肪酸の濃度を高め本発明の組成物に使用することができる。これらの脂肪酸エチルエステル混合物からｎ−４系及び／又はｎ−７系高度不飽和脂肪酸のエチルエステルを単離するには、カラムクロマトグラフィー、低温結晶化法、尿素包接法、液々交流分配クロマトグラフィー等を単独で、又は組み合わせて使用することができる。 This mixture can be used in the composition of the present invention as it is or after increasing the concentration of the fatty acid of the present invention. To isolate ethyl esters of n-4 and / or n-7 polyunsaturated fatty acids from these fatty acid ethyl ester mixtures, column chromatography, low temperature crystallization, urea inclusion, liquid-liquid exchange Chromatography and the like can be used alone or in combination.
こうして単離されたｎ−４系及び／又はｎ−７系高度不飽和脂肪酸のエチルエステルから遊離のｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を得るには、アルカリで加水分解した後、エーテル、酢酸エチル等の有機溶媒で抽出すればよい。また、ｎ−４系及び／又はｎ−７系高度不飽和脂肪酸をそのエチルエステルを経ないで摂取するには、前記の抽出脂質をアルカリ分解（例えば5%水酸化ナトリウムにより室温にて2 〜3 時間）した後、この分解液から、脂肪酸の抽出・精製に常用されている方法により抽出・精製することができる。得られた遊離の本発明の脂肪酸及びその塩もまた本発明の組成物のために使用することができる。 In order to obtain free n-4 and / or n-7 polyunsaturated fatty acid from the ethyl ester of n-4 and / or n-7 polyunsaturated fatty acid thus isolated, hydrolysis with alkali is required. Then, extraction with an organic solvent such as ether or ethyl acetate may be performed. In addition, in order to take n-4 series and / or n-7 series highly unsaturated fatty acid without passing through its ethyl ester, the extracted lipid is subjected to alkaline decomposition (for example, 5% sodium hydroxide at room temperature). After 3 hours), the decomposition solution can be extracted and purified by a method commonly used for extraction and purification of fatty acids. The resulting free fatty acids of the invention and their salts can also be used for the compositions of the invention.
本発明のｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を含有する脂質又はトリグリセリドは炭素数18以上で二重結合数が２以上であるｎ−４系及び／又はｎ−７系高度不飽和脂肪酸を豊富に含有しており、その用途に関しては無限の可能性があり、食品、飲料、化粧品、医薬品、動物用飼料などの原料並びに添加物として使用することがでる。例えば、一般食品、飲料、機能性食品、栄養補助食品、調製乳、化粧水、乳液、経腸栄養剤、粉末、顆粒、トローチ、シロップ、錠剤、カプセル剤、輸液、注射剤、塗布用ゲル、湿布、粉末飼料、固形飼料、液状飼料などを挙げることができるが、これらに限定するものではなく、その使用目的、使用量、加工形態に関して何ら制限を受けるものではない。 The lipid or triglyceride containing the n-4 series and / or n-7 series highly unsaturated fatty acid of the present invention is an n-4 series and / or an n-7 series having 18 or more carbon atoms and 2 or more double bonds. It is rich in polyunsaturated fatty acids, has unlimited possibilities for its use, and can be used as a raw material and additive for foods, beverages, cosmetics, pharmaceuticals, animal feeds and the like. For example, general foods, beverages, functional foods, dietary supplements, formulas, lotions, emulsions, enteral nutrients, powders, granules, troches, syrups, tablets, capsules, infusions, injections, gels for application, Examples thereof include compresses, powdered feeds, solid feeds, liquid feeds, and the like, but are not limited thereto, and there are no restrictions on the purpose of use, the amount used, and the form of processing.
次に、実施例により、本発明をさらに具体的に説明する。しかし、本発明は、実施例に限定されない。
実施例１． モルティエレラ・アルピナ IFO8568の変異処理によるパルミチン酸からステアリン酸への脂肪酸鎖長延長活性が低下した菌株の取得
モルティエレラ・アルピナ（Mortierella alpina）IFO8568 をCzapek寒天培地（0.2% NaNO3、0.1% K2HPO4 、0.05% MgSO4・7H2O、0.05% KCl 、0.01% FeSO4・7H2O、3%シュークロース、2%寒天、pH6.0 ）300mL を含む大型スラント瓶に植菌し、28℃で２週間培養した。
Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples.
