JPH07252493A - Method for separating and purifying docosahexaenoic ester from marine fine algae - Google Patents
Method for separating and purifying docosahexaenoic ester from marine fine algaeInfo
- Publication number
- JPH07252493A JPH07252493A JP6045426A JP4542694A JPH07252493A JP H07252493 A JPH07252493 A JP H07252493A JP 6045426 A JP6045426 A JP 6045426A JP 4542694 A JP4542694 A JP 4542694A JP H07252493 A JPH07252493 A JP H07252493A
- Authority
- JP
- Japan
- Prior art keywords
- dha
- ester
- fatty acid
- separating
- purifying
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 20
- 241000195493 Cryptophyta Species 0.000 title claims abstract description 10
- 150000002148 esters Chemical class 0.000 title abstract 2
- 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 73
- 235000020669 docosahexaenoic acid Nutrition 0.000 claims abstract description 50
- 229940090949 docosahexaenoic acid Drugs 0.000 claims abstract description 45
- 229930195729 fatty acid Natural products 0.000 claims abstract description 29
- 239000000194 fatty acid Substances 0.000 claims abstract description 29
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 28
- -1 fatty acid esters Chemical class 0.000 claims abstract description 27
- 150000002632 lipids Chemical class 0.000 claims abstract description 17
- 238000004810 partition chromatography Methods 0.000 claims abstract description 17
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 60
- 239000007788 liquid Substances 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000005191 phase separation Methods 0.000 claims description 3
- DVSZKTAMJJTWFG-SKCDLICFSA-N (2e,4e,6e,8e,10e,12e)-docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCC\C=C\C=C\C=C\C=C\C=C\C=C\C(O)=O DVSZKTAMJJTWFG-SKCDLICFSA-N 0.000 claims description 2
- GZJLLYHBALOKEX-UHFFFAOYSA-N 6-Ketone, O18-Me-Ussuriedine Natural products CC=CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O GZJLLYHBALOKEX-UHFFFAOYSA-N 0.000 claims description 2
- 241000199912 Crypthecodinium cohnii Species 0.000 claims description 2
- KAUVQQXNCKESLC-UHFFFAOYSA-N docosahexaenoic acid (DHA) Natural products COC(=O)C(C)NOCC1=CC=CC=C1 KAUVQQXNCKESLC-UHFFFAOYSA-N 0.000 claims description 2
- 150000001298 alcohols Chemical class 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 11
- 238000000926 separation method Methods 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 23
- 230000005526 G1 to G0 transition Effects 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- ITNKVODZACVXDS-YNUSHXQLSA-N ethyl (4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoate Chemical compound CCOC(=O)CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC ITNKVODZACVXDS-YNUSHXQLSA-N 0.000 description 9
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 8
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 8
- 239000002904 solvent Substances 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000005886 esterification reaction Methods 0.000 description 4
- 235000021323 fish oil Nutrition 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 241000199913 Crypthecodinium Species 0.000 description 3
- 230000001133 acceleration Effects 0.000 description 3
- 125000004494 ethyl ester group Chemical group 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000005192 partition Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000002798 polar solvent Substances 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- QDRKDTQENPPHOJ-UHFFFAOYSA-N sodium ethoxide Chemical compound [Na+].CC[O-] QDRKDTQENPPHOJ-UHFFFAOYSA-N 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 229910015900 BF3 Inorganic materials 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000144181 Thraustochytrium aureum Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 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
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 1
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 1
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000000199 molecular distillation Methods 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 102220201851 rs143406017 Human genes 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Fats And Perfumes (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、医薬品としての開発が
期待されている高純度ドコサヘキサエン酸エステル（以
下ＤＨＡエステルという）の製造方法にかかわり、さら
に詳しくは微細藻を培地に培養して得られる藻体を原料
とする新規なＤＨＡエステルの製造方法に関する。ドコ
サヘキサエン酸（以下ＤＨＡということがある）は、近
年、コレステロール低下作用、抗血液凝固作用、学習機
能向上作用など多彩な生理作用が報告されている高度不
飽和脂肪酸である。FIELD OF THE INVENTION The present invention relates to a method for producing high-purity docosahexaenoic acid ester (hereinafter referred to as DHA ester), which is expected to be developed as a drug, and more specifically, it is obtained by culturing microalgae in a medium. The present invention relates to a novel method for producing a DHA ester using algae as a raw material. Docosahexaenoic acid (hereinafter sometimes referred to as DHA) is a highly unsaturated fatty acid, which has recently been reported to have various physiological actions such as a cholesterol lowering action, an anticoagulant action, and a learning function improving action.
