JPH0595792A - Production of oil and fat containing concentrated highly unsaturated fatty acid - Google Patents
Production of oil and fat containing concentrated highly unsaturated fatty acidInfo
- Publication number
- JPH0595792A JPH0595792A JP3283560A JP28356091A JPH0595792A JP H0595792 A JPH0595792 A JP H0595792A JP 3283560 A JP3283560 A JP 3283560A JP 28356091 A JP28356091 A JP 28356091A JP H0595792 A JPH0595792 A JP H0595792A
- Authority
- JP
- Japan
- Prior art keywords
- fatty acid
- highly unsaturated
- unsaturated fatty
- lipase
- oil
- 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.)
- Granted
Links
- 235000021122 unsaturated fatty acids Nutrition 0.000 title claims abstract description 111
- 150000004670 unsaturated fatty acids Chemical class 0.000 title claims abstract description 105
- 238000004519 manufacturing process Methods 0.000 title claims description 7
- 108090001060 Lipase Proteins 0.000 claims abstract description 99
- 239000004367 Lipase Substances 0.000 claims abstract description 99
- 102000004882 Lipase Human genes 0.000 claims abstract description 99
- 235000019421 lipase Nutrition 0.000 claims abstract description 99
- 238000000034 method Methods 0.000 claims abstract description 65
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 claims abstract description 57
- -1 alcohol ester Chemical class 0.000 claims abstract description 23
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims abstract description 13
- 241000589516 Pseudomonas Species 0.000 claims abstract description 12
- 235000020660 omega-3 fatty acid Nutrition 0.000 claims description 67
- 239000000047 product Substances 0.000 claims description 67
- 235000019197 fats Nutrition 0.000 claims description 66
- 239000002904 solvent Substances 0.000 claims description 53
- 238000006243 chemical reaction Methods 0.000 claims description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 41
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 claims description 35
- 239000012141 concentrate Substances 0.000 claims description 29
- 238000006460 hydrolysis reaction Methods 0.000 claims description 29
- 230000007062 hydrolysis Effects 0.000 claims description 23
- 239000011261 inert gas Substances 0.000 claims description 17
- 230000003301 hydrolyzing effect Effects 0.000 claims description 13
- 238000006555 catalytic reaction Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 239000006227 byproduct Substances 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims 1
- 235000019198 oils Nutrition 0.000 abstract description 88
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract description 71
- 239000002994 raw material Substances 0.000 abstract description 20
- 239000003957 anion exchange resin Substances 0.000 abstract description 15
- 235000021323 fish oil Nutrition 0.000 abstract description 11
- 230000003100 immobilizing effect Effects 0.000 abstract description 10
- 238000001640 fractional crystallisation Methods 0.000 abstract description 9
- 239000004925 Acrylic resin Substances 0.000 abstract description 7
- 229920000178 Acrylic resin Polymers 0.000 abstract description 7
- 244000005700 microbiome Species 0.000 abstract description 6
- 235000013376 functional food Nutrition 0.000 abstract description 3
- 230000003449 preventive effect Effects 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 239000003921 oil Substances 0.000 description 83
- 239000003925 fat Substances 0.000 description 64
- 235000014113 dietary fatty acids Nutrition 0.000 description 58
- 229930195729 fatty acid Natural products 0.000 description 58
- 239000000194 fatty acid Substances 0.000 description 58
- 230000005484 gravity Effects 0.000 description 58
- 150000004665 fatty acids Chemical class 0.000 description 56
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 48
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 34
- 235000011187 glycerol Nutrition 0.000 description 34
- 238000000354 decomposition reaction Methods 0.000 description 32
- 239000000203 mixture Substances 0.000 description 26
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound 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 description 22
- 239000000758 substrate Substances 0.000 description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 20
- 238000003786 synthesis reaction Methods 0.000 description 20
- 235000019512 sardine Nutrition 0.000 description 18
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 16
- 241001125048 Sardina Species 0.000 description 16
- 230000009471 action Effects 0.000 description 16
- 230000015572 biosynthetic process Effects 0.000 description 16
- 238000000926 separation method Methods 0.000 description 15
- 150000003626 triacylglycerols Chemical class 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 description 12
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 description 12
- 238000003756 stirring Methods 0.000 description 12
- 235000020669 docosahexaenoic acid Nutrition 0.000 description 11
- 229940090949 docosahexaenoic acid Drugs 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 10
- 239000007795 chemical reaction product Substances 0.000 description 10
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 108010093096 Immobilized Enzymes Proteins 0.000 description 8
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 8
- 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 8
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 8
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 8
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 8
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 description 8
- 125000005456 glyceride group Chemical group 0.000 description 8
- 241000588881 Chromobacterium Species 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000235395 Mucor Species 0.000 description 6
- 230000001476 alcoholic effect Effects 0.000 description 6
- 230000018044 dehydration Effects 0.000 description 6
- 238000006297 dehydration reaction Methods 0.000 description 6
- 238000006911 enzymatic reaction Methods 0.000 description 6
- 238000001704 evaporation Methods 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 6
- 235000014593 oils and fats Nutrition 0.000 description 6
- 238000005192 partition Methods 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 229940013317 fish oils Drugs 0.000 description 5
- YUFFSWGQGVEMMI-JLNKQSITSA-N (7Z,10Z,13Z,16Z,19Z)-docosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCCCC(O)=O YUFFSWGQGVEMMI-JLNKQSITSA-N 0.000 description 4
- 208000005156 Dehydration Diseases 0.000 description 4
- 235000021294 Docosapentaenoic acid Nutrition 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 4
- 230000002411 adverse Effects 0.000 description 4
- 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 4
- 229910052786 argon Inorganic materials 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 125000004432 carbon atom Chemical group C* 0.000 description 4
- 239000001569 carbon dioxide Substances 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 229910001873 dinitrogen Inorganic materials 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000032050 esterification Effects 0.000 description 4
- 238000005886 esterification reaction Methods 0.000 description 4
- 125000004494 ethyl ester group Chemical group 0.000 description 4
- 235000013402 health food Nutrition 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 4
- 150000002978 peroxides Chemical class 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- 235000019688 fish Nutrition 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 150000004671 saturated fatty acids Chemical class 0.000 description 3
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 241000555825 Clupeidae Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 2
- 108010048733 Lipozyme Proteins 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 241000269821 Scombridae Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 239000002156 adsorbate Substances 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000021342 arachidonic acid Nutrition 0.000 description 2
- 229940114079 arachidonic acid Drugs 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 150000002170 ethers Chemical class 0.000 description 2
- 235000021588 free fatty acids Nutrition 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 229930195733 hydrocarbon Natural products 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000006317 isomerization reaction Methods 0.000 description 2
- 150000002576 ketones Chemical class 0.000 description 2
- 235000010445 lecithin Nutrition 0.000 description 2
- 239000000787 lecithin Substances 0.000 description 2
- 229940067606 lecithin Drugs 0.000 description 2
- 235000020778 linoleic acid Nutrition 0.000 description 2
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 2
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical compound NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 description 2
- 235000020640 mackerel Nutrition 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000008239 natural water Substances 0.000 description 2
- 229910052754 neon Inorganic materials 0.000 description 2
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
- 229920005862 polyol Polymers 0.000 description 2
- 150000003077 polyols Chemical class 0.000 description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 2
- CASUWPDYGGAUQV-UHFFFAOYSA-M potassium;methanol;hydroxide Chemical compound [OH-].[K+].OC CASUWPDYGGAUQV-UHFFFAOYSA-M 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000007127 saponification reaction Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 235000003441 saturated fatty acids Nutrition 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000003760 tallow Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 1
- 239000003026 cod liver oil Substances 0.000 description 1
- 235000012716 cod liver oil Nutrition 0.000 description 1
- 239000010779 crude oil Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000010685 fatty oil Substances 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Fats And Perfumes (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は濃縮された高度不飽和脂
肪酸油脂、特に、濃縮されたω−３高度不飽和脂肪酸含
有油脂の製造方法に関する。なお、本明細書におけるω
−３高度不飽和脂肪酸とは、炭素鎖の末端メチルから第
３番目と第４番目の炭素の結合が二重結合であり、かつ
炭素鎖中の全二重結合数が２個以上の不飽和脂肪酸を意
味する。また、高度不飽和脂肪酸含有油脂とは、全二重
結合数が２以上の不飽和脂肪酸トリグリセリドを含む油
脂を意味する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a concentrated polyunsaturated fatty acid oil and fat, and more particularly to a method for producing a concentrated omega-3 highly unsaturated fatty acid-containing oil and fat. In this specification, ω
-3 Polyunsaturated fatty acid is a unsaturated bond in which the third and fourth carbon bonds from the terminal methyl of the carbon chain are double bonds, and the total number of double bonds in the carbon chain is 2 or more. Means fatty acid. The polyunsaturated fatty acid-containing fat or oil means a fat or oil containing an unsaturated fatty acid triglyceride having a total number of double bonds of 2 or more.