Example 1. Mortierella alpina acquisition Mortierella alpina strains fatty-acid-chain elongase activity from palmitic acid to stearic acid was reduced by mutagenesis of IFO8568 (Mortierella alpina) IFO8568 a Czapek agar medium (0.2% NaNO 3, 0.1% K 2 HPO 4 , 0.05% MgSO 4 · 7H 2 O, 0.05% KCl, 0.01% FeSO 4 · 7H 2 O, 3% sucrose, 2% agar, pH6.0) Inoculated into a large slant bottle containing 300mL, 28 Culturing was carried out at 2 ° C. for 2 weeks.
培養後、滅菌水50 mLを加え振り混ぜ、４重のガーゼで濾過し、8,000 ×g で10分間遠心した後、50mM トリス緩衝溶液（pH7.5 ）に懸濁して胞子懸濁液を調製した。１×106 ／mLの胞子懸濁液1.5 mLに、0.5％NTG （Ｎ−メチル−Ｎ'−ニトロ−Ｎ−ニトロソグアジニン）溶液0.5 mLを加えて、28℃で15分間変異処理を行った。10％ Na2S2O3 を３mL加え、5,500 ×g で10分間遠心した後、滅菌水で洗浄し、NTG 処理胞子懸濁液を得た。 After culturing, 50 mL of sterilized water was added, shaken, filtered through quadruple gauze, centrifuged at 8,000 xg for 10 minutes, and then suspended in 50 mM Tris buffer solution (pH 7.5) to prepare a spore suspension. . Add 0.5 mL of 0.5% NTG (N-methyl-N′-nitro-N-nitrosoguanidine) solution to 1.5 mL of 1 × 10 6 / mL spore suspension and perform mutation treatment at 28 ° C. for 15 minutes. It was. After adding 3 mL of 10% Na 2 S 2 O 3 and centrifuging at 5,500 × g for 10 minutes, it was washed with sterilized water to obtain an NTG-treated spore suspension.
NTG 処理胞子懸濁液を、GY寒天培地（１％グルコース、0.5％酵母エキス、0.005 ％トリトンX-100 、１．５％寒天、pH6.0 ）に塗布し、28℃で生育したコロニーを別のGY寒天培地に移した。生育した菌体の一部を乾燥させ、常法に従い、塩酸メタノールで菌体内の脂肪酸をメチルエステル化した後、ヘキサンで抽出し、ヘキサンを留去して得られた脂肪酸メチルエステルをガスクロマトグラフィーで分析した。約1,000 個のコロニーを調べた結果、パルミチン酸からステアリン酸への脂肪酸鎖長延長活性が低下した菌株SAM 2268（FERM P-17762）が得られた。 Apply NTG-treated spore suspension to GY agar medium (1% glucose, 0.5% yeast extract, 0.005% Triton X-100, 1.5% agar, pH 6.0), and separate colonies grown at 28 ° C. Transferred to the GY agar medium. A portion of the grown cells are dried, and in accordance with a conventional method, the fatty acid in the cells is methyl esterified with hydrochloric acid methanol, then extracted with hexane, and the hexane is distilled off to obtain the fatty acid methyl ester obtained by gas chromatography. Analyzed with As a result of examining about 1,000 colonies, a strain SAM 2268 (FERM P-17762) in which the activity of extending the fatty acid chain length from palmitic acid to stearic acid was reduced was obtained.
実施例２． アラキドン酸生産能を有する微生物によるｎ−４系、ｎ−７系高度不飽和脂肪酸の製造方法
下記のアラキドン酸生産能を有する微生物の一白金耳を、10mLエルレンマイヤーフラスコに入れた液体培地２mL( グルコース２％、酵母エキス１％、パルミトレイン酸１％又はなし) に植菌し、28℃、120rpmで７日間振とう培養した。培養後、菌体を濾過により集め、乾燥した。常法に従い、塩酸メタノールで菌体内の脂肪酸をメチルエステル化した後、ヘキサンで抽出し、ヘキサンを留去して得られた脂肪酸メチルエステルをガスクロマトグラフィーで分析した。結果を表１に示す。
Example 2 Method for producing n-4 and n-7 polyunsaturated fatty acids by microorganisms capable of producing arachidonic acid 2 mL of liquid medium containing one platinum loop of microorganism having the ability to produce arachidonic acid in a 10 mL Erlenmeyer flask (2% glucose, 1% yeast extract, 1% palmitoleic acid or none) was inoculated and cultured with shaking at 28 ° C. and 120 rpm for 7 days. After cultivation, the cells were collected by filtration and dried. According to a conventional method, the fatty acid in the microbial cells was methyl esterified with methanolic hydrochloric acid, extracted with hexane, and the fatty acid methyl ester obtained by distilling off hexane was analyzed by gas chromatography. The results are shown in Table 1.