【０００２】[0002]
【従来の技術】多彩な生理作用が報告されている高度不
飽和脂肪酸であるＤＨＡは、古くから魚油中に含まれる
ことが知られている。この魚油中に含まれるＤＨＡは、
尿素付加法、硝酸銀付加法、クロマト分離法、超臨界抽
出法、分子蒸留法等により分離精製されている。しか
し、原料が魚油の場合には、いずれの精製方法を採用し
ても、高純度のＤＨＡエステルを高収率で回収すること
は困難である。一方、これとは別に微生物などに選択的
にＤＨＡを産生させる検討が行なわれてきた。プラチマ
・バッパイらによる検討では、下等な菌類に属するスラ
ウストキトリウム・オーレウム(Thraustochytrium aure
um) にＤＨＡを産生させることが報告されている（App
l.Microbiol.Biotechnol., 35, 706(1991) 参照）が、
これは培養に光を必要とし、さらに物性の似通ったアラ
キドン酸、エイコサペンタエン酸といった他の高度不飽
和脂肪酸を同時に１０〜２０％産生するなどの点で、特
殊な大掛かりな培養装置や高度な分離精製設備を必要と
するなどの問題点があった。また、Ｒ．Ｊ．ヘンダーソ
ンらによる検討では、海洋性微細藻類のクリプテコディ
ニウム・コーニーが高度不飽和脂肪酸としてほぼＤＨＡ
のみを全脂肪酸に対して９％程度産生させることが報告
されている（Phytochemistry, 27 (6), 1697(1988)参
照）が、培養方法が大量培養に向かない静置培養である
点とＤＨＡの含量が低いなどの問題点があった。一方、
本発明者らは、海洋性微細藻類を液体振盪培養や液体深
部培養といった方法で培養することにより、藻体生産性
を高めたばかりでなく、高度不飽和脂肪酸としてはほぼ
ＤＨＡのみを選択的に産生させ、かつ脂質中のＤＨＡの
含量を飛躍的に増大させることを見いだし、これを提案
している（特願平４−３４４２７９号）。しかしなが
ら、ＤＨＡを含有した微生物や藻類を原料とした場合で
も、これから高純度のＤＨＡエステルを高収率で回収し
たという報告例は見当たらない。2. Description of the Related Art DHA, which is a highly unsaturated fatty acid for which various physiological actions have been reported, has long been known to be contained in fish oil. The DHA contained in this fish oil is
It is separated and purified by the urea addition method, silver nitrate addition method, chromatographic separation method, supercritical extraction method, molecular distillation method and the like. However, when the raw material is fish oil, it is difficult to recover a high-purity DHA ester in a high yield, whichever purification method is adopted. On the other hand, separately from this, studies have been conducted to selectively cause DHA to produce DHA. In the study by Platima Bupai et al., Thraustochytrium aureum belonging to the lower fungi
um) has been reported to produce DHA (App
l.Microbiol.Biotechnol., 35 , 706 (1991)),
This requires light for cultivation, and simultaneously produces 10 to 20% of other highly unsaturated fatty acids such as arachidonic acid and eicosapentaenoic acid, which have similar physical properties. There were problems such as the need for refining equipment. In addition, R. J. According to a study by Henderson et al., The marine microalga Crypthecodinium cornie is almost DHA as a highly unsaturated fatty acid.
It has been reported that only about 9% of total fatty acid is produced (see Phytochemistry, 27 (6), 1697 (1988)), but the culturing method is static culture which is not suitable for large-scale culture and DHA. There was a problem such as low content. on the other hand,
The present inventors not only enhance algal productivity by culturing marine microalgae by a method such as liquid shaking culture or liquid submerged culture, but also selectively produce almost only DHA as a highly unsaturated fatty acid. In addition, the inventors have found that the content of DHA in the lipid is drastically increased and proposed this (Japanese Patent Application No. 4-344279). However, even when a microorganism or algae containing DHA is used as a raw material, there is no report that high-purity DHA ester was recovered from this in high yield.
【０００３】[0003]
【発明が解決しようとする課題】本発明の目的は、海洋
性微細藻類に属し、ＤＨＡを産生する能力を有する藻類
を、好ましくは、振盪培養または深部通気撹拌培養して
増殖させて得られた海洋性微細藻類の藻体から、高純度
のＤＨＡエステルを高回収率で取得する分離精製方法を
提供することにある。The object of the present invention was obtained by proliferating algae belonging to marine microalgae and having the ability to produce DHA, preferably by shaking culture or deep aeration stirring culture. An object of the present invention is to provide a separation and purification method for obtaining a highly pure DHA ester with a high recovery rate from an algal body of a marine microalga.
【０００４】[0004]
【課題を解決するための手段】本発明者らは、さきにＤ
ＨＡを産生する能力を有する海洋性微細藻類を、振盪培
養または深部通気撹拌培養することにより、脂質中のＤ
ＨＡの含量が飛躍的に上昇することを見いだし、また、
その脂肪酸組成が非常に特徴的で、高度不飽和脂肪酸と
して、他の不飽和脂肪酸の含有量が少なくほとんどＤＨ
Ａのみである培養法を開発した。本発明者らは、この開
発した培養法の特徴を生かしたＤＨＡの分離精製方法を
検討し、本発明をなすに至った。DISCLOSURE OF THE INVENTION The present inventors have previously described D
By subjecting a marine microalgae capable of producing HA to shaking culture or deep aeration stirring culture,
We have found that the content of HA rises dramatically,
Its fatty acid composition is very characteristic, and as a highly unsaturated fatty acid, the content of other unsaturated fatty acids is low and it is almost DH.