【０００２】[0002]
【従来の技術及びその問題点】ω−３高度不飽和脂肪酸
含有油脂は、高級飽和脂肪酸油脂が主成分を占める牛脂
などとは異り、各種魚類の油、肝油などの海産食品に多
く含まれており、最近では、健康食品の成分として知ら
れるアラキドン酸やリノール酸などのω−６高度不飽和
脂肪酸とバランス良く摂取することが必要であると言わ
れている。このバランスをとるため、ω−３高度不飽和
脂肪酸のエチルエステルが使用されている。しかしなが
らこの脂肪酸のエチルエステルは、原油脂すなわち脂肪
酸トリグリセリドと比較して消化吸収の程度が低い〔例
えばＣａｒｏｌ Ｍ． Ｗｏｊｅｎｓｈｉ氏らのＢｉｏ
ｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ａｃｔａ １０８１（１９
９１）３３〜３８参照〕。一方、魚油等の原料油脂を加
水分解してω−３高度不飽和脂肪酸を濃縮生成させる手
段として、酵素を用いる方法が考えられ、リパーゼを用
いた場合の不飽和脂肪酸と飽和脂肪酸との分解速度の差
を利用する方法〔Ｔ．Ｈｏｓｈｉｎｏ，Ｔ．Ｙａｍａｎ
ｅおよびＳ．Ｓｈｉｍｉｚｕ氏らのＡｇｒｉｃ．Ｂｉｏ
ｌ．Ｃｈｅｍ．５４（１９９０）、１４５９−１４６
７〕あるいはキャンディダ属菌に由来するリパーゼを用
いてω−３高度不飽和脂肪酸油脂を選択的に加水分解
後、この脂肪酸とグリセリンとを、クロモバクテリウム
属の細菌に由来するリパーゼの作用で反応させてトリグ
リセリドを合成することでω−３高度不飽和脂肪酸を濃
縮する方法〔田中、今村および小菅氏らによる「実戦バ
イオリアクター」、食品産業バイオリアクターシステム
技術研究組合成果論文集（１９９０年）第３９１〜４１
１頁〕が知られているが、これらの方法においてω−３
高度不飽和脂肪酸を濃縮するプロセスはいずれも原料油
脂をリパーゼの作用で選択的に加水分解する工程のみに
置かれ、方法全体としては効率的とは言い難い。さらに
リパーゼを用いる方法として、いわし油を加水分解し、
ω−３高度不飽和脂肪酸を濃縮し、次いでグリセリンを
用いて同じくクロモバクテリウム属の細菌に由来するリ
パーゼの作用によりこの脂肪酸のトリグリセリドを合成
する方法〔長田、高橋、羽田野および細川氏らによる日
水誌５７（１９９１年）、第１１９〜１２５頁〕が知ら
れているが、この方法によるトリグリセリドの収率は５
０％以下であって効率的な方法とは言えない。ω−３高
度不飽和脂肪酸含有油脂は前述したように健康食品とし
て摂取することが望まれるが、このものは分子中の不飽
和結合部分で酸化され易く、また、二重結合の移動、異
性化が起り易くまた価格的にも非常に高価である。した
がってω−３高度不飽和脂肪酸を一層高度な割合で含有
する油脂を得ることが望まれている。2. Description of the Related Art ω-3 Polyunsaturated fatty acid-containing fats and oils are different from beef tallow in which higher saturated fatty acid fats are the main component, and are often contained in marine foods such as various fish oils and liver oils. Recently, it has been said that it is necessary to take a well-balanced intake with ω-6 highly unsaturated fatty acids such as arachidonic acid and linoleic acid which are known as components of health foods. To balance this, ethyl esters of omega-3 highly unsaturated fatty acids have been used. However, the ethyl ester of this fatty acid has a lower degree of digestion and absorption as compared to crude oil and fat, ie fatty acid triglyceride [eg Carol M. et al. Bio by Wojenshi et al.
chem. Biophys. Acta 1081 (19
91) 33-38]. On the other hand, as a means for hydrolyzing raw material fats such as fish oil to concentrate and produce ω-3 highly unsaturated fatty acids, a method using an enzyme is considered, and the decomposition rate of unsaturated fatty acids and saturated fatty acids when lipase is used. Method using the difference of [T. Hoshino, T .; Yaman
e and S.E. Shimizu et al., Agric. Bio
l. Chem. 54 (1990), 1459-146.
7] Alternatively, after selectively hydrolyzing ω-3 highly unsaturated fatty acid oil and fat using a lipase derived from Candida, the fatty acid and glycerin are treated by the action of lipase derived from a bacterium of the genus Chromobacterium. A method of concentrating ω-3 highly unsaturated fatty acids by reacting to synthesize triglyceride [“actual bioreactor” by Tanaka, Imamura and Kosuge et al., Food Industry Bioreactor System Technology Research Association Results Collection (1990) 391st to 41st
1 page] is known, but in these methods, ω-3
All of the processes for concentrating highly unsaturated fatty acids are placed only in the step of selectively hydrolyzing the raw material fats and oils by the action of lipase, and it cannot be said that the method as a whole is efficient. Furthermore, as a method using lipase, hydrolyzing sardine oil,
A method of concentrating ω-3 highly unsaturated fatty acids and then using glycerin to synthesize triglycerides of these fatty acids by the action of a lipase derived from bacteria of the genus Chromobacterium [Nagata, Takahashi, Hatano and Hosokawa et al. Water magazine 57 (1991), pp. 119-125] is known, but the yield of triglyceride by this method is 5
It is less than 0% and cannot be said to be an efficient method. As described above, ω-3 highly unsaturated fatty acid-containing fats and oils are desired to be ingested as health foods, but these are easily oxidized at the unsaturated bond portion in the molecule, and the double bond migration and isomerization are also likely to occur. Is likely to occur and is very expensive in price. Therefore, it is desired to obtain an oil / fat containing a higher proportion of ω-3 highly unsaturated fatty acid.
【０００３】[0003]
【発明が解決しようとする課題】本発明は、高度不飽和
脂肪酸含有油脂、特に、ω−３高度不飽和脂肪酸含有油
脂を、この脂肪酸の変性を起すことなく効率的に加水分
解し、さらに加水分解生成物からこの高度不飽和脂肪酸
を濃縮し、次いでグリセリンを作用させて高度に濃縮さ
れた高度不飽和脂肪酸のトリグリセリドを製造する方法
を提供することをその課題とする。SUMMARY OF THE INVENTION The present invention efficiently hydrolyzes highly unsaturated fatty acid-containing fats and oils, particularly ω-3 highly unsaturated fatty acid-containing fats and oils without causing modification of the fatty acid, and further hydrolyzes the fatty acid. It is an object of the present invention to provide a method for concentrating this highly unsaturated fatty acid from a decomposition product and then allowing glycerin to act thereon to produce a highly concentrated triglyceride of highly unsaturated fatty acid.
【０００４】[0004]
【課題を解決するための手段】本発明者らは前記課題を
解決するため鋭意検究を重ねた結果、本発明を完成し
た。The present inventors have completed the present invention as a result of intensive investigations to solve the above-mentioned problems.