（１）モルティエレラ・アルピナ（Mortierella alpina）IFO8568
（２）モルティエレラ・フィグロフィラ（Mortierella hygrophila）IFO5941
（３）エキノスポランジウム・トランスベルサレ（Echinosporangium transversale ）NRRL3116
（４）サプロレグニア・ラポニカ（Saprolegnia lapponica ）CBS313.81
(1) Mortierella alpina IFO8568
(2) Mortierella hygrophila IFO5941
(3) Echinosporangium transversale NRRL3116
(4) Saprolegnia lapponica CBS313.81
実施例３． パルミトレイン酸誘導体の効果
モルティエレラ・アルピナ（Mortierella alpina）IFO8568 の一白金耳を、10mLエルレンマイヤーフラスコに入れた液体培地２mL（グルコース２％、酵母エキス１％、表２に示したパルミトレイン酸誘導体１％）に植菌し、28℃で、120rpmで７日間振とう培養した。培養後、実施例２と同様にメチルエステル化し、得られた脂肪酸メチルエステルをガスクロマトグラフィーで分析した。結果を表２に示す。
Example 3 Effect of palmitoleic acid derivative 2 mL of liquid medium (2% glucose, 1% yeast extract, palmitoleic acid derivative 1 shown in Table 2) in which a platinum loop of Mortierella alpina IFO8568 is placed in a 10 mL Erlenmeyer flask %) And cultured with shaking at 28 rpm at 120 rpm for 7 days. After the culture, methyl esterification was performed in the same manner as in Example 2, and the obtained fatty acid methyl ester was analyzed by gas chromatography. The results are shown in Table 2.
実施例４． 脂肪酸鎖長延長活性低下株による製造方法
モルティエレラ・アルピナ（Mortierella alpina）SAM 2268（FERM P-17762）の一白金耳を、10mLエルレンマイヤーフラスコに入れた液体培地２mL（グルコース４％、酵母エキス１％）に植菌し、28℃で、120rpmで６、10、12及び14日間振とう培養した。培養後、実施例２と同様にメチルエステル化し、得られた脂肪酸メチルエステルをガスクロマトグラフィーで分析した。また、同条件で６、10、12及び14日間培養した別の菌体を回収し、乾燥後、クロロホルム−メタノール抽出法により脂質を抽出した。
Example 4 Production method using a reduced fatty acid chain extension activity Morterella alpina SAM 2268 (FERM P-17762) one platinum loop in a 10 mL Erlenmeyer flask 2 mL of liquid medium (glucose 4%, yeast extract 1%) and cultured with shaking at 28 ° C. at 120 rpm for 6, 10, 12, and 14 days. After the culture, methyl esterification was performed in the same manner as in Example 2, and the obtained fatty acid methyl ester was analyzed by gas chromatography. In addition, other cells cultured for 6, 10, 12 and 14 days under the same conditions were collected, dried, and then extracted with a chloroform-methanol extraction method.
さらに常法に従い、抽出した脂質を、薄層クロマトグラフィー、ケイ酸カラムクロマトで分画してトリグリセリドを得た。得られたトリグリセリドは上記の方法でメチルエステル化し、ガスクロマトグラフィーで分析した。経時変化を表３及び表４に示す。なお、14日間培養した菌体から抽出した脂質の組成を調べたところ、中性脂質が90重量％（トリグリセリドは抽出した脂質の89重量％）、極性脂質が10重量％であった。 Further, according to a conventional method, the extracted lipid was fractionated by thin layer chromatography and silicic acid column chromatography to obtain triglyceride. The obtained triglyceride was methyl esterified by the above method and analyzed by gas chromatography. The changes with time are shown in Tables 3 and 4. The composition of the lipid extracted from the cells cultured for 14 days was 90% by weight of neutral lipid (triglyceride was 89% by weight of the extracted lipid) and 10% by weight of polar lipid.
実施例５． 基質の添加効果
モルティエレラ・アルピナ（Mortierella alpina）SAM 2268（FERM P-17762）の一白金耳を、10mLエルレンマイヤーフラスコに入れた液体培地２mL（グルコース２％、酵母エキス１％、表４に示した化合物１％）に植菌し、28℃で、120rpmで７日間振とう培養した。培養後、実施例２と同様にメチルエステル化し、得られた脂肪酸メチルエステルをガスクロマトグラフィーで分析した。結果を表５に示す。
Example 5 FIG. Effect of addition of substrate 2 mL of liquid medium (2% glucose, 1% yeast extract, 1%) in a 10 mL Erlenmeyer flask with one platinum loop of Mortierella alpina SAM 2268 (FERM P-17762) The indicated compound (1%) was inoculated and cultured with shaking at 28 rpm at 120 rpm for 7 days. After the culture, methyl esterification was performed in the same manner as in Example 2, and the obtained fatty acid methyl ester was analyzed by gas chromatography. The results are shown in Table 5.
実施例６． ｎ−７系高度不飽和脂肪酸を含みｎ−４系高度不飽和脂肪酸を含まない脂質の製造方法
モルティエレラ・アルピナ（Mortierella alpina）IFO8568 又はΔ１２不飽和化反応欠損株であるモルティエレラ・アルピナ（Mortierella alpina）SAM 1861（微工研条寄第3590号）の一白金耳を、10mLエルレンマイヤーフラスコに入れた液体培地２mL（グルコース５％、酵母エキス１％、パルミトレイン酸１％）に植菌し、12〜28℃で、120rpmで７日間振とう培養した。培養後、実施例２と同様にメチルエステル化し、得られた脂肪酸メチルエステルをガスクロマトグラフィーで分析した。結果を表６に示す。
Example 6 Method for producing lipid containing n-7 polyunsaturated fatty acid and not containing n-4 polyunsaturated fatty acid Mortierella alpina IFO8568 or Mortierella alpina deficient in Δ12 desaturation reaction (Mortierella alpina) Inoculate one platinum loop of SAM 1861 (Novel 3570) into 2 mL of liquid medium (5% glucose, 1% yeast extract, 1% palmitoleic acid) in a 10 mL Erlenmeyer flask. And cultured at 12 to 28 ° C. with shaking at 120 rpm for 7 days. After the culture, methyl esterification was performed in the same manner as in Example 2, and the obtained fatty acid methyl ester was analyzed by gas chromatography. The results are shown in Table 6.
Δ12不飽和化反応欠損株であるSAM 1861株を用いると、ｎ−７系からｎ−４系への変換が起こらないため、本菌株から得られる脂質には、ｎ−７系高度不飽和脂肪酸が含まれているが、ｎ−４系高度不飽和脂肪酸は含まれていなかった。 When the SAM 1861 strain, which is a Δ12 desaturation-deficient strain, is used, no conversion from the n-7 system to the n-4 system occurs, so the lipid obtained from this strain contains n-7 highly unsaturated fatty acids. However, n-4 type polyunsaturated fatty acid was not contained.
実施例７． カプセル剤の調製
ゼラチン100 重量部及び食品添加用グリセリン35重量部に水を加え50〜60℃で溶解し、粘度20000cpsのゼラチン皮膜を調製した。次に実施例３に記した方法で得た乾燥菌体を破砕した後、ヘキサンで抽出したヘキサン抽出油又はこれを脱酸、脱臭、脱ガム処理したトリグリセリドに、３重量％のビタミンＥ油を混合し、内容物を調製した。これらを用いて、常法に従い、カプセル成型及び乾燥を行い、１粒当たり180 mgの内容物を含有するソフトカプセルを製造した。
Example 7 Preparation of capsules Water was added to 100 parts by weight of gelatin and 35 parts by weight of glycerin for food addition and dissolved at 50 to 60 ° C. to prepare a gelatin film having a viscosity of 20000 cps. Next, after crushing the dried microbial cells obtained by the method described in Example 3, hexane extracted oil extracted with hexane or triglyceride obtained by deoxidizing, deodorizing and degumming this was added 3 wt% vitamin E oil. The contents were prepared by mixing. Using these, capsules were molded and dried according to a conventional method to produce soft capsules containing 180 mg of content per grain.
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