A culture method that is A only was developed. The present inventors have conducted a study on a method for separating and purifying DHA that makes the best use of the characteristics of the developed culture method, and completed the present invention.
【０００５】すなわち、本発明は、ドコサヘキサエン酸
（ＤＨＡ）を含有する脂質を産生する海洋性微細藻類を
培養して得られた該海洋性微細藻類の藻体から脂質を抽
出し、該脂質中の脂肪酸をアルコール類でエステル化し
脂肪酸エステルを得、次いで、これを遠心向流分配クロ
マトグラフィーで処理し、ＤＨＡエステルを含む画分を
分取することを特徴とするＤＨＡエステルの分離精製方
法を提供する。That is, according to the present invention, a lipid is extracted from an alga body of the marine microalgae obtained by culturing a marine microalgae that produces a lipid containing docosahexaenoic acid (DHA), and A method for separating and purifying a DHA ester, which comprises esterifying a fatty acid with an alcohol to obtain a fatty acid ester, which is then subjected to centrifugal countercurrent partition chromatography to fractionate a fraction containing the DHA ester. .
【０００６】さらに、上記海洋性微細藻類が、クリプテ
コディニウム・コーニー（Crypthecodinium cohnii）に
属する藻類であるのが好ましい。Further, it is preferable that the above-mentioned marine microalgae belong to Crypthecodinium cohnii.
【０００７】また、遠心向流分配クロマトグラフィーで
使用する２相分離液が、ヘキサン／アセトニトリルおよ
び／またはヘキサン／メタノール水溶液であるのが好ま
しい。Also, the two-phase separation liquid used in centrifugal countercurrent partition chromatography is preferably an aqueous solution of hexane / acetonitrile and / or hexane / methanol.
【０００８】本発明のＤＨＡエステルの製造に用いる好
ましい微生物は、海洋性微細藻類で、ＤＨＡを生成する
能力のある菌株である。具体例としては、クリプテコデ
ィニウム・コーニーに属する藻類、特に、クリプテコデ
ィニウム・コーニーＡＴＣＣ３００２１、３０５４３、
３０５５６、３０５７１、３０５７２、３０７７５、５
００５１、５００５３、５００５５、５００５６、５０
０５８、５００６０等が挙げられる。しかし、本発明で
精製するＤＨＡエステルは、上記のものによって製造さ
れるものに限定されるものでなく、ＤＨＡが脂質中の脂
肪酸として含まれるものであればいかなるものでもよ
い。The preferred microorganisms used to produce the DHA esters of the present invention are marine microalgae, which are strains capable of producing DHA. Specific examples include algae belonging to Crypthecodinium cornie, particularly Crypthecodinium cornie ATCC 30021, 30543,
30556, 30571, 30572, 30775, 5
0051, 50053, 50055, 50056, 50
058, 50060 and the like. However, the DHA ester to be purified in the present invention is not limited to the one produced by the above, and any DHA ester may be used so long as DHA is contained as a fatty acid in the lipid.
【０００９】上述の海洋性微細藻類の菌体を液体振盪培
養または液体深部培養して、藻体を得る。このような藻
体は、高度不飽和脂肪酸としてＤＨＡを極めて特異的に
産出させるばかりでなく、意外にも脂質中のＤＨＡの割
合が４０％程度にまで上昇するので好ましい。The cells of the above-mentioned marine microalgae are subjected to liquid shaking culture or liquid submerged culture to obtain algal cells. Such an algal body is preferable because it not only produces DHA as a highly unsaturated fatty acid very specifically, but also the ratio of DHA in the lipid is unexpectedly increased to about 40%.
【００１０】本発明の分離精製方法では、上述の藻体、
あるいは藻体を乾燥させた乾燥藻体からＤＨＡエステル
を分離精製するものである。まず、エタノール、ヘキサ
ンおよびその混合物を用いて、上述の藻体から脂質を抽
出する。得られた脂質と低級アルコールとを反応させて
ＤＨＡエステルを得る。低級アルコールとしてはメタノ
ール、エタノール、プロピルアルコール、ブチルアルコ
ールなどを用いることができる。反応にあたっては、エ
ステル化触媒の存在下に行なうのが望ましく、例えば三
フッ化ホウ素、塩酸、硫酸、ナトリウムメトキシド、ナ
トリウムエトキシドなど公知のエステル化触媒を用いる
ことができる。In the separation and purification method of the present invention, the above-mentioned algal cells,
Alternatively, the DHA ester is separated and purified from the dried algal cells obtained by drying the algal cells. First, ethanol, hexane, and a mixture thereof are used to extract lipids from the alga. The DHA ester is obtained by reacting the obtained lipid with a lower alcohol. As the lower alcohol, methanol, ethanol, propyl alcohol, butyl alcohol or the like can be used. It is desirable to carry out the reaction in the presence of an esterification catalyst, and known esterification catalysts such as boron trifluoride, hydrochloric acid, sulfuric acid, sodium methoxide, sodium ethoxide can be used.
【００１１】エステル化反応させた後、脂肪酸エステル
を溶剤抽出などにより回収し、抽出溶剤を留去すること
で、脂肪酸エステルを回収する。次にこの脂肪酸エステ
ルを遠心向流分配クロマトグラフィーにより精製する。After the esterification reaction, the fatty acid ester is recovered by solvent extraction or the like, and the extraction solvent is distilled off to recover the fatty acid ester. The fatty acid ester is then purified by centrifugal countercurrent partition chromatography.
【００１２】遠心向流分配クロマトグラフィーは特開昭
５９−６２３１２号公報に開示されているもので、相互
に自由には混和せず、２相に分離する２種類またはそれ
以上の溶媒の混合液のうち、一方を固定相として遠心力
により保持しつつ、他方を移動相として連続的に固定相
内を通過させて、移動相内に注入された試料を２層間の
物質の分配係数の差を利用して連続的に分画する向流分
配クロマトグラフィーである。遠心向流分配クロマトグ
ラフィーに用いる２相分離液は、ＤＨＡエステルと他の
不純物との分配係数の値を参考にして決めることができ
る。本発明においては、特に、ヘキサン／アセトニトリ
ル、ヘキサン／メタノール水溶液を用いるのが適してい
る。Centrifugal countercurrent partition chromatography is disclosed in Japanese Patent Laid-Open No. 59-62312, and it is a mixed solution of two or more kinds of solvents which do not freely mix with each other but separate into two phases. Among them, one is held as a stationary phase by centrifugal force, while the other is continuously passed through the stationary phase as a mobile phase, and the sample injected into the mobile phase is subjected to a difference in distribution coefficient between the two layers. It is a counter-current partition chromatography that utilizes continuous fractionation. The two-phase separation liquid used for centrifugal countercurrent partition chromatography can be determined with reference to the value of the partition coefficient between the DHA ester and other impurities. In the present invention, it is particularly suitable to use hexane / acetonitrile or hexane / methanol aqueous solution.
【００１３】本発明では比重および極性が異なり、２相
に分離する２種の溶媒の一方を固定相、他方を移動相と
し、遠心力の作用により固定相中を移動相を移動させ、
移動相中に含まれる試料中の各成分を分配係数の差を利
用して多段分配平衡によりクロマトグラフィー的に分画
する。この方法では、試料中の極性の高い成分が順次極
性の高い溶媒に分配され、また極性の低い成分が順次極
性の低い溶媒に分配される。In the present invention, one of two kinds of solvents having different specific gravities and polarities and separated into two phases is used as a stationary phase and the other is used as a mobile phase, and the mobile phase is moved in the stationary phase by the action of centrifugal force.
Each component in the sample contained in the mobile phase is chromatographically fractionated by a multistage partition equilibrium utilizing the difference in partition coefficient. In this method, highly polar components in a sample are sequentially distributed to a highly polar solvent, and low polar components are sequentially distributed to a less polar solvent.
【００１４】図１は本発明で用いられる遠心向流分配ク
ロマトグラフィーの系統図であり、その原理は以下の通
りである。遠心機ローターＲ上に多数の分配管Ｃが遠心
加速度ｇの方向と平行に配列され、相互に直列に導管Ｔ
で接続されている。導管Ｔの両端は遠心機回転軸の両端
に設置された回転送液ジョイントＪ1 、Ｊ2 に接続され
ている。回転送液ジョイントＪ1 、Ｊ2 は４方バルブＶ
3 、６方バルブ（試料インジェクター）Ｖ2 、定流量ポ
ンプＰおよび４方ロータリーバルブＶ1 を介して固定相
ＳＰ、移動相ＭＰ、洗浄液ＷＳの容器に接続され、また
４方バルブＶ3 、フローセルモニターＭおよび３方ロー
タリーバルブＶ4 を介してフラクションコレクターＦお
よび廃液ＷＴの容器に接続している。またＳＬは６方バ
ルブＶ2 を介して試料を移動相ＭＰ中に導入するための
サンプリング回路である。ＲＣは移動相中の試料成分を
検出するフローセルモニターＭで検出されたデーターを
記録するレコーダーである。FIG. 1 is a systematic diagram of centrifugal countercurrent partition chromatography used in the present invention, the principle of which is as follows. A large number of distribution pipes C are arranged on the centrifuge rotor R in parallel with the direction of the centrifugal acceleration g, and the conduits T are arranged in series with each other.