【０００５】本発明によれば、高度不飽和脂肪酸含有油
脂または高度不飽和脂肪酸のアルコールエステルを固定
化されたリパーゼを用いて加水分解する工程、得られた
加水分解生成物に含まれる高度不飽和脂肪酸を濃縮する
工程及び高度不飽和脂肪酸濃縮物を固定化されたリパー
ゼを用いてトリグリセリドに変換させる工程からなる濃
縮された高度不飽和脂肪酸含有油脂の製造方法が提供さ
れる。高度不飽和脂肪酸は化学的に不安定なために前記
の各工程は不活性気体の雰囲気中で実施するのが好まし
い。According to the present invention, a step of hydrolyzing a fat or oil containing a polyunsaturated fatty acid or an alcohol ester of a polyunsaturated fatty acid using an immobilized lipase, a polyunsaturation contained in the obtained hydrolysis product, Provided is a method for producing a concentrated highly unsaturated fatty acid-containing fat or oil, which comprises a step of concentrating a fatty acid and a step of converting a highly unsaturated fatty acid concentrate into a triglyceride using immobilized lipase. Since the polyunsaturated fatty acids are chemically unstable, it is preferable to carry out the above steps in an atmosphere of an inert gas.
【０００６】本発明は、高度不飽和脂肪酸含有油脂を微
生物由来のリパーゼの作用により選択的に加水分解し、
得られたω−３高度不飽和脂肪酸を濃縮し、次いで微生
物由来のリパーゼの作用によりグリセリンと反応させて
トリグリセライドとすることにより濃縮されたω−３高
度不飽和脂肪酸含有油脂を得ることを意図するものであ
る。The present invention selectively hydrolyzes oils and fats containing highly unsaturated fatty acids by the action of lipase derived from microorganisms,
It is intended to obtain a concentrated ω-3 highly unsaturated fatty acid-containing oil or fat by concentrating the obtained ω-3 highly unsaturated fatty acid and then reacting it with glycerin by the action of lipase derived from a microorganism to form triglyceride. It is a thing.
【０００７】本発明において原料として使用される油脂
は、高度不飽和脂肪酸トリグリセリド、特に、ω−３高
度不飽和脂肪酸トリグリセリドを高割合例えば１０重量
％以上含有する油脂であり、このような油脂は海産性ま
たは淡水産性のプランクトンを捕食している生物、例え
ば魚類の油脂、一般的に魚油と呼ばれるもので、大量に
捕獲できる鰯、さば等の魚油、またはこれら魚類の肝油
である。また鰯油のようなω−３高度不飽和脂肪酸を含
有する油脂をエタノールのようなアルコールでアルコー
ル分解したアルコールエステルも本発明の原料として使
用できる。The fats and oils used as a raw material in the present invention are highly unsaturated fatty acid triglycerides, particularly fats and oils containing a high proportion of ω-3 highly unsaturated fatty acid triglycerides, for example, 10% by weight or more. Organisms that eat plankton that are natural or freshwater, for example, fish oils and fats, generally called fish oil, which are fish oils such as sardines, mackerel, and the like, or liver oils of these fishes. Further, an alcohol ester obtained by alcoholyzing an oil or fat containing ω-3 highly unsaturated fatty acid such as sardine oil with an alcohol such as ethanol can also be used as the raw material of the present invention.
【０００８】本発明における前記油脂の加水分解は、ア
ルカリ等の化学的手段を用いることなく、リパーゼ等の
酵素を用いて生化学的手段で実施する。リパーゼは、油
脂のアシル基を加水分解し、脂肪酸とグリセリンを生成
させる。本発明で使用するリパーゼは微生物由来のも
の、例えば、シュウドモナス属菌、キャンディダ属菌、
クロモバクテリゥム属菌等のリパーゼで、特に、シュウ
ドモナス属菌のリパーゼが好適に使用できる。リパーゼ
は通常担体に固定して使用するのが好適であり、この担
体としては陰イオン交換樹脂が好適であり、リパーゼを
陰イオン交換樹脂に固定化した固定化リパーゼは市販品
も入手できる。中でもシュウドモナス属の菌に由来する
リパーゼを陰イオン交換樹脂に固定したリパーゼは加水
分解の反応物質が流動性を持つ５０〜６０℃の温度にお
いても長時間安定であり、魚油等の油脂の分解性もよ
く、後記する実施例１に記載したように分解率６６％以
上に加水分解を行うとω−３高度不飽和脂肪酸を効率よ
く生成するので望ましい分解酵素である。魚油分解の際
の化学組成は未分解のトリグリセリド、一部分解のジグ
リセリドおよび完全分解した脂肪酸であり、モノグリセ
リドの割合は極めて少ない。このモノグリセリドは界面
活性作用が大きいので生成した脂肪酸の分離を因難にす
るので好ましくないが、このモノグリセリドの量が少な
いことは分離効率を高めることになる。また未分解のグ
リセリドにはエイコサンペンタエン酸が濃縮される傾向
を示す。油脂の加水分解率は、脂肪酸にまで分解された
油脂の重量％で表わして、７０％以上、好ましくは８０
〜９９重量％である。The hydrolysis of the fat or oil in the present invention is carried out by a biochemical means using an enzyme such as lipase without using a chemical means such as an alkali. Lipase hydrolyzes the acyl groups of fats and oils to produce fatty acids and glycerin. The lipase used in the present invention is of microbial origin, for example, Pseudomonas spp, Candida spp,
Lipases of the genus Chromobacterium and the like, particularly lipases of the genus Pseudomonas, can be preferably used. Usually, it is preferable to use the lipase immobilized on a carrier, and an anion exchange resin is suitable for this carrier. An immobilized lipase obtained by immobilizing the lipase on the anion exchange resin is also commercially available. Among them, lipase obtained by fixing lipase derived from Pseudomonas spp. To anion exchange resin is stable for a long time even at a temperature of 50 to 60 ° C at which the reaction product of hydrolysis has fluidity and is degradable to oils and fats such as fish oil. In addition, as described in Example 1 described later, hydrolysis is performed at a decomposition rate of 66% or more to efficiently produce ω-3 highly unsaturated fatty acid, which is a desirable degrading enzyme. The chemical composition during decomposition of fish oil is undecomposed triglyceride, partially decomposed diglyceride and completely decomposed fatty acid, and the ratio of monoglyceride is extremely small. This monoglyceride is not preferable because it has a large surface-active effect and makes separation of the produced fatty acid difficult. However, a small amount of this monoglyceride increases the separation efficiency. Further, eicosampentapentanoic acid tends to be concentrated in undegraded glyceride. The degree of hydrolysis of fats and oils is 70% or more, preferably 80% or less, expressed by weight% of fats and oils decomposed to fatty acids.
~ 99% by weight.
【０００９】この加水分解を実施するには、反応器に固
定したリパーゼを満たし軽質の原料油脂と水とを向流的
に接触させる。この際生成した脂肪酸を含む流れとグリ
セリンおよび水を含む流れとがよく分離するように容器
は直立型のものとし、重力によってこれらの流れが向流
的に流れるようにするとよい。この際の反応温度は一般
に４０〜７０℃であり、５０〜６０℃の温度に保持する
のが好ましい。この加水分解反応を実施する際、反応器
を不活性気体の雰囲気に保持することが好ましく、不活
性気体としては窒素、アルゴン、炭酸ガス、ネオン等を
使用することができ、反応器の空気をこれら不活性気体
のいずれかで置換する。炭酸ガスおよびアルゴンは比重
が大きいので置換処理には便利な気体である。反応帯域
を不活性気体の雰囲気にすることにより、高度不飽和脂
肪酸中の二重結合が酸素の作用により過酸化結合を生じ
過酸化物価が上昇するのが防止できる。また、この反応
の際、水より軽質成分の脂肪酸、未反応グリセリド等を
分離するのを助長するために水非混和性軽質溶剤を用い
ることができる。本明細書において「軽質」とは比重的
に水より小さい、すなわち水より軽いことを意味する。
この水非混合性軽質溶剤には、例えば、インオクタン、
オクタン、ペンタン、ヘプタン、ヘキサン等の炭化水素
及びその他の疎水性溶剤が挙げられる。この溶剤は反応
前に油脂に添加してもよく、また反応の任意の時点で反
応系中に加えることができる。この溶剤を添加すること
により、低比重生成物として水より分離される生成物の
分離が容易になる。反応は連続的にまたは非連続的に実
施することができ、回分式で加水分解反応を実施した場
合、一般に１５〜２０時間を必要とする。高度不飽和脂
肪酸を含む生成物は低比重生産物として、水およびグリ
セリンを含む高比重生成物から分離される。To carry out this hydrolysis, the lipase fixed to the reactor is filled and the light raw material oil and fat is brought into countercurrent contact with water. It is advisable that the container is of an upright type so that the flow containing the fatty acid and the flow containing glycerin and water produced at this time are well separated, and these flows flow countercurrently by gravity. The reaction temperature at this time is generally 40 to 70 ° C, and it is preferable to maintain the temperature at 50 to 60 ° C. When carrying out this hydrolysis reaction, it is preferable to maintain the reactor in an atmosphere of an inert gas, and nitrogen, argon, carbon dioxide, neon or the like can be used as the inert gas, and the air in the reactor is Substitute with one of these inert gases. Since carbon dioxide and argon have a large specific gravity, they are gases convenient for the substitution process. By making the reaction zone an inert gas atmosphere, it is possible to prevent the double bond in the polyunsaturated fatty acid from forming a peroxide bond due to the action of oxygen and increasing the peroxide value. Further, in this reaction, a water-immiscible light solvent can be used in order to promote separation of light components such as fatty acid and unreacted glyceride from water. As used herein, “light” means smaller in specific gravity than water, that is, lighter than water.
This water-immiscible light solvent includes, for example, inoctane,
Hydrocarbons such as octane, pentane, heptane, hexane and other hydrophobic solvents are mentioned. This solvent may be added to the fat or oil before the reaction, or may be added to the reaction system at any point in the reaction. The addition of this solvent facilitates the separation of products that are separated from water as low specific gravity products. The reaction can be carried out continuously or discontinuously, and when the hydrolysis reaction is carried out batchwise, it generally requires 15 to 20 hours. The product containing polyunsaturated fatty acids is separated from the high density product containing water and glycerin as a low density product.
【００１０】加水分解反応によって得られ、高比重生成
物から分離された高度不飽和脂肪酸を含有する低比重生
成物は、これを軽質溶剤に溶解してその脂肪酸の濃縮を
行うことができる。軽質溶剤の例としては、脂肪酸エス
テルを生成する恐れのあるアルコール性ＯＨ基を分子内
に持たない溶剤、例えば、アセトン、メチルエチルケト
ンのようなケトン、ペンタン、ヘキサン、ペプタン、オ
クタン、イソオクタンのような炭化水素、その他エーテ
ル類がある。加水分解生成物中に高度不飽和脂肪酸のア
ルコールエステルが存在すると、最終工程で高度不飽和
脂肪酸をトリグリセリドに変換する際の収率が低下する
ので、軽質溶剤としてはアルコール性ＯＨ基を分子内に
持たない前記のような溶剤を使用する。The low specific gravity product containing the highly unsaturated fatty acid obtained by the hydrolysis reaction and separated from the high specific gravity product can be dissolved in a light solvent to concentrate the fatty acid. Examples of the light solvent include a solvent having no alcoholic OH group in the molecule which may form a fatty acid ester, such as acetone, ketone such as methyl ethyl ketone, pentane, hexane, peptane, octane and isooctane. There are hydrogen and other ethers. The presence of alcohol ester of highly unsaturated fatty acid in the hydrolysis product lowers the yield when converting the highly unsaturated fatty acid to triglyceride in the final step. Therefore, as a light solvent, an alcoholic OH group is incorporated in the molecule. Use a solvent as described above that does not have.
【００１１】高度不飽和脂肪酸の濃縮はそれ自体周知の
方法で実施され、例えば低温分別結晶法、塩形成法、尿
素付加法、吸着分離法などが使用される。この濃縮工程
も不活性気体の雰囲気で実施して高度不飽和脂肪酸の酸
化による変成を防止することが望ましい。Concentration of the polyunsaturated fatty acid is carried out by a method known per se, and for example, a low temperature fractional crystallization method, a salt formation method, a urea addition method, an adsorption separation method and the like are used. It is desirable to carry out this concentration step also in an atmosphere of an inert gas to prevent denaturation of highly unsaturated fatty acids due to oxidation.
【００１２】加水分解時に水非混合性軽質溶剤を用いた
場合には、溶剤の回収を簡単にするために、濃縮時に用
いる溶剤は、それと同一の溶剤であることが望ましい
が、加水分解時と濃縮時とで異った溶剤を用いることも
可能である。この濃縮工程において高度不飽和脂肪酸の
濃度は濃縮前の濃度の約２〜６倍に濃縮することができ
る。When a water-immiscible light solvent is used at the time of hydrolysis, the solvent used at the time of concentration is preferably the same solvent as the solvent used at the time of concentration in order to simplify the recovery of the solvent. It is also possible to use a different solvent for the concentration. In this concentration step, the concentration of highly unsaturated fatty acid can be concentrated to about 2 to 6 times the concentration before concentration.
【００１３】溶剤を蒸発させることによって得られた高
度不飽和脂肪酸濃縮物は次にグリセリンと反応（エステ
ル化）させてトリグリセリドに変換させる。このトリグ
リセリド合成において生成した水はエステル化と同時に
脱水処理によって除去する。この合成反応は固定化され
たリパーゼとの接触反応によって実施される。この固定
化されたリパーゼは水分含有量が１００ｐｐｍ以下のほ
ぼ無水状態でも活性を維持でき、高度不飽和脂肪酸トリ
グリセリドの合成に好適なものである。このような固定
化されたリパーゼには、例えばカンデイダ属菌に由来す
るリパーゼをアクリル樹脂に固定化したもの、ムコール
属菌に由来するリパーゼをマクロポーラスな陰イオン交
換樹脂に固定化したもの等が挙げられる。またほぼ無水
の状態でも活性が維持できるようにレシチンやポリオー
ルの存在下に固定化したシュウドモナス属菌やクロモバ
クテリウム属菌由来のリパーゼも使用することができ
る。ムコール属菌に由来するリパーゼも使用することが
できるが、ドコサヘキサエン酸トリグリセリドの合成は
良くない。なお高度不飽和脂肪酸濃縮物中にアルコール
エステルが存在する場合にはリパーゼの基質特異性によ
り、遊離脂肪酸を用いた場合と比較してトリグリセリド
の収率が悪くなる。したがって本発明における加水分解
工程、濃縮工程で用いる溶剤の選択には注意を要する。The highly unsaturated fatty acid concentrate obtained by evaporating the solvent is then reacted (esterified) with glycerin to convert it into triglycerides. The water generated in this triglyceride synthesis is removed by dehydration at the same time as esterification. This synthetic reaction is carried out by a catalytic reaction with immobilized lipase. This immobilized lipase can maintain the activity even in a substantially anhydrous state having a water content of 100 ppm or less, and is suitable for the synthesis of highly unsaturated fatty acid triglyceride. Such immobilized lipases include, for example, lipases derived from Candida spp. Immobilized on an acrylic resin, lipases derived from Mucor spp. Immobilized on a macroporous anion exchange resin, and the like. Can be mentioned. In addition, lipases derived from Pseudomonas spp. Or Chromobacterium spp. Immobilized in the presence of lecithin or polyol so that the activity can be maintained even in an almost anhydrous state can also be used. Lipases derived from Mucor can be used, but the synthesis of docosahexaenoic acid triglyceride is not good. When alcohol esters are present in the polyunsaturated fatty acid concentrate, the substrate specificity of the lipase results in a poor yield of triglycerides as compared with the case where free fatty acids are used. Therefore, care must be taken in selecting the solvent used in the hydrolysis step and the concentration step in the present invention.
【００１４】このトリグリセリドの合成に際して使用さ
れるグリセリンは、この反応の化学量論量付近である。
高度不飽和脂肪酸が反応中に変成するのを考慮して脂肪
酸の分子量から計算すると、使用する高度不飽和脂肪酸
濃縮物の８〜１０重量％の量のグリセリンが使用され
る。The glycerin used in the synthesis of this triglyceride is near the stoichiometric amount of this reaction.
Glycerin is used in an amount of 8 to 10% by weight of the polyunsaturated fatty acid concentrate used, calculated from the molecular weight of the fatty acids, taking into account that the polyunsaturated fatty acids are modified during the reaction.
【００１５】本発明における固定化リパーゼの接触反応
と同時に行われる脱水処理は真空脱水方式または乾燥不
活性気体の通気方式が採用される。この際反応系に存在
する水分および固化されたリパ−ゼの作用でグリセリン
と高度不飽和脂肪酸濃縮物との反応によって生成した水
分が共に脱水され、反応系内の水分濃度をほぼ無水の１
００ｐｐｍ以下にする。このトリグリセリド合成反応に
おいてアルコ−ルエステルが存在すると前述したように
リパ−ゼの基質特異性によりトリグリセリドの収率が低
下するため、原料を活性炭充填カラムに通して脱アルコ
−ル処理することが必要となる。The dehydration treatment carried out at the same time as the catalytic reaction of the immobilized lipase in the present invention employs a vacuum dehydration method or a dry inert gas aeration method. At this time, the water present in the reaction system and the water produced by the reaction between the glycerin and the polyunsaturated fatty acid concentrate are dehydrated by the action of the solidified lipase, so that the water concentration in the reaction system becomes almost anhydrous.
It should be below 00 ppm. The presence of alcohol ester in this triglyceride synthesis reaction lowers the yield of triglyceride due to the substrate specificity of lipase as described above, so it is necessary to pass the raw material through a column packed with activated carbon for de-alcohol treatment. Become.
【００１６】前記のトリグリセリド合成反応に際して、
反応温度は高度不飽和脂肪酸中の二重結合の移動等を防
止するため低い方が好ましいが、酵素反応であることを
考慮し、その効率性から３０〜６０℃、望ましくは４０
〜６０℃である。反応に要する時間は温度その他の条件
にもよるが一般に２４〜４８時間である。In the above triglyceride synthesis reaction,
The reaction temperature is preferably low in order to prevent migration of double bonds in the highly unsaturated fatty acid, but considering that it is an enzymatic reaction, its temperature is 30 to 60 ° C., preferably 40 ° C.
~ 60 ° C. The time required for the reaction depends on the temperature and other conditions, but it is generally 24 to 48 hours.
【００１７】前記の反応により得られた高度不飽和脂肪
酸トリグリセリドは種々の方法、例えば、カラム処理に
より精製できる。溶剤としてエーテルやヘキサンを用
い、塩基性アルミナ充填カラムまたはマグネシウムで活
性化したシリカゲル充填カラムにより精製を行うと過酸
化物も除去されたトリグリセリドが得られるので好都合
である。前記のトリグリセリド合成反応によって得られ
る生成物の８０％以上が高度不飽和脂肪酸トリグリセリ
ドである。この生成物をエ−テルを溶剤として塩基性ア
ルミナ充填カラムで処理し、吸着物をエーテル溶出液と
して処理し、この溶出液を蒸発させると約９９％以上の
高純度の高度不飽和脂肪酸トリグリセリドが得られる。
高度不飽和脂肪酸中にアルコ−ルエステルが混在すると
トリグリセリドの収量が悪くなり、また、トリグリセリ
ド合成も脱アルコール処理を同時に行う必要が生じるよ
うになり、反応操作が複雑になる。この工程も不活性気
体の雰囲気中で実施するのが望ましい。The polyunsaturated fatty acid triglyceride obtained by the above reaction can be purified by various methods such as column treatment. It is convenient to use ether or hexane as a solvent and perform purification with a column packed with basic alumina or a column packed with silica gel activated with magnesium to obtain triglyceride from which the peroxide is removed. 80% or more of the products obtained by the above triglyceride synthesis reaction are highly unsaturated fatty acid triglycerides. The product was treated with a basic alumina packed column using ether as a solvent, the adsorbate was treated as an ether eluent, and the eluate was evaporated to obtain highly unsaturated polyunsaturated fatty acid triglyceride of about 99% or more. can get.
When alcohol ester is mixed in the polyunsaturated fatty acid, the yield of triglyceride is deteriorated, and it becomes necessary to carry out dealcoholization treatment for triglyceride synthesis at the same time, which complicates the reaction operation. It is desirable to carry out this step also in an atmosphere of an inert gas.
【００１８】本発明方法の好適な一例によれば、シュウ
ドモナス属の細菌に由来するリパ−ゼを陰イオン交換樹
脂に固定化して製造した固定化リパ−ゼの作用により、
鰯油のようなω−３高度不飽和脂肪酸含有油脂を分解率
８０％以上まで加水分解することにより遊離のω−３高
度不飽和脂肪酸を８０％以上含有する低比重分解産物が
得られる。この際未分解のグリセリドには界面活性の大
きいモノグリセリドが含まれずしかもエイコサペンタエ
ン酸が濃縮されていた。また原料のω−３高度不飽和脂
肪酸含有油脂を水非混和性軽質溶剤としてイソオクタン
に溶解して加水分解を行うと、酵素的油脂加水分解に悪
影響を与えずに油分と水分との分離が促進され、工程を
連続的に実施した場合、生成物である低比重分解産物の
回収過程が容易になる。こうして得られたω−３高度不
飽和脂肪酸を含む生成物をアルコ−ル性ＯＨ基を持たな
い溶剤に溶解し、濃縮を行うことによりその脂肪酸のエ
ステル化を防止することができる。次にキャンデイタ属
菌の生産したリパ−ゼを例えばアクリル樹脂に固定化し
て製造した固定化リパ−ゼを用いて濃縮されたω−３高
度不飽和脂肪酸とグリセリンを反応させるとドコサヘキ
サエン酸のようなω−３高度不飽和脂肪酸トリグリセラ
イドが効率よく合成できる。According to a preferred example of the method of the present invention, the lipase derived from a bacterium of the genus Pseudomonas is immobilized on an anion exchange resin by the action of the immobilized lipase,
By hydrolyzing an oil / fat containing ω-3 highly unsaturated fatty acid such as sardine oil to a decomposition rate of 80% or more, a low specific gravity decomposition product containing 80% or more of free ω-3 highly unsaturated fatty acid can be obtained. At this time, undegraded glyceride did not contain monoglyceride having large surface activity and eicosapentaenoic acid was concentrated. When the raw material ω-3 highly unsaturated fatty acid-containing fats and oils are dissolved in isooctane as a water-immiscible light solvent and hydrolyzed, the separation of oil and water is promoted without adversely affecting the enzymatic fat and oil hydrolysis. In the case where the steps are continuously performed, the recovery process of the low specific gravity decomposition product, which is a product, becomes easy. Esterification of the fatty acid can be prevented by dissolving the product containing the ω-3 highly unsaturated fatty acid thus obtained in a solvent having no alcoholic OH group and concentrating the product. Then, the immobilized lipase produced by immobilizing the lipase produced by the genus Candida on an acrylic resin is reacted with concentrated ω-3 highly unsaturated fatty acid and glycerin to produce docosahexaenoic acid. ω-3 highly unsaturated fatty acid triglyceride can be efficiently synthesized.
【００１９】[0019]
【実施例】次に本発明の方法を実施例を挙げてさらに詳
しく説明する。EXAMPLES The method of the present invention will now be described in more detail with reference to examples.
【００２０】実施例１ シュウドモナス フルオレセンス（Pseudomonus fluore
scence)バイオタイプΙにより生産されたリパ−ゼを陰
イオン交換樹脂に固定化したもの（エンチロンPF、洛東
化成社製)1g、鰯油1gおよび水1gを50mlの三角フラスコ
にとり、シリコン栓をつけて50℃で振とうした。各反応
時間で脂肪酸画分をガスクロマトグラフィ−（WCOT CP-
Sil88 Chrompack社製）にかけ脂肪酸分析した結果を表
１に示す。分解率は酸価と鹸化価との比より求めた。表
１をみると、分解率６６％以上分解すればメタノール分
解と同じ程度の炭素数２０で、二重結合を５個有するエ
イコサペンタエン酸（Ｃ２０：５）および炭素数２２
で、二重結合を６個有するドコサヘキサエン酸（Ｃ２
２：６）が得られることが解った。Example 1 Pseudomonus fluore
Scence) Lipase produced by biotype Ι was immobilized on anion exchange resin (Enchiron PF, manufactured by Rakuto Kasei Co., Ltd.) 1 g, sardine oil 1 g and water 1 g were placed in a 50 ml Erlenmeyer flask and a silicon stopper was placed on it. Shake it at 50 ° C. Gas chromatography- (WCOT CP-
The results of fatty acid analysis by Sil88 Chrompack) are shown in Table 1. The decomposition rate was obtained from the ratio of the acid value and the saponification value. Table 1 shows that if the decomposition rate is 66% or more, eicosapentaenoic acid (C20: 5) having 20 carbon atoms, which has the same carbon number as methanol decomposition, and has 5 double bonds, and 22 carbon atoms.
And docosahexaenoic acid (C2 having 6 double bonds)
2: 6) was obtained.
【００２１】[0021]
【表１】 [Table 1]
【００２２】実施例２ 図１に示す反応器を用意した。この反応器は向流接触型
の二相系反応器である。仕切り板５及びエマルジョン破
壊装置１０は７５ミクロンの編目を持つステンレススチ
ールメッシュの付切り板である。図面には示していない
が反応槽はマントルにより囲まれていて恒温の液体を導
入、排出させることにより、温度が一定に保たれるよう
になっている。上端と下端に円錐状の静置槽２，８を設
け、上端の静置槽２の上端部に低比重生成物排出管１が
あり、下端の静置槽８には高比重生産物排出管９があ
る。中間の６個の撹拌槽のうち上端の撹拌槽には高比重
基質供給管３があり、下端の撹拌槽には低比重基質供給
管７がある。各槽の直径が５０ｍｍ、高さは２６〜３０
ｍｍで上下の静置槽まで含めた反応器内の体積は４２１
ｍｌであった。６個の撹拌槽４に加えた固定化酵素（エ
ンチロンＰＦ）量は、合計で６４．６ｇであった。高比
重生成物排出管９には１２０度の方向に三方に分かれた
分岐管を有する継ぎ手がその分岐管の１つを介してつな
いであり、残りの一方の分岐管１１はパルス発生機６に
つながれ、残りの他方の分岐管１２には送液ポンプが接
続されている。パルス発生機６は１分間に１５回ずつ脈
流が流れるようになっている。１回の脈流の大きさは約
１５ｍｌである。この脈流は撹拌槽内容物を混合させる
作用を示す。高比重生成物排出管９を閉じて高比重基質
供給管３より水、低比重基質供給管７より鰯油を両者が
約１：１になるように反応器内に基質を満たし、送液ポ
ンプは止めたままパルス発生機６のみを一晩動かすと二
相分離が達成される。なお図示していないが基質は窒素
ガスで飽和にしたのち窒素ガス加圧下の容器に保存す
る。パルス発生機６を作動しながら低比重基質供給管７
より鰯油を５ｍｌ／時間、高比重基質供給管３より水を
２．５ｍｌ／時間で供給し、高比重生成物排出管９より
１２０〜１９０ｍｇ／ｍｌのグリセリンを含む高比重生
成物を１〜３ｍｌ／時間で回収してやると、低比重生成
物が３〜５ｍｌ／時間で回収された。低比重生成物は窒
素で容器中の大気を置換した−９０℃の容器に保存し
た。上記のような条件で反応器を８４０時間運転したの
ち、条件を変えて鰯油に水非混和性軽質溶剤として３０
％のヘキサンを加えて２９６時間、３０％のイソオクタ
ンを加えて２４０時間および最初と同じ条件であるが反
応時間を９６時間にして運転した。その結果を表２に示
す。Example 2 The reactor shown in FIG. 1 was prepared. This reactor is a countercurrent contact type two-phase system reactor. The partition plate 5 and the emulsion breaking device 10 are stainless steel mesh partition plates having 75 micron stitches. Although not shown in the drawing, the reaction tank is surrounded by a mantle, and the temperature is kept constant by introducing and discharging a constant temperature liquid. Conical stationary tanks 2 and 8 are provided at the upper and lower ends, a low specific gravity product discharge pipe 1 is provided at the upper end of the upper stationary tank 2, and a high specific gravity product discharge pipe is provided at the lower stationary tank 8. There is 9. Among the six stirring tanks in the middle, a high specific gravity substrate supply pipe 3 is provided at the upper end stirring tank, and a low specific gravity substrate supply pipe 7 is provided at the lower end stirring tank. Each tank has a diameter of 50 mm and a height of 26-30
The volume in the reactor including the upper and lower stationary tanks in mm is 421.
It was ml. The total amount of immobilized enzyme (ENTILON PF) added to the six stirring tanks 4 was 64.6 g. The high specific gravity product discharge pipe 9 is connected to a joint having branch pipes divided in three directions in the direction of 120 degrees through one of the branch pipes, and the other branch pipe 11 is connected to the pulse generator 6. A liquid feed pump is connected to the other branch pipe 12 connected to the other. The pulse generator 6 has a pulsating flow of 15 times per minute. The size of one pulsating flow is about 15 ml. This pulsating flow has the function of mixing the contents of the stirring tank. The high specific gravity product discharge pipe 9 is closed, water is supplied from the high specific gravity substrate supply pipe 3, and sardine oil is supplied from the low specific gravity substrate supply pipe 7 in the reactor so that the ratio of both is about 1: 1. Two-phase separation is achieved by moving only the pulse generator 6 overnight while it is stopped. Although not shown, the substrate is saturated with nitrogen gas and then stored in a container under pressure of nitrogen gas. Low specific gravity substrate supply pipe 7 while operating the pulse generator 6
More sardine oil is supplied at 5 ml / hour, water is supplied at 2.5 ml / hour from the high specific gravity substrate supply pipe 3, and high specific gravity products containing 120 to 190 mg / ml glycerin are supplied from the high specific gravity product discharge pipe 9 to When it was collected at 3 ml / hour, low specific gravity products were collected at 3 to 5 ml / hour. The low specific gravity product was stored in a container at −90 ° C. in which the atmosphere in the container was replaced with nitrogen. After operating the reactor under the above conditions for 840 hours, the conditions were changed to 30% as a water-immiscible light solvent in sardine oil.
% Hexane was added for 296 hours, 30% isooctane was added for 240 hours, and the same conditions as at the beginning but with a reaction time of 96 hours. The results are shown in Table 2.
【００２３】[0023]
【表２】 [Table 2]
【００２４】表２のＴＧは未反トリグセリド、ＤＧはジ
グリセリド、ＭＧはモノグリセリド、ＦＡは脂肪酸を表
し、ＨＰＬＣカラム（ＳＨＯＤＥＸ ＧＰＣ Ｋｆ−８
０２昭和電工社製 ３００ｘ３）で求めた。イソオクタ
ンを添加すると無溶媒に比較すると６〜７％分解率も上
昇し油水分離も良好であった。また前記の固定化リパー
ゼによると油水分離に悪影響を及ぼす界面活性作用の強
いモノグリセリドの生成も少なかった。In Table 2, TG represents un-triglyceride, DG represents diglyceride, MG represents monoglyceride, FA represents fatty acid, and HPLC column (SHODEX GPC Kf-8).
02: Showa Denko Co., Ltd. 300 × 3). When isooctane was added, the decomposition rate was increased by 6 to 7% as compared with the case where no solvent was used, and oil-water separation was also good. Further, the immobilized lipase produced less monoglyceride having a strong surface-active effect, which adversely affects oil-water separation.
【００２５】また以下の表３には鰯油及び無溶媒の分解
産物（分解率８１％）中の脂肪酸組成を示す。グリセリ
ド及び脂肪酸画分は、分解産物を薄層クロマトグラフィ
ーにより分画した。表２および表３をみると未分解のジ
グリセリド部分（ＤＣ）にはω−３のエイコサペンタン
酸（Ｃ２０：５）、ドコサペンタエン酸（Ｃ２２：
５）、およびドコサヘキサエン酸（Ｃ２２：６）が濃縮
されているが、これは低比重生産物中に回収される。な
お表３中、下線を付した数値はω−３高度不飽和脂肪酸
についてのものである。Table 3 below shows the fatty acid composition in sardine oil and solvent-free decomposition products (decomposition rate 81%). For the glyceride and fatty acid fractions, the degradation products were fractionated by thin layer chromatography. As shown in Tables 2 and 3, the undegraded diglyceride moiety (DC) contained ω-3 eicosapentaenoic acid (C20: 5) and docosapentaenoic acid (C22: 5).
5), and docosahexaenoic acid (C22: 6) are concentrated but are recovered in the low specific gravity product. In Table 3, the underlined numerical values are for ω-3 highly unsaturated fatty acids.
【００２６】[0026]
【表３】 [Table 3]
【００２７】実施例３ 実施例２の無溶媒の低比重生成物（分解率８１％）にヘ
キサンを加え無水硫酸ナトリウムで脱水し、ヘキサンを
蒸散させた分解産物３５ｇに、低温分別結晶法による濃
縮のため軽質溶剤として７倍量のアセトンを加え、−５
０℃で一晩放置後濾過し、−５０℃にしたアセトンで洗
浄し濾液及び洗液のアセトンを蒸散させ１次濃縮物１
８．７２ｇを得た。再び７倍量のアセトを加え−８０℃
で一晩放置後同様にして２次濃縮物１０．３１ｇを得
た。２次濃縮物１ｇにグリセリン０．０７１４ｇをリボ
ザイムＩＭ６０を０．１ｇ加え、１０〜２０トル（Ｔｏ
ｒｒ）まで脱気し真空乾燥しながら、６０℃で振とう反
応した。リポザイムＩＭ６０（Ｎｏｖｏ社製）はムコー
ル・ミーヘイの生産したリパーゼをマクロ細孔の陰イオ
ン交換樹脂に固定化したものである。４８時間後の反応
物の組成は、トリグリセリド６１．２％、ジグリセリド
２２．０％、モノグリセリド２．８％および脂肪酸１
３．９％であった。この反応物をエチルエーテルに溶か
し固定化酵素を濾別した後、塩基性アルミナカラム（ア
ルミニウムオキシド９０（メルク社製製品番号１０７
６）およびアルカリ脱酸法により精製した。各段階の組
成および脂肪酸組成を、表４に示す。表中、下線を付し
た数値はいずれもω−３高度不飽和脂肪酸についてのも
のであり、特にエイコサペンタエン酸およびドコサペン
タエン酸の場合にはアセトンによる低温分別結晶法によ
る濃縮の結果、第２次濃縮物にいずれも２倍濃縮されて
いることが判る。またこれらの脂肪酸はグリセリンとの
反応によりドコヘキサエン酸（Ｃ22：６）の取込みが若
干悪いがトリグリセリドに変換されることを示してい
る。Example 3 Hexane was added to the solventless low specific gravity product (decomposition rate 81%) of Example 2 and dehydrated with anhydrous sodium sulfate, and 35 g of the decomposition product obtained by evaporating hexane was concentrated by the low temperature fractional crystallization method. Therefore, add 7 times the amount of acetone as a light solvent, and
The mixture was left at 0 ° C overnight, filtered, washed with acetone at -50 ° C, and the acetone in the filtrate and washings was evaporated to obtain a primary concentrate 1
8.72 g was obtained. Add 7 times the amount of aceto again at -80 ° C.
After being left overnight at 10 ° C., 10.31 g of a secondary concentrate was similarly obtained. Glycerin (0.0714 g) and ribozyme IM60 (0.1 g) were added to the secondary concentrate (1 g) to obtain 10 to 20 Torr (To
The mixture was degassed to rr) and vacuum-dried while reacting with shaking at 60 ° C. Lipozyme IM60 (manufactured by Novo) is obtained by immobilizing lipase produced by Mucor Mihay on an anion exchange resin having macropores. The composition of the reaction product after 48 hours was as follows: triglyceride 61.2%, diglyceride 22.0%, monoglyceride 2.8% and fatty acid 1
It was 3.9%. This reaction product was dissolved in ethyl ether and the immobilized enzyme was filtered off, followed by a basic alumina column (aluminum oxide 90 (product number 107 manufactured by Merck).
6) and the alkaline deoxidation method. Table 4 shows the composition of each stage and the fatty acid composition. In the table, all underlined numbers are for ω-3 highly unsaturated fatty acids, and particularly in the case of eicosapentaenoic acid and docosapentaenoic acid, the result of concentration by the low temperature fractional crystallization method with acetone was It can be seen that each of the subsequent concentrates was twice concentrated. Further, it is shown that these fatty acids are converted into triglycerides by the reaction with glycerin, although the uptake of docohexaenoic acid (C 22 : 6) is slightly poor.
【００２８】[0028]
【表４】 [Table 4]
【００２９】実施例４ 実施例２のイソオクタン添加の低比重生成物（分解率８
１％）にイソオクタンを蒸散させた加水分解物１９０ｇ
に塩形成法による濃縮のために軽質溶剤として７倍量の
アセトン１３３０ｇおよび４規定の水酸化カリウムメタ
ノール溶液２０２ｍｌを加え３０分間室温で撹拌し−２
０℃に１６時間放置した。次いでカリウム塩として沈殿
する部分を除き濾液５８．２ｇに対し、５８２ｇのアセ
トンを加えて、−２０℃に１６時間放置した。沈殿する
部分を除き濾液を４規定の塩酸で酸性にしアセトンを蒸
散させたのちへキサンを加え洗液がｐＨ７なるまで水洗
した。ヘキサンを蒸散させて２４．４ｇのω−３高度不
飽和脂肪酸濃縮物を得た。この濃縮物１ｇにグリセリン
０．０９ｇおよび固定化リパーゼｓｐ３８２を０．１ｇ
を加え、６０℃で２４時間１０〜２０トル（Ｔｏｒｒ）
まで脱気し、真空乾燥しながら６０℃で振とう反応し
た。固定化リパーゼｓｐ３８２（Ｎｏｖｏ社製）はキャ
リデダ・アンタクチカの生産するリパーゼをアクリル樹
脂に固定化したものである。２４時間後のこの反応物の
組成は、トリグリセリド６８．３％、ジグリセリド２
４．７％、モノグリセリド３．７％および脂肪酸３．３
％であった。この反応物をエチルエーテルに溶かし固定
化酵素を濾別した後、塩基性アルミナカラムにより精製
した。各段階の組成および脂肪酸組成を、表５に示す。Example 4 A low specific gravity product (decomposition rate: 8) obtained by adding isooctane in Example 2
190 g of hydrolyzate obtained by evaporating isooctane to 1%)
For concentration by the salt-forming method, 1330 g of 7 times amount of acetone as a light solvent and 202 ml of 4N potassium hydroxide methanol solution were added and stirred at room temperature for 30 minutes.
It was left at 0 ° C. for 16 hours. Then, 582 g of acetone was added to 58.2 g of the filtrate except the portion precipitated as a potassium salt, and the mixture was allowed to stand at -20 ° C for 16 hours. After removing the precipitated portion, the filtrate was acidified with 4N hydrochloric acid to evaporate acetone, and then hexane was added to the solution and the solution was washed with water until the pH became 7. Hexane was evaporated to obtain 24.4 g of ω-3 highly unsaturated fatty acid concentrate. To 1 g of this concentrate, 0.09 g of glycerin and 0.1 g of immobilized lipase sp382
10 to 20 Torr at 60 ° C. for 24 hours
The mixture was degassed, and the mixture was shaken at 60 ° C. while being dried under vacuum. Immobilized lipase sp382 (manufactured by Novo) is obtained by immobilizing lipase produced by Calideda antactica on acrylic resin. After 24 hours, the composition of this reaction product was: triglyceride 68.3%, diglyceride 2
4.7%, monoglyceride 3.7% and fatty acid 3.3
%Met. This reaction product was dissolved in ethyl ether, the immobilized enzyme was filtered off, and then purified by a basic alumina column. Table 5 shows the composition of each stage and the fatty acid composition.
【００３０】[0030]
【表５】 [Table 5]
【００３１】表中、下線を付した数値はω−３高度不飽
和脂肪酸についてのものである。本方法によると、トリ
グリセライド合成の際の酵素としてキャンデイダ属の菌
由来のリパーゼを用いているので、ドコサヘキサエン酸
（Ｃ22：６）のトリグリセリドへの取り込みが良くトリ
グリセリド合成量も６８．３％と良いものであった。In the table, the underlined numbers are for ω-3 highly unsaturated fatty acids. According to this method, since lipase derived from a bacterium belonging to the genus Candida is used as an enzyme for the synthesis of triglyceride, the incorporation of docosahexaenoic acid (C 22 : 6) into triglyceride is good and the amount of triglyceride synthesis is as good as 68.3%. It was a thing.
【００３２】[0032]
【発明の効果】ω−３高度不飽和脂肪酸は、循環器系疾
病の予防効果、ラットの学習能力の向上効果等重要な生
理的意義がある。本発明によれば、ω−３高度不飽和脂
肪酸を豊富に含み多量に供給入手が可能な魚油等の油脂
を原料として、酵素反応を利用してω−３高度不飽和脂
肪酸含有油脂を一旦加水分解して脂肪酸とし、これを低
温分別結晶法などの濃縮方法により他の脂肪酸などから
分離濃縮し、再度酵素反応を利用してグリセリンと反応
させることにより、ω−３高度不飽和脂肪酸トリグリセ
リド油脂を効率良く得ることができる。この際の原料油
脂は安価であり、反応はすべて緩和な条件下で実施で
き、しかもω−３高度不飽和脂肪酸を実質的な変成を起
すことなく高度に濃縮できる。従って本発明は単に機能
性食品としての利用ばかりでなく医学的および薬学的な
応用にたいしても寄与するものである。EFFECT OF THE INVENTION ω-3 highly unsaturated fatty acids have important physiological significance such as a preventive effect on cardiovascular diseases and an effect of improving learning ability of rats. According to the present invention, an oil or fat such as fish oil, which is rich in ω-3 highly unsaturated fatty acids and can be supplied and supplied in a large amount, is used as a raw material to hydrolyze an ω-3 highly unsaturated fatty acid-containing oil or fat by utilizing an enzymatic reaction. By decomposing it into fatty acid, separating and concentrating it from other fatty acids by a concentrating method such as low temperature fractional crystallization, and reacting again with glycerin using an enzymatic reaction, ω-3 highly unsaturated fatty acid triglyceride It can be obtained efficiently. In this case, the raw material fats and oils are inexpensive, all the reactions can be carried out under mild conditions, and the ω-3 highly unsaturated fatty acid can be highly concentrated without causing substantial alteration. Therefore, the present invention not only serves as a functional food but also contributes to medical and pharmaceutical applications.
【図１】向流接触型の二相系固定化酵素充填反応器の説
明断面図である。FIG. 1 is an explanatory cross-sectional view of a countercurrent contact type two-phase system immobilized enzyme packed reactor.
１．低比重生産物排出管 ２．上端の静置層 ３．高比重基質供給管 ４．撹拌槽 ５．仕切り板 ６．パルス発生機 ７．低比重基質供給管 ８．下端の静置層 ９．高比重生産物排出管 １０．エマルジョン破壊装置 １１．パルス発生機につながれた分岐管 １２．定量ポンプにつながれた分岐管 1. Low specific gravity product discharge pipe 2. Top stationary layer 3. High specific gravity substrate supply pipe 4. Stirring tank 5. Partition plate 6. Pulse generator 7. Low specific gravity substrate supply tube 8. Bottom static layer 9. High specific gravity product discharge pipe 10. Emulsion breaker 11. Branch pipe connected to pulse generator 12. Branch pipe connected to metering pump
─────────────────────────────────────────────────────
─────────────────────────────────────────────────── ───
【手続補正書】[Procedure amendment]
【提出日】平成３年１２月２日[Submission date] December 2, 1991
【手続補正１】[Procedure Amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】全文[Name of item to be corrected] Full text
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【書類名】 明細書[Document name] Statement
【発明の名称】 濃縮された高度不飽和脂肪酸含有油脂
の製造方法Title: Method for producing concentrated highly unsaturated fatty acid-containing fats and oils
【特許請求の範囲】[Claims]
【発明の詳細な説明】Detailed Description of the Invention
【０００１】[0001]
【産業上の利用分野】本発明は濃縮された高度不飽和脂
肪酸油脂、特に、濃縮されたω−３高度不飽和脂肪酸含
有油脂の製造方法に関する。なお、本明細書におけるω
−３高度不飽和脂肪酸とは、炭素鎖の末端メチルから第
３番目と第４番目の炭素の結合が二重結合であり、かつ
炭素鎖中の全二重結合数が２個以上の不飽和脂肪酸を意
味する。また、高度不飽和脂肪酸含有油脂とは、全二重
結合数が２以上の不飽和脂肪酸トリグリセリドを含む油
脂を意味する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a concentrated polyunsaturated fatty acid oil and fat, and more particularly to a method for producing a concentrated omega-3 highly unsaturated fatty acid-containing oil and fat. In this specification, ω
-3 Polyunsaturated fatty acid is a unsaturated bond in which the third and fourth carbon bonds from the terminal methyl of the carbon chain are double bonds, and the total number of double bonds in the carbon chain is 2 or more. Means fatty acid. The polyunsaturated fatty acid-containing fat or oil means a fat or oil containing an unsaturated fatty acid triglyceride having a total number of double bonds of 2 or more.
【０００２】[0002]
【従来の技術及びその問題点】ω−３高度不飽和脂肪酸
含有油脂は、Ｃ18脂肪酸油脂が主成分を占める牛脂など
とは異り、各種魚類の油、肝油などの海産食品に多く含
まれており、最近では、健康食品の成分として知られる
アラキドン酸やリノール酸などのω−６高度不飽和脂肪
酸とバランス良く摂取することが必要であると言われて
いる。このバランスをとるため、ω−３高度不飽和脂肪
酸のエチルエステルが使用されている。しかしながらこ
の脂肪酸のエチルエステルは、原料油脂すなわち脂肪酸
トリグリセリドと比較して消化吸収の程度が低い〔例え
ばＣａｒｏｌ Ｍ． Ｗｏｊｅｎｓｈｉ氏らのＢｉｏｃ
ｈｅｍ．Ｂｉｏｐｈｙｓ．Ａｃｔａ １０８１（１９９
１）３３〜３８参照〕。一方、魚油等の原料油脂を加水
分解してω−３高度不飽和脂肪酸を濃縮生成させる手段
として、酵素を用いる方法が考えられ、リパーゼを用い
た場合の不飽和脂肪酸と飽和脂肪酸との分解速度の差を
利用する方法〔Ｔ．Ｈｏｓｈｉｎｏ，Ｔ．Ｙａｍａｎｅ
およびＳ．Ｓｈｉｍｉｚｕ氏らのＡｇｒｉｃ．Ｂｉｏ
ｌ．Ｃｈｅｍ．５４（１９９０）、１４５９−１４６
７〕あるいはキャンディダ属菌に由来するリパーゼを用
いてω−３高度不飽和脂肪酸油脂を選択的に加水分解
後、この脂肪酸とグリセリンとを、クロモバクテリウム
属の細菌に由来するリパーゼの作用で反応させてトリグ
リセリドを合成することでω−３高度不飽和脂肪酸を濃
縮する方法〔田中、今村および小菅氏らによる「実践バ
イオリアクター」、食品産業バイオリアクターシステム
技術研究組合成果論文集（１９９０年）第３９１〜４１
１頁〕が知られているが、これらの方法においてω−３
高度不飽和脂肪酸を濃縮するプロセスはいずれも原料油
脂をリパーゼの作用で選択的に加水分解する工程のみに
置かれ、方法全体としては効率的とは言い難い。さらに
リパーゼを用いる方法として、いわし油を加水分解し、
ω−３高度不飽和脂肪酸を濃縮し、次いでグリセリンを
用いて同じくクロモバクテリウム属の細菌に由来するリ
パーゼの作用によりこの脂肪酸のトリグリセリドを合成
する方法〔長田、高橋、羽田野および細川氏らによる日
水誌５７（１９９１年）、第１１９〜１２５頁〕が知ら
れているが、この方法によるトリグリセリドの収率は５
０％以下であって効率的な方法とは言えない。ω−３高
度不飽和脂肪酸含有油脂は前述したように健康食品とし
て摂取することが望まれるが、このものは分子中の不飽
和結合部分で酸化され易く、また、二重結合の移動、異
性化が起り易くまた価格的にも非常に高価である。した
がってω−３高度不飽和脂肪酸を一層高度な割合で含有
する油脂を得ることが望まれている。BACKGROUND OF THE INVENTION omega-3 highly unsaturated fatty acid-containing fats and oils, etc. The beef tallow C 18 fatty oil occupies the principal component Ili, a wide variety of oils of fish, contained in many seafood such as cod liver oil Recