JP2024004585A - METHOD FOR PRODUCING ω-3 POLYUNSATURATED FATTY ACID-CONTAINING COMPOSITION - Google Patents
METHOD FOR PRODUCING ω-3 POLYUNSATURATED FATTY ACID-CONTAINING COMPOSITION Download PDFInfo
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- 239000000203 mixture Substances 0.000 title claims abstract description 48
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 22
- 235000020660 omega-3 fatty acid Nutrition 0.000 claims abstract description 66
- MBMBGCFOFBJSGT-KUBAVDMBSA-N docosahexaenoic acid Natural products CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 claims abstract description 61
- -1 docosahexaenoic acid ester Chemical class 0.000 claims abstract description 49
- 235000020669 docosahexaenoic acid Nutrition 0.000 claims abstract description 47
- 108090001060 Lipase Proteins 0.000 claims abstract description 31
- 102000004882 Lipase Human genes 0.000 claims abstract description 31
- 239000004367 Lipase Substances 0.000 claims abstract description 31
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- 235000021122 unsaturated fatty acids Nutrition 0.000 claims description 67
- 150000004670 unsaturated fatty acids Chemical class 0.000 claims description 36
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- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 2
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
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- 208000024827 Alzheimer disease Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- TYLNXKAVUJJPMU-DNKOKRCQSA-N Docosahexaenoic acid ethyl ester Chemical compound CCCCCCCCC\C=C\C=C\C=C\C=C\C=C\C=C\C(=O)OCC TYLNXKAVUJJPMU-DNKOKRCQSA-N 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
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- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 1
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- RKAKAVQIMCZXPE-AAQCHOMXSA-N ethyl (6z,9z,12z,15z,18z)-henicosa-6,9,12,15,18-pentaenoate Chemical compound CCOC(=O)CCCC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC RKAKAVQIMCZXPE-AAQCHOMXSA-N 0.000 description 1
- VCSQUSNNIFZJAP-AAQCHOMXSA-N ethyl (7Z,10Z,13Z,16Z,19Z)-docosapentaenoate Chemical compound CCOC(=O)CCCCC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC VCSQUSNNIFZJAP-AAQCHOMXSA-N 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
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- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 1
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- 150000002617 leukotrienes Chemical class 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical compound NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
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- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
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- 239000011541 reaction mixture Substances 0.000 description 1
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
本発明は、ω－３高度不飽和脂肪酸含有組成物の製造方法に関する。
詳細には、ω－３高度不飽和脂肪酸エステルを含む原料を、リパーゼ、好ましくはRhizomucor miehei（リゾムコール・ミエヘイ）由来リパーゼで処理することによる、ドコサヘキサエン酸エステルおよびヘンエイコサペンタエン酸エステルが選択的に濃縮されて、ドコサヘキサエン酸エステルおよびヘンエイコサペンタエン酸エステルをともに十分な量含有するω－３高度不飽和脂肪酸含有組成物の製造方法及びドコサヘキサエン酸エステルおよびヘンエイコサペンタエン酸エステルをともに十分な量含有するω－３高度不飽和脂肪酸含有組成物に関する。
The present invention relates to a method for producing a composition containing ω-3 highly unsaturated fatty acids.
Specifically, docosahexaenoic acid ester and heneicosapentaenoic acid ester are selectively concentrated by treating a raw material containing omega-3 highly unsaturated fatty acid ester with lipase, preferably lipase derived from Rhizomucor miehei. A method for producing an ω-3 highly unsaturated fatty acid-containing composition containing sufficient amounts of both docosahexaenoic acid ester and heneicosapentaenoic acid ester, and a method for producing an ω-3 polyunsaturated fatty acid-containing composition containing sufficient amounts of both docosahexaenoic acid ester and heneicosapentaenoic acid ester. -3 Concerning a highly unsaturated fatty acid-containing composition.
高度不飽和脂肪酸は、不飽和結合を２つ以上持つ脂肪酸であり、ω－６不飽和脂肪酸のリノール酸（ＬＡ、１８：２ｎ－６）、γ－リノレン酸（ＧＬＡ、１８：３ｎ－６）、アラキドン酸（ＡＲＡ、２０：４ｎ－６）、ω－３不飽和脂肪酸のα－リノレン酸（ＡＬＡ、１８：３ｎ－３）、エイコサペンタエン酸（ＥＰＡ、２０：５ｎ－３）、ドコサヘキサエン酸（ＤＨＡ、２２：６ｎ－３）などが挙げられる。高度不飽和脂肪酸は、生体膜の主要構成成分として膜の流動性の調節に関与するほか、生体機能性成分の前駆体としても重要である。 Highly unsaturated fatty acids are fatty acids with two or more unsaturated bonds, and include the ω-6 unsaturated fatty acids linoleic acid (LA, 18:2n-6) and γ-linolenic acid (GLA, 18:3n-6). , arachidonic acid (ARA, 20:4n-6), ω-3 unsaturated fatty acids α-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid ( DHA, 22:6n-3) and the like. Polyunsaturated fatty acids are involved in regulating membrane fluidity as major constituents of biological membranes, and are also important as precursors of biologically functional components.
例えば、ω－３不飽和脂肪酸のＥＰＡは、高等動物内においてプロスタグランジン、トロンボキサン、ロイコトリエンなどの前駆体であり、血小板凝集阻害作用、血中中性脂肪低下作用、抗動脈硬化作用、血液粘度低下作用、血圧降下作用、抗炎症作用、抗腫瘍作用等の生理作用を有し、医薬品、食品、化粧品、飼料等の様々な分野に利用されている。また、ＤＨＡは脳内に最も多量に存在する高度不飽和脂肪酸であり、学習機能の向上、抗腫瘍、抗アレルギー等の優れた効果を有することが知られている。さらに、ＥＰＡ又はＤＨＡはアトピー性等の皮膚炎に対しても有効であることが知られている。そのため、ＥＰＡ、ＤＨＡ等のω-３高度不飽和脂肪酸はその広範な有効性から、食品、健康食品、医薬品として市販されている。 For example, EPA, an omega-3 unsaturated fatty acid, is a precursor of prostaglandins, thromboxanes, leukotrienes, etc. in higher animals, and has an inhibitory effect on platelet aggregation, a blood neutral fat lowering effect, an antiarteriosclerosis effect, and a blood It has physiological effects such as viscosity lowering effect, blood pressure lowering effect, anti-inflammatory effect, and antitumor effect, and is used in various fields such as medicine, food, cosmetics, and feed. Furthermore, DHA is a highly unsaturated fatty acid that exists in the largest amount in the brain, and is known to have excellent effects such as improving learning function, antitumor, and antiallergy. Furthermore, EPA or DHA is known to be effective against dermatitis such as atopic dermatitis. Therefore, ω-3 polyunsaturated fatty acids such as EPA and DHA are commercially available as foods, health foods, and medicines due to their wide range of effectiveness.
ω－３高度不飽和脂肪酸は、二重結合を複数有するため、化学合成によって得ることは容易ではない。したがって、工業利用されるω－３高度不飽和脂肪酸のほとんどは、ω－３高度不飽和脂肪酸を豊富に含む海洋生物由来原料、例えば魚油などから抽出又は精製することによって製造されている。しかしながら、生物由来原料は、炭素数、二重結合の数や位置、さらには立体異性体の構成比などが異なる多種の脂肪酸の混合物であるため、目的とするＥＰＡやＤＨＡの含有量は必ずしも高くない。 Since ω-3 highly unsaturated fatty acids have multiple double bonds, it is not easy to obtain them by chemical synthesis. Therefore, most of the ω-3 highly unsaturated fatty acids used industrially are produced by extracting or refining raw materials derived from marine organisms, such as fish oil, which are rich in ω-3 highly unsaturated fatty acids. However, biological raw materials are a mixture of various fatty acids with different numbers of carbon atoms, number and position of double bonds, and even composition ratios of stereoisomers, so the target content of EPA and DHA is not necessarily high. do not have.
そのため、従来、生物由来原料からＥＰＡやＤＨＡを選択的に精製することが求められていた。特に、食品や医薬品の原料としてＥＰＡやＤＨＡを用いる場合、極力不純物がない高純度のＥＰＡやＤＨＡが求められている。また、海洋生物由来原料から精製したＥＰＡやＤＨＡは、微量でも不純物を含有すると強い魚臭を発散するため、従来のＥＰＡやＤＨＡを含有する食品や医薬品は、ＥＰＡやＤＨＡをカプセルに密封したり、マスキング剤とともに製剤化するなど、臭いの対策を施す必要があった。したがって、医薬品や健康食品の原料となり得るＥＰＡやＤＨＡを高純度に含有する組成物を簡便に製造することができる方法が求められている。 Therefore, it has conventionally been required to selectively purify EPA and DHA from biological raw materials. In particular, when EPA and DHA are used as raw materials for foods and medicines, highly pure EPA and DHA with as few impurities as possible are required. In addition, EPA and DHA purified from raw materials derived from marine organisms emit a strong fishy odor if they contain even a small amount of impurities. It was necessary to take measures against odor, such as formulating it with a masking agent. Therefore, there is a need for a method that can easily produce a composition containing highly purified EPA and DHA that can be used as raw materials for pharmaceuticals and health foods.
ω－３高度不飽和脂肪酸類の分離方法としては、例えば蒸留による方法が知られているが、炭素鎖が類似したω－３高度不飽和脂肪酸を分離するのは非効率であった。また、蒸
留における加熱によって熱異性体が増加するという問題があった。
As a method for separating ω-3 highly unsaturated fatty acids, for example, a method using distillation is known, but it is inefficient to separate ω-3 highly unsaturated fatty acids having similar carbon chains. Additionally, there was a problem in that thermal isomers increased due to heating during distillation.
ＤＨＡを選択的に濃縮するための蒸留以外の方法としては、以下の酵素を使用した方法が知られている。 As a method other than distillation for selectively concentrating DHA, the following method using an enzyme is known.
特許文献１には、遊離脂肪酸としてＥＰＡおよびＤＨＡを含有する海産油組成物を、リパーゼ触媒存在下でグリセロールでエステル化して、ＥＰＡのグリセリド体とＤＨＡの遊離体を得る方法が記載されている。 Patent Document 1 describes a method of obtaining a glyceride form of EPA and a free form of DHA by esterifying a marine oil composition containing EPA and DHA as free fatty acids with glycerol in the presence of a lipase catalyst.
また、特許文献２には、ドコサヘキサエン酸をグリセリドの構成脂肪酸として含有する組成物の製造方法として、ドコサヘキサエン酸を構成脂肪酸として含むグリセリドを含有する原料油に複数のリパーゼを作用させて加水分解反応を行い、グリセリド画分中のドコサヘキサエン酸の割合を高める工程を含む方法が開示されている Further, Patent Document 2 describes a method for producing a composition containing docosahexaenoic acid as a constituent fatty acid of glyceride, in which a plurality of lipases are caused to act on a raw material oil containing glyceride containing docosahexaenoic acid as a constituent fatty acid to carry out a hydrolysis reaction. Disclosed is a method comprising the step of increasing the proportion of docosahexaenoic acid in the glyceride fraction.
さらに、特許文献３には、ω－３系高度不飽和脂肪酸を含有する長鎖脂肪酸と低級アルコールから構成される脂肪酸エステルを、リパーゼを用いて直鎖高級アルコールと選択的アルコリシス反応させることを特徴とするω－３系高度不飽和脂肪酸エステルの精製方法において、高度不飽和脂肪酸エステルは未反応のままでそれ以外の脂肪酸エステルが優先的に高級アルコールとアルコール交換されて、容易にワックスエステルが生成することが開示されている。 Furthermore, Patent Document 3 discloses that a fatty acid ester composed of a long chain fatty acid containing an ω-3 highly unsaturated fatty acid and a lower alcohol is subjected to a selective alcoholysis reaction with a straight chain higher alcohol using lipase. In the purification method for omega-3 highly unsaturated fatty acid esters, the highly unsaturated fatty acid esters remain unreacted and other fatty acid esters are preferentially exchanged with higher alcohols to easily generate wax esters. It is disclosed that
近年、ω－３不飽和脂肪酸のひとつであるヘンエイコサペンタエン酸（ＨＰＡ、２１：５ｎ－３）についても、アルツハイマー病の治療においてＤＨＡ誘導体と関連して重要な役割を担っている可能性があるとの報告（非特許文献１）があり、その機能性に注目が集まっていることから、ＤＨＡとともにＨＰＡを摂取することに意義があると考えられ、ＤＨＡとＨＰＡをともに十分な量含有する組成物が求められている。しかし、ＤＨＡとＨＰＡを併せて選択的かつ簡便に濃縮する方法は現在までに知られていない。 In recent years, heneicosapentaenoic acid (HPA, 21:5n-3), an ω-3 unsaturated fatty acid, may also play an important role in the treatment of Alzheimer's disease in conjunction with DHA derivatives. There is a report that (Non-patent Document 1), and its functionality is attracting attention, so it is thought that it is meaningful to ingest HPA together with DHA, and a composition containing sufficient amounts of both DHA and HPA things are wanted. However, a method for selectively and conveniently concentrating DHA and HPA together is not known to date.
本発明の課題は、濃縮工程における熱負荷が少なく、類似構造を効率的に分離し、ＤＨＡおよびＨＰＡを選択的に濃縮して、ＤＨＡとＨＰＡをともに十分な量含有する組成物を提供することである。 An object of the present invention is to provide a composition that requires less heat load in the concentration step, efficiently separates similar structures, selectively concentrates DHA and HPA, and contains sufficient amounts of both DHA and HPA. It is.
本発明者らは、ω－３高度不飽和脂肪酸エステルを含む原料を、リパーゼで処理することにより、ドコサヘキサエン酸エステルおよびヘンエイコサペンタエン酸エステルが選択的に濃縮され、ＤＨＡとＨＰＡをともに十分な量含有する組成物が得られることを見出し、本発明に到達することができた。 The present inventors have demonstrated that by treating raw materials containing omega-3 highly unsaturated fatty acid esters with lipase, docosahexaenoic acid esters and heneicosapentaenoic acid esters are selectively concentrated, and sufficient amounts of both DHA and HPA are obtained. It has been discovered that a composition containing the following can be obtained, and the present invention has been achieved.
したがって、上記の課題は、以下の製造方法及び組成物によって解決することができる。
（１）ω－３高度不飽和脂肪酸エステルを含む原料を、リパーゼにより処理して、ドコサヘキサエン酸エステルおよびヘンエイコサペンタエン酸エステルを濃縮することを含む、ドコサヘキサエン酸エステルおよびヘンエイコサペンタエン酸エステルが選択的に濃縮されたω－３高度不飽和脂肪酸又はそのエステル含有組成物の製造方法。
（２）リパーゼがRhizomucor miehei（リゾムコール・ミエヘイ）由来のものである（１）の製造方法。
（３）ω－３高度不飽和脂肪酸エステルを含む原料が、魚油由来のものである（１）又は（２）の製造方法。
（４）ω－３高度不飽和脂肪酸エステルがエチルエステルである（１）～（３）の製造方法。
（５）濃縮後のω－３高度不飽和脂肪酸又はそのエステル含有組成物にドコサヘキサエン酸又はそのエステル８０面積％以上およびヘンエイコサペンタエン酸又はそのエステル８面積％以上が含有される（１）～（４）の製造方法。
（６）ドコサヘキサエン酸又はそのエステル８０面積％以上およびヘンエイコサペンタエン酸又はそのエステル８面積％以上を含有する、ω－３高度不飽和脂肪酸又はそのエステル含有組成物。
Therefore, the above problems can be solved by the following manufacturing method and composition.
(1) Selective treatment of docosahexaenoic acid esters and heneicosapentaenoic acid esters, including treating a raw material containing omega-3 highly unsaturated fatty acid esters with lipase to concentrate docosahexaenoic acid esters and heneicosapentaenoic acid esters. A method for producing a composition containing concentrated omega-3 highly unsaturated fatty acids or esters thereof.
(2) The method for producing (1), wherein the lipase is derived from Rhizomucor miehei.
(3) The production method of (1) or (2), wherein the raw material containing the ω-3 highly unsaturated fatty acid ester is derived from fish oil.
(4) The production methods of (1) to (3), wherein the ω-3 highly unsaturated fatty acid ester is ethyl ester.
(5) The concentrated omega-3 highly unsaturated fatty acid or its ester-containing composition contains 80 area % or more of docosahexaenoic acid or its ester and 80 area % or more of heneicosapentaenoic acid or its ester (1) ~ ( 4) Manufacturing method.
(6) An ω-3 highly unsaturated fatty acid or ester-containing composition containing 80 area % or more of docosahexaenoic acid or its ester and 8 area % or more of heneicosapentaenoic acid or its ester.
本発明の製造方法は、濃縮工程における熱負荷が少なく、類似構造を効率的に分離して、ＤＨＡおよびＨＰＡを選択的に濃縮することができるため、近年、それらの機能性から望まれている、ＤＨＡとＨＰＡをともに十分な量含有する組成物を効率的に提供することができる。 The production method of the present invention has been desired in recent years due to its functionality because it requires less heat load in the concentration step and can efficiently separate similar structures and selectively concentrate DHA and HPA. , a composition containing sufficient amounts of both DHA and HPA can be efficiently provided.
本発明のω－３高度不飽和脂肪酸含有組成物の製造方法について、以下に説明する。 The method for producing the ω-3 highly unsaturated fatty acid-containing composition of the present invention will be explained below.
（ω－３高度不飽和脂肪酸エステル含有原料）
本発明で用いるω－３高度不飽和脂肪酸含有組成物の原料としては、主として天然物由来の油脂混合物であって、上述したω－３高度不飽和脂肪酸が含まれているものが挙げられる。そのような原料の例としては、魚類等の海産動物やプランクトン由来の油脂、藻類等の微生物由来の油脂などが挙げられ、中でもイワシ、ハマチ等の魚類由来の油脂が好ましい。
(Raw material containing ω-3 highly unsaturated fatty acid ester)
The raw material for the ω-3 highly unsaturated fatty acid-containing composition used in the present invention includes oil and fat mixtures mainly derived from natural products, which contain the above-mentioned ω-3 highly unsaturated fatty acids. Examples of such raw materials include oils and fats derived from marine animals such as fish and plankton, and oils and fats derived from microorganisms such as algae, among which oils and fats derived from fish such as sardines and yellowtail are preferred.
本発明で用いるω－３高度不飽和脂肪酸含有組成物の原料は、ＤＨＡ及びＨＰＡの合計含有量ができるだけ高いものであることが好ましく、ＤＨＡ及びＨＰＡを、含有する脂肪酸の全量に対して２５面積％以上、より好ましくは６５面積％以上含有する油脂である。該原料中のω－３高度不飽和脂肪酸は、遊離脂肪酸の形態で存在していてもよく、又はモノ、ジ若しくはトリグリセリド等のエステル化脂肪酸の形態で存在していてもよい。 The raw material for the ω-3 highly unsaturated fatty acid-containing composition used in the present invention preferably has a total content of DHA and HPA as high as possible, and DHA and HPA are contained in an amount of 25% by area based on the total amount of fatty acids contained. % or more, more preferably 65 area % or more. The omega-3 highly unsaturated fatty acids in the raw material may be present in the form of free fatty acids or in the form of esterified fatty acids such as mono-, di- or triglycerides.
本発明の方法において、当該原料中のω－３高度不飽和脂肪酸はアルキルエステル化されている。ω－３高度不飽和脂肪酸アルキルエステルは、例えば、ω－３高度不飽和脂肪酸を含有する油脂と所望のアルキル基を有する酸とを公知の方法によりエステル化反応させることにより製造することができる。あるいは、該油脂に含まれるグリセリド中のω－３高度不飽和脂肪酸を、触媒又は酵素の存在下で低級アルコールと反応させてアルキルエステル化することにより、ω－３高度不飽和脂肪酸アルキルエステルを得ることができる。 In the method of the present invention, the ω-3 highly unsaturated fatty acids in the raw material are alkyl esterified. The ω-3 highly unsaturated fatty acid alkyl ester can be produced, for example, by subjecting an oil or fat containing the ω-3 highly unsaturated fatty acid to an esterification reaction with an acid having a desired alkyl group by a known method. Alternatively, an ω-3 highly unsaturated fatty acid alkyl ester is obtained by reacting the ω-3 highly unsaturated fatty acid in the glyceride contained in the fat with a lower alcohol in the presence of a catalyst or enzyme to alkyl esterify the ω-3 highly unsaturated fatty acid. be able to.
本発明において、原料のω－３高度不飽和脂肪酸アルキルエステルのアルキルエステル
化の程度は高いほど好適であり、該原料油中に含まれるω－３不飽和脂肪酸（遊離体を含む）の全量のうち、好ましくは９０質量％以上、より好ましくは９５質量％以上がアルキルエステル化されているとよい。該ω－３高度不飽和脂肪酸のアルキルエステルを構成するアルキル基としては、炭素数１～５の直鎖状又は分岐鎖状のアルキル基が挙げられ、好ましくはメチル基又はエチル基であり、より好ましくはエチル基である。
In the present invention, the higher the degree of alkyl esterification of the raw material ω-3 highly unsaturated fatty acid alkyl ester is, the more preferable it is, and the total amount of ω-3 unsaturated fatty acids (including free forms) contained in the raw material oil is Of these, preferably 90% by mass or more, more preferably 95% by mass or more is alkyl esterified. The alkyl group constituting the alkyl ester of the ω-3 highly unsaturated fatty acid includes a linear or branched alkyl group having 1 to 5 carbon atoms, preferably a methyl group or an ethyl group, and more preferably a methyl group or an ethyl group. Preferably it is an ethyl group.
本発明における原料として用いられるω－３高度不飽和脂肪酸アルキルエステルを含有する油脂としては、市販されている油脂類を用いてもよい。例えば、含有するω－３高度不飽和脂肪酸の種類や量が規格化された市販の魚油由来の油脂類などを用いることが好ましい。また、高純度の高度不飽和脂肪酸エステル、例えば、ＥＰＡエチルエステル含有組成物を製造した際に得られる副産物を用いてもよい。 As the fat and oil containing the omega-3 highly unsaturated fatty acid alkyl ester used as a raw material in the present invention, commercially available fats and oils may be used. For example, it is preferable to use commercially available fish oil-derived fats and oils whose type and amount of ω-3 highly unsaturated fatty acids are standardized. Alternatively, a by-product obtained when producing a composition containing highly pure highly unsaturated fatty acid ester, for example, EPA ethyl ester, may be used.
（リパーゼ）
本発明に使用するリパーゼは、リゾムコール属（Rhizomucor属）に属する微生物由来のリパーゼであり、リゾムコール・ミエヘイ（Rhizomucor miehei）由来リパーゼが好ましく、これに該当する市販品としては、例えば、Sigma-Aldrich社製 Lipase from Rhizomucor miehei（製品番号Ｌ４２７７）、ノボザイムジャパン社製 Palatase 20000LやLipozyme RMなどが挙げられる。
(Lipase)
The lipase used in the present invention is a lipase derived from a microorganism belonging to the genus Rhizomucor, preferably lipase derived from Rhizomucor miehei. Examples include Lipase from Rhizomucor miehei (product number L4277), Palatase 20000L and Lipozyme RM manufactured by Novozyme Japan.
本発明に使用するリパーゼの使用形態については、特に制限されるものではなく、そのまま使用しても固定化剤に固定して使用しても良い。 The form of use of the lipase used in the present invention is not particularly limited, and it may be used as it is or after being fixed on a fixative.
（リパーゼ反応液）
本発明において、リパーゼ反応液におけるリパーゼの使用量は、特に制限されるものではなく、適宜決定することができるが、例えば、原料油脂に対して１５～７５質量％が好ましく、２３～４５質量％がさらに好ましい。また、水分量については、原料油脂に対して１００～９１０質量％が好ましく、２１０～９１０質量％がさらに好ましい 。
(Lipase reaction solution)
In the present invention, the amount of lipase used in the lipase reaction solution is not particularly limited and can be determined as appropriate, but for example, it is preferably 15 to 75% by mass, and 23 to 45% by mass based on the raw material fat. is even more preferable. Furthermore, the water content is preferably 100 to 910% by mass, more preferably 210 to 910% by mass based on the raw material fat.
（リパーゼ反応条件）
本発明において、リパーゼ反応は、通常行われているリパーゼ反応の条件で行うことができ、特に限定されるものではなく、適宜決定することができるが、例えば、３５～５０℃、好ましくは４０～４５℃の温度条件で、脂肪酸エステルの酸化を防止するため真空下で、２．５～１０時間、好ましくは５～１０時間、等の反応条件を例示することができる。
(Lipase reaction conditions)
In the present invention, the lipase reaction can be carried out under commonly used lipase reaction conditions, which are not particularly limited and can be determined as appropriate, for example, 35 to 50°C, preferably 40 to 50°C. Examples of reaction conditions include 2.5 to 10 hours, preferably 5 to 10 hours, at a temperature of 45° C. and under vacuum to prevent oxidation of the fatty acid ester.
（精製）
リパーゼ反応後の反応液中には、ω－３高度不飽和脂肪酸エステルが加水分解することで生じる遊離脂肪酸とアルコールと、目的のＤＨＡエステル及びＨＰＡエステルを含む未反応のω－３高度不飽和脂肪酸エステルが含まれている。したがって、目的のＤＨＡエステルおよびＨＰＡエステルを分取するために、加水分解された遊離脂肪酸とアルコールを除去し精製する必要がある。目的のω－３不飽和脂脂肪酸エステルを分取する方法には、例えばカラム精製による方法、液液分配による方法が挙げられる。
(purification)
The reaction solution after the lipase reaction contains free fatty acids and alcohol produced by hydrolysis of the ω-3 highly unsaturated fatty acid ester, and unreacted ω-3 highly unsaturated fatty acid including the target DHA ester and HPA ester. Contains ester. Therefore, in order to separate the desired DHA ester and HPA ester, it is necessary to remove and purify the hydrolyzed free fatty acids and alcohols. Methods for fractionating the desired ω-3 unsaturated fat fatty acid ester include, for example, a column purification method and a liquid-liquid distribution method.
また、得られた目的のＤＨＡエステルおよびＨＰＡエステルを含有する組成物を公知の方法である、酸やアルカリを用いた加水分解やリチウム金属を用いた加水分解、または脂肪酸選択能がない酵素を用いた加水分解等を行うことによって、ＤＨＡおよびＨＰＡの遊離体を含有する組成物とすることもできる。 In addition, the obtained composition containing the target DHA ester and HPA ester can be subjected to known methods of hydrolysis using acid or alkali, hydrolysis using lithium metal, or using an enzyme that does not have fatty acid selectivity. A composition containing free forms of DHA and HPA can also be obtained by performing hydrolysis or the like.
以下、実施例を挙げて、本発明をさらに詳細に説明するが、本発明は実施例に限定され
るものではない。
（実施例１～１２）
EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the Examples.
(Examples 1 to 12)
（脂肪酸組成比の分析）
測定試料１２．５ｍｇをｎ－ヘキサン１ｍＬに希釈し、ガスクロマトグラフィー分析装置（Ｔｙｐｅ ７８９０ ＧＣ；Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ製）を用いて、以下の条件にて全脂肪酸中における各脂肪酸の含有比を分析した。結果は、クロマトグラムの面積から換算した面積％として表した。
＜注入口条件＞
注入口温度：２５０℃、スプリット比：１０
＜カラム条件＞
カラム：Ｊ＆Ｗ社製ＤＢ－ＷＡＸ ０．２５ ｍｍ×３０ ｍ、カラム温度：２１０℃、
Ｈｅ流量：１．０ ｍＬ／ｍｉｎ、Ｈｅ圧力：２０ ＰＳＩ
＜検出条件＞
Ｈ2流量：４０ ｍＬ／ｍｉｎ、Ａｉｒ流量：４５０ ｍＬ／ｍｉｎ
Ｈｅ流量：１．００ ｍＬ／ｍｉｎ、ＤＥＴ温度：２６０℃
(Analysis of fatty acid composition ratio)
12.5 mg of the measurement sample was diluted with 1 mL of n-hexane, and the content ratio of each fatty acid in the total fatty acids was analyzed using a gas chromatography analyzer (Type 7890 GC; manufactured by Agilent Technologies) under the following conditions. The results were expressed as area % converted from the area of the chromatogram.
<Inlet conditions>
Inlet temperature: 250℃, split ratio: 10
<Column conditions>
Column: J&W DB-WAX 0.25 mm x 30 m, column temperature: 210°C,
He flow rate: 1.0 mL/min, He pressure: 20 PSI
<Detection conditions>
H2 flow rate: 40 mL/min, Air flow rate: 450 mL/min
He flow rate: 1.00 mL/min, DET temperature: 260°C
なお、 分析した脂肪酸は、以下のとおりである：ＥＴＡ－Ｅ：エイコサテトラエン酸エチルエステル、ＥＰＡ－Ｅ：エイコサペンタエン酸エチルエステル、ＨＰＡ－Ｅ：ヘンエイコサペンタエン酸エチルエステル、ＤＰＡ－Ｅ：ドコサペンタエン酸エチルエステル、ＤＨＡ－Ｅ：ドコサヘキサエン酸エチルエステル。 The fatty acids analyzed are as follows: ETA-E: eicosapentaenoic acid ethyl ester, EPA-E: eicosapentaenoic acid ethyl ester, HPA-E: heneicosapentaenoic acid ethyl ester, DPA-E: Docosapentaenoic acid ethyl ester, DHA-E: Docosahexaenoic acid ethyl ester.
（試料調製方法）
実施例１～３
油脂に対し、水を総投入量の半分量加え、酵素（Lipase from Rhizomucor miehei Sigma-Aldrich社製 製品番号：Ｌ４２７７）を対油脂１５質量％または２３質量％投入し、反応系中を真空状態(真空圧０．９ＭＰａ以下）としながら、３５℃、２．５時間攪拌した。攪拌後、水を総投入量の半分量加え、真空状態で更に２．５時間攪拌して反応物を得た後、遠心分離により油脂と酵素を分離し、生成物を得た。得られた生成物をシリカゲルカラムクロマトグラフィーを用いてω－３高度不飽和脂肪酸エステル画分と遊離脂肪酸画分に分離し、ω－３高度不飽和脂肪酸エステル画分を減圧下、濃縮乾固し、組成物を得た。
(Sample preparation method)
Examples 1 to 3
Half of the total amount of water was added to the fat and oil, and an enzyme (Lipase from Rhizomucor miehei, manufactured by Sigma-Aldrich, product number: L4277) was added at 15% or 23% by weight based on the fat and oil, and the reaction system was placed in a vacuum ( The mixture was stirred at 35° C. for 2.5 hours while maintaining a vacuum pressure of 0.9 MPa or less. After stirring, half of the total input amount of water was added, and the mixture was further stirred in vacuum for 2.5 hours to obtain a reaction product.The oil and fat and enzyme were separated by centrifugation to obtain a product. The obtained product was separated into an ω-3 highly unsaturated fatty acid ester fraction and a free fatty acid fraction using silica gel column chromatography, and the ω-3 highly unsaturated fatty acid ester fraction was concentrated to dryness under reduced pressure. , a composition was obtained.
実施例４～６
油脂に対し、水を総投入量の半分量加え、酵素（Lipase from Rhizomucor miehei Sigma-Aldrich社製 製品番号：Ｌ４２７７）を対油脂４５質量％投入し、反応系中を真空状態(真空圧０．９ＭＰａ以下）としながら、各反応温度で２．５時間攪拌した。攪拌後、水を総投入量の半分量加えて真空状態でさらに２．５時間攪拌し、反応物を得た後、遠心分離により油脂と酵素を分離し、生成物を得た。得られた生成物をシリカゲルカラムクロマトグラフィーを用いてω－３高度不飽和脂肪酸エステル画分と遊離脂肪酸画分に分離し、ω－３高度不飽和脂肪酸エステル画分を減圧下、濃縮乾固し、組成物を得た。
Examples 4-6
Half of the total input amount of water was added to the fat and oil, and 45% by mass of enzyme (Lipase from Rhizomucor miehei, manufactured by Sigma-Aldrich, product number: L4277) was added to the fat and oil, and the reaction system was placed in a vacuum state (vacuum pressure 0. 9 MPa or less) and stirring at each reaction temperature for 2.5 hours. After stirring, half of the total input amount of water was added and the mixture was further stirred in vacuum for 2.5 hours to obtain a reaction product. After that, oil and fat and enzyme were separated by centrifugation to obtain a product. The obtained product was separated into an ω-3 highly unsaturated fatty acid ester fraction and a free fatty acid fraction using silica gel column chromatography, and the ω-3 highly unsaturated fatty acid ester fraction was concentrated to dryness under reduced pressure. , a composition was obtained.
実施例７～８
油脂に対し、水を総投入量の半分量加え、酵素（Lipase from Rhizomucor miehei Sigma-Aldrich社製 製品番号：Ｌ４２７７）を対油脂４５質量％または７５質量％投入し、反応系中を真空状態(真空圧０．９ＭＰａ以下）としながら、各反応温度で２．５時間攪拌した。攪拌後、水を総投入量の半分量加え、真空状態でさらに２．５時間攪拌し、反応物を得た後、遠心分離により油脂と酵素を分離し、生成物を得た。得られた生成物をシリカゲルカラムクロマトグラフィーを用いてω－３高度不飽和脂肪酸エステル画分と遊離脂
肪酸画分に分離し、ω－３高度不飽和脂肪酸エステル画分を減圧下、濃縮乾固し、組成物を得た。
Examples 7-8
Half of the total amount of water was added to the fat and oil, and an enzyme (Lipase from Rhizomucor miehei, manufactured by Sigma-Aldrich, product number: L4277) was added to the fat and oil at 45% or 75% by mass, and the reaction system was placed in a vacuum ( The reaction mixture was stirred for 2.5 hours at each reaction temperature while maintaining a vacuum pressure of 0.9 MPa or less. After stirring, half of the total input amount of water was added, and the mixture was further stirred in vacuum for 2.5 hours to obtain a reaction product. The oil and fat and enzyme were separated by centrifugation to obtain a product. The obtained product was separated into an ω-3 highly unsaturated fatty acid ester fraction and a free fatty acid fraction using silica gel column chromatography, and the ω-3 highly unsaturated fatty acid ester fraction was concentrated to dryness under reduced pressure. , a composition was obtained.
実施例９～１１
各油脂に対し、水を総投入量の１／４量加え、酵素（Lipase from Rhizomucor miehei
Sigma-Aldrich社製 製品番号：Ｌ４２７７）を対油脂４５質量％投入し、反応系中を真空状態(真空圧０．９ＭＰａ以下）としながら、４５℃、２．５時間攪拌した。攪拌後、水を総投入量の１／４量加えて真空状態でさらに２．５時間攪拌し、この操作をさらに２回繰り返して、合計１０時間反応させて反応物を得た。その後、遠心分離により油脂と酵素を分離し、生成物を得た。得られた生成物をシリカゲルカラムクロマトグラフィーを用いてω－３高度不飽和脂肪酸エステル画分と遊離脂肪酸画分に分離し、ω－３高度不飽和脂肪酸エステル画分を減圧下、濃縮乾固し、組成物を得た。
Examples 9-11
Add 1/4 amount of water to each fat and oil, and add enzyme (Lipase from Rhizomucor miehei).
Sigma-Aldrich product number: L4277) was added in an amount of 45% by mass based on oil and fat, and the mixture was stirred at 45° C. for 2.5 hours while the reaction system was kept in a vacuum state (vacuum pressure 0.9 MPa or less). After stirring, water was added in an amount of 1/4 of the total input amount, and the mixture was further stirred in a vacuum for 2.5 hours. This operation was repeated two more times to react for a total of 10 hours to obtain a reaction product. Thereafter, fats and oils and enzymes were separated by centrifugation to obtain a product. The obtained product was separated into an ω-3 highly unsaturated fatty acid ester fraction and a free fatty acid fraction using silica gel column chromatography, and the ω-3 highly unsaturated fatty acid ester fraction was concentrated to dryness under reduced pressure. , a composition was obtained.
実施例１２
油脂に対し、水を総投入量の１／４量加え、酵素（Lipase from Rhizomucor miehei Sigma-Aldrich社製 製品番号：Ｌ４２７７）を対油脂４５質量％投入し、反応系中を真空状態(真空圧０．９ＭＰａ以下）としながら、４５℃、２．５時間攪拌した。攪拌後、水を総投入量の１／４量加え、真空状態でさらに２．５時間攪拌し、この操作をさらに２回繰り返して、合計１０時間反応させて反応物を得た。その後、遠心分離により油脂と酵素を分離し、生成物を得た。得られた生成物を、ＨＰＬＣを用いてω－３高度不飽和脂肪酸エステル画分中のＤＨＡ及びＨＰＡエステル画分を分取精製し、減圧下、濃縮乾固し、組成物を得た。
Example 12
Add 1/4 of the total amount of water to the fat and oil, add 45% by mass of enzyme (Lipase from Rhizomucor miehei, manufactured by Sigma-Aldrich, product number: L4277) to the fat and oil, and place the reaction system in a vacuum state (vacuum pressure). The mixture was stirred at 45° C. for 2.5 hours while maintaining a pressure of 0.9 MPa or less. After stirring, water was added in an amount of 1/4 of the total input amount, and the mixture was further stirred for 2.5 hours in a vacuum state. This operation was repeated two more times to react for a total of 10 hours to obtain a reaction product. Thereafter, fats and oils and enzymes were separated by centrifugation to obtain a product. The DHA and HPA ester fractions in the ω-3 highly unsaturated fatty acid ester fraction were separated and purified using HPLC and concentrated to dryness under reduced pressure to obtain a composition.
＜HPLC分取精製条件＞
カラム ：Ｃ１８ ２５０ｍｍ×２０ｍｍ×５μｍ
温度 ：４０℃
流速 ：１８ｍＬ／ｍｉｎ
波長 ：２０６ｎｍ
移動相 ：メタノール：水＝９０：１０
サンプル濃度 ：サンプル／メタノール＝５０／５０
注入量 ：２００ μＬ
<HPLC preparative purification conditions>
Column: C18 250mm x 20mm x 5μm
Temperature: 40℃
Flow rate: 18mL/min
Wavelength: 206nm
Mobile phase: methanol:water = 90:10
Sample concentration: sample/methanol = 50/50
Injection volume: 200 μL
表中、「油脂」はω－３高度不飽和脂肪酸エステルを含む原料を、「酵素」はリパーゼを意味する。 In the table, "oil" means a raw material containing omega-3 highly unsaturated fatty acid ester, and "enzyme" means lipase.
表１～４に示すように、ω－３高度不飽和脂肪酸エステルを含有する原料をリパーゼによって処理する本発明の方法によって、ＤＨＡとＨＰＡの含有量が増加したω－３高度不飽和脂肪酸エステル含有組成物を製造することができた。また、表５に示すように、さらにＨＰＬＣ分取精製を施すことによって、ＤＨＡとＨＰＡの含有量が一層高いω－３高度不飽和脂肪酸エステル含有組成物を得ることができた。 As shown in Tables 1 to 4, the ω-3 highly unsaturated fatty acid ester containing increased content of DHA and HPA is obtained by the method of the present invention in which the raw material containing the ω-3 highly unsaturated fatty acid ester is treated with lipase. The composition could be manufactured. Moreover, as shown in Table 5, by further performing HPLC preparative purification, it was possible to obtain a composition containing ω-3 highly unsaturated fatty acid ester with higher contents of DHA and HPA.
本発明の製造方法により、優れた生体機能性が知られているＤＨＡとＨＰＡをともに十分な量含有するω－３高度不飽和脂肪酸又はそのエステルを含有する組成物を提供することができるので、食品、健康食品、医薬品等の製造において、極めて利用性が高いものである。 By the production method of the present invention, it is possible to provide a composition containing omega-3 highly unsaturated fatty acids or esters thereof, which contain sufficient amounts of both DHA and HPA, which are known to have excellent biological functionality. It is extremely useful in the production of foods, health foods, pharmaceuticals, etc.
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