Connected by. Both ends of the conduit T are connected to retransfer liquid joints J 1 and J 2 installed at both ends of the rotary shaft of the centrifuge. Transfer liquid joint J 1 , J 2 is a 4-way valve V
Connected to the stationary phase SP, mobile phase MP, and washing solution WS via a 3- and 6-way valve (sample injector) V 2 , a constant flow pump P and a 4-way rotary valve V 1 , and a 4-way valve V 3 and a flow cell. It is connected to the fraction collector F and the waste liquid WT container via a monitor M and a three-way rotary valve V 4 . SL is a sampling circuit for introducing the sample into the mobile phase MP via the 6-way valve V 2 . RC is a recorder for recording the data detected by the flow cell monitor M for detecting the sample components in the mobile phase.
【００１５】この装置を用いた遠心向流分配クロマトグ
ラフィーは次のような手順で行なわれる。比重および極
性が異なり、２相に分離する任意の溶媒を混合、静置
後、重液相、軽液相にそれぞれ分離し容器に貯える。い
ずれか一方の液相（図面では重液相）を固定相ＳＰとし
て分配管Ｃに充填した後、ローターＲを回転させ、一定
の遠心加速度ｇを与えつつ、他方の液相（図面では軽液
相）を移動相ＭＰとして回転軸の一端にある回転送液ジ
ョイントＪ1 を通して連続的に送液する。移動相ＭＰは
遠心加速度ｇの作用により、固定相ＳＰ中を微細な液滴
となって分配管Ｃ中を順次通過し、回転軸の他端に位置
する回転送液ジョイントＪ2 より連続的に流出する。遠
心機外部に設置されたサンプリング回路ＳＬにより、送
液初期の一定量の移動相ＭＰ中に導入された試料成分
は、上記過程中に遠心向流分配クロマトグラフィーによ
りそれぞれ目的成分に分離され、その後必要に応じてフ
ラクションコレクターＦで分画される。このようにし
て、遠心向流分配クロマトグラフィーにより回収される
ＤＨＡエステルは極めて高純度であり、健康食品用、試
薬用、医薬用に十分利用できる。遠心向流分配クロマト
グラフィーは、必要により複数回行ってもよい。Centrifugal countercurrent partition chromatography using this apparatus is performed in the following procedure. After mixing an arbitrary solvent having different specific gravities and polarities and separating into two phases and allowing to stand, it is separated into a heavy liquid phase and a light liquid phase and stored in a container. After filling one of the liquid phases (heavy liquid phase in the drawing) into the distribution pipe C as the stationary phase SP, the rotor R is rotated to give a constant centrifugal acceleration g, while the other liquid phase (light liquid in the drawing). Phase) as the mobile phase MP, and the liquid is continuously sent through the transfer liquid joint J 1 at one end of the rotary shaft. The mobile phase MP becomes fine droplets in the stationary phase SP by the action of the centrifugal acceleration g and sequentially passes through the distribution pipe C, and continuously from the transfer liquid joint J 2 located at the other end of the rotating shaft. leak. By the sampling circuit SL installed outside the centrifuge, the sample components introduced into the fixed amount of mobile phase MP at the initial stage of liquid transfer are separated into the target components by centrifugal countercurrent distribution chromatography during the above process, and thereafter, Fractionate with Fraction Collector F as needed. Thus, the DHA ester recovered by centrifugal countercurrent partition chromatography has an extremely high purity and can be sufficiently used for health foods, reagents, and medicines. Centrifugal countercurrent partition chromatography may be performed multiple times if necessary.
【００１６】[0016]
【実施例】以下に本発明を実施例によりさらに詳しく説
明するが、これらの実施例が本発明の範囲を限定するも
のではないことはいうまでもない。以下の実施例におい
て、脂肪酸エステル成分はガスクロマトグラフィーで定
量することにより測定した。また、％は重量％である。
また、精製収率は下記の値を示す。* 精製収率＝〔得られた脂肪酸エチルエステルの重量×
ＤＨＡエチルエステルの純度％／粗製脂肪酸エチルエス
テルの重量×ＤＨＡの組成％〕×１００（％）EXAMPLES The present invention will be described in more detail with reference to examples below, but it goes without saying that these examples do not limit the scope of the present invention. In the following examples, the fatty acid ester component was measured by quantifying by gas chromatography. Further,% is% by weight.
Further, the purification yield shows the following values. * Purification yield = [weight of fatty acid ethyl ester obtained x
Purity% of DHA ethyl ester / weight of crude fatty acid ethyl ester × DHA composition%] × 100 (%)
【００１７】（実施例１）乾燥藻体（水分５％）１００
ｇにヘキサン４４０ｍＬを加え、室温で２時間攪拌し、
藻体に含有されるＤＨＡ含有脂質をヘキサン中に抽出し
た。攪拌終了後、吸引濾過により廃藻体を濾別し、ＤＨ
Ａ含有脂質が溶解したヘキサン溶液を回収した。回収し
たヘキサン溶液から、減圧下、エバポレーターにてヘキ
サンを留去し、橙色のＤＨＡ含有脂質２０ｇを得た。次
にこのＤＨＡ含有脂質２０ｇにエタノール２５．６ｍＬ
およびナトリウムエトキシド０．４ｇを加え、窒素雰囲
気下、温度６０℃で２時間攪拌し、エステル化反応を行
なった。反応終了後、反応液を室温まで冷却した。この
反応液にヘキサン４０ｍＬおよび水８ｍＬを加え、５分
間攪拌し、ヘキサン層に脂肪酸エチルエステルを抽出し
た。攪拌終了後、静置分離でヘキサン層を回収した。次
に、このヘキサン層を水２ｍＬで、２回水洗した。水洗
したヘキサン層より、ヘキサンを減圧下、エバポレータ
ーにて留去したところ、橙色の粗製脂肪酸エチルエステ
ル１８ｇが得られた。得られた粗製脂肪酸エチルエステ
ルの組成を表１に示す。(Example 1) 100 dry algal cells (water content 5%)
440 mL of hexane was added to g, and the mixture was stirred at room temperature for 2 hours,
The DHA-containing lipid contained in the alga was extracted into hexane. After completion of stirring, waste algal cells are filtered off by suction filtration, and DH
A hexane solution in which the A-containing lipid was dissolved was collected. From the recovered hexane solution, hexane was distilled off under reduced pressure with an evaporator to obtain 20 g of orange DHA-containing lipid. Next, 25.6 mL of ethanol was added to 20 g of this DHA-containing lipid.
And 0.4 g of sodium ethoxide were added, and the mixture was stirred under a nitrogen atmosphere at a temperature of 60 ° C. for 2 hours to carry out an esterification reaction. After completion of the reaction, the reaction solution was cooled to room temperature. 40 mL of hexane and 8 mL of water were added to this reaction solution, and the mixture was stirred for 5 minutes to extract the fatty acid ethyl ester in the hexane layer. After completion of stirring, the hexane layer was recovered by stationary separation. Next, this hexane layer was washed twice with 2 mL of water. Hexane was distilled off from the washed hexane layer under reduced pressure with an evaporator to obtain 18 g of orange crude fatty acid ethyl ester. The composition of the obtained crude fatty acid ethyl ester is shown in Table 1.
【００１８】 [0018]
【００１９】次にこの粗製脂肪酸エチルエステルを遠心
向流分配クロマトグラフィーにより精製した。用いた装
置は、三鬼エンジニアリング（株）製ＣＰＣ−ＬＬＮで
ある。分配液としては、ヘキサンおよびアセトニトリル
を混合後静置して分離した軽液を固定相として２５０ｍ
Ｌ充填し、重液を移動相とした。次に表１の組成のサン
プル１．０ｇを５ｍＬとなるように移動相に溶解した溶
液を注入し、その後、移動相を流量３．５ｍＬ／分で
５．５時間流通させた。その時のローターの回転数は７
００ｒｐｍ、操作温度は２０℃であった。上記により、
溶出した移動相（重液）から、減圧下、エバポレーター
にて溶剤を留去したところ、わずかに黄色味を帯びた脂
肪酸エチルエステル０．３２５ｇが回収された。回収さ
れた脂肪酸エチルエステル中のＤＨＡエチルエステルの
純度は、９６．７％であり、ＤＨＡエチルエステルの精
製収率* は９１．５％であった。The crude fatty acid ethyl ester was then purified by centrifugal countercurrent partition chromatography. The apparatus used was CPC-LLN manufactured by Miki Engineering Co., Ltd. As a distribution liquid, a light liquid separated by mixing hexane and acetonitrile and then allowing to stand is 250 m as a stationary phase.
L was filled and the heavy liquid was used as a mobile phase. Next, a solution prepared by dissolving 1.0 g of the sample having the composition shown in Table 1 in the mobile phase was injected to 5 mL, and then the mobile phase was circulated at a flow rate of 3.5 mL / min for 5.5 hours. Rotor speed at that time is 7
The operating temperature was 00 rpm and 20 ° C. By the above,
When the solvent was distilled off from the eluted mobile phase (heavy liquid) under reduced pressure with an evaporator, 0.325 g of a slightly yellowish fatty acid ethyl ester was recovered. The purity of DHA ethyl ester in the recovered fatty acid ethyl ester was 96.7%, and the purification yield * of DHA ethyl ester was 91.5%.
【００２０】（実施例２）実施例１で精製したＤＨＡエ
チルエステルを、再度、遠心向流分配クロマトグラフィ
ーで精製した。用いた装置は実施例１と同じである。分
配液としては、ヘキサンおよび９０％メタノール水溶液
を混合後静置して分離した軽液を固定相として２５０ｍ
Ｌ充填し、重液を移動相とした。次に実施例１で回収し
た脂肪酸エチルエステル（ＤＨＡエチルエステル純度：
９６．７％）１．０ｇを５ｍＬとなるように移動相に溶
解した溶液を注入し、その後、移動相（重液）を流量
３．３ｍＬ／分で１７．５時間流通させた。次に、送液
方向を反転して、軽液を流量３．３ｍＬ／分で２時間流
通させた。この時のローターの回転数は６００ｒｐｍ、
操作温度は２０℃であった。溶出した固定相（軽液）か
ら、減圧下、エバポレーターにて溶剤を留去したとこ
ろ、無色透明な脂肪酸エチルエステル０．９２０ｇが回
収された。回収された脂肪酸エチルエステル中のＤＨＡ
エチルエステルの純度は、９９．９％であり、ＤＨＡエ
チルエステルの精製収率* は９５．０％であった。Example 2 The DHA ethyl ester purified in Example 1 was purified again by centrifugal countercurrent partition chromatography. The apparatus used is the same as in Example 1. As a distribution liquid, a light liquid separated by standing still after mixing hexane and a 90% aqueous methanol solution was used as a stationary phase at 250 m.
L was filled and the heavy liquid was used as a mobile phase. Next, fatty acid ethyl ester recovered in Example 1 (DHA ethyl ester purity:
A solution prepared by dissolving 1.0 g of 96.7%) in 5 mL was injected into the mobile phase, and then the mobile phase (heavy liquid) was circulated for 17.5 hours at a flow rate of 3.3 mL / min. Next, the liquid feeding direction was reversed, and the light liquid was circulated for 2 hours at a flow rate of 3.3 mL / min. The rotation speed of the rotor at this time is 600 rpm,
The operating temperature was 20 ° C. When the solvent was distilled off from the eluted stationary phase (light liquid) under reduced pressure with an evaporator, 0.920 g of colorless and transparent fatty acid ethyl ester was recovered. DHA in recovered fatty acid ethyl ester
The purity of ethyl ester was 99.9%, and the purification yield * of DHA ethyl ester was 95.0%.
【００２１】（実施例３）実施例１の表１に示した組成
の粗製脂肪酸エチルエステル１．０ｇを、実施例２と同
じ条件の下で遠心向流配分クロマトグラフィーにより精
製した。無色透明な脂肪酸エチルエステル０．４１２ｇ
が回収された。回収された脂肪酸エチルエステル中のＤ
ＨＡエチルエステルの純度は、７５．０％であり、ＤＨ
Ａエチルエステルの精製収率* は９０．０％であった。Example 3 1.0 g of crude fatty acid ethyl ester having the composition shown in Table 1 of Example 1 was purified by centrifugal countercurrent distribution chromatography under the same conditions as in Example 2. Colorless and transparent fatty acid ethyl ester 0.412g
Was recovered. D in recovered fatty acid ethyl ester
The purity of HA ethyl ester is 75.0%, DH
The purification yield * of A ethyl ester was 90.0%.
【００２２】（実施例４）実施例３で精製したＤＨＡエ
チルエステル１．０ｇを、実施例１と同じ条件の下で遠
心向流分配クロマトグラフィーにより精製した。無色透
明な脂肪酸エチルエステル０．７１３ｇが回収された。
回収された脂肪酸エチルエステル中のＤＨＡエチルエス
テルの純度は、９９．９％であり、ＤＨＡエチルエステ
ルの精製収率* は９５．０％であった。(Example 4) 1.0 g of DHA ethyl ester purified in Example 3 was purified by centrifugal countercurrent partition chromatography under the same conditions as in Example 1. 0.713 g of colorless and transparent fatty acid ethyl ester was recovered.
The purity of DHA ethyl ester in the recovered fatty acid ethyl ester was 99.9%, and the purification yield * of DHA ethyl ester was 95.0%.
【００２３】[0023]
【発明の効果】本発明はＤＨＡを産生する能力を有する
海洋性微細藻類を、振盪培養または深部通気攪拌培養す
ることにより、他の高度不飽和脂肪酸をほとんど含ま
ず、ＤＨＡの含量のみを上昇させることを見いだしたこ
とを基に、従来は原料の供給が不安定で品質が一定せ
ず、独特の臭気をもつ魚油からの抽出と高度な分離精製
技術により得ていた高純度ＤＨＡエステルを簡便な精製
で製造できる点、およびＤＨＡエステルの回収率が極め
て高い点で、その経済的効果は非常に大である。INDUSTRIAL APPLICABILITY According to the present invention, marine microalgae capable of producing DHA are shake-cultured or submerged-agitated and agitated to increase the content of DHA without containing other polyunsaturated fatty acids. Based on these findings, the high-purity DHA ester that was previously obtained by the extraction of fish oil with a unique odor and the advanced separation and purification technology was unstable because the supply of raw materials was unstable and the quality was simple. The economical effect is very large because it can be produced by purification and the recovery rate of DHA ester is extremely high.
【図１】 遠心向流分配クロマトグラフィーの系統図で
ある。FIG. 1 is a systematic diagram of centrifugal countercurrent partition chromatography.
ＳＰ 固定相 ＭＰ 移動相 ＷＳ 洗浄液 ＷＴ 廃液 Ｖ1 ４方ロータリーバルブ Ｖ2 ６方バルブ Ｖ3 ４方バルブ Ｖ4 ３方ロータリーバルブ ＳＬ サンプリング回路 Ｐ 定量ポンプ Ｊ1 ，Ｊ2 回転送液ジョイント Ｒ ローター Ｃ 分配管 Ｔ 導管 Ｍ フローセルモニター ＲＣ レコーダー Ｆ フラクションコレクターSP Stationary phase MP Mobile phase WS Wash solution WT Waste solution V 1 4-way rotary valve V 2 6-way valve V 3 4-way valve V 4 3-way rotary valve SL Sampling circuit P Metering pump J 1 , J 2 times transfer liquid joint R rotor C Distribution pipe T Conduit M Flow cell monitor RC recorder F Fraction collector
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 //(Ｃ１２Ｐ 7/64 Ｃ１２Ｒ 1:89） ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification number Office reference number FI technical display area // (C12P 7/64 C12R 1:89)
Claims (3)
脂質を産生する海洋性微細藻類を培養して得られた該海
洋性微細藻類の藻体から脂質を抽出し、該脂質中の脂肪
酸をアルコール類でエステル化し脂肪酸エステルを得、
次いで、これを遠心向流分配クロマトグラフィーで処理
し、ドコサヘキサエン酸エステルを含む画分を分取する
ことを特徴とするドコサヘキサエン酸エステルの分離精
製方法。1. A lipid is extracted from an algal body of a marine microalga obtained by culturing a marine microalga that produces a lipid containing docosahexaenoic acid (DHA), and fatty acids in the lipid are alcohols. To obtain a fatty acid ester
Then, this is subjected to centrifugal countercurrent partition chromatography to separate a fraction containing docosahexaenoic acid ester, and a method for separating and purifying docosahexaenoic acid ester.
ウム・コーニー（Crypthecodiniumcohnii）に属する藻
類である請求項１に記載のドコサヘキサエン酸エステル
の分離精製方法。2. The method for separating and purifying docosahexaenoic acid ester according to claim 1, wherein the marine microalgae are algae belonging to Crypthecodinium cohnii.
用する２相分離液が、ヘキサン／アセトニトリルおよび
／またはヘキサン／メタノール水溶液である請求項１ま
たは２に記載のドコサヘキサエン酸エステルの分離精製
方法。3. The method for separating and purifying docosahexaenoic acid ester according to claim 1 or 2, wherein the two-phase separation liquid used in the centrifugal countercurrent partition chromatography is an aqueous solution of hexane / acetonitrile and / or hexane / methanol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6045426A JPH07252493A (en) | 1994-03-16 | 1994-03-16 | Method for separating and purifying docosahexaenoic ester from marine fine algae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6045426A JPH07252493A (en) | 1994-03-16 | 1994-03-16 | Method for separating and purifying docosahexaenoic ester from marine fine algae |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07252493A true JPH07252493A (en) | 1995-10-03 |
Family
ID=12718964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6045426A Pending JPH07252493A (en) | 1994-03-16 | 1994-03-16 | Method for separating and purifying docosahexaenoic ester from marine fine algae |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07252493A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103086873A (en) * | 2013-01-11 | 2013-05-08 | 国家海洋局第三海洋研究所 | Preparation method of high-purity DHA (Docosahexaenoic Acid) by means of high-speed counter-current chromatography separation |
| JP2014514924A (en) * | 2011-04-06 | 2014-06-26 | ヘリアエ デベロップメント、 エルエルシー | Extraction of neutral lipids by two-solvent method |
| CN104017734A (en) * | 1996-03-28 | 2014-09-03 | Dsmip资产有限公司 | Process for the preparation of granular microbial biomass and isolation of valuable compounds therefrom |
-
1994
- 1994-03-16 JP JP6045426A patent/JPH07252493A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017734A (en) * | 1996-03-28 | 2014-09-03 | Dsmip资产有限公司 | Process for the preparation of granular microbial biomass and isolation of valuable compounds therefrom |
| JP2014514924A (en) * | 2011-04-06 | 2014-06-26 | ヘリアエ デベロップメント、 エルエルシー | Extraction of neutral lipids by two-solvent method |
| CN103086873A (en) * | 2013-01-11 | 2013-05-08 | 国家海洋局第三海洋研究所 | Preparation method of high-purity DHA (Docosahexaenoic Acid) by means of high-speed counter-current chromatography separation |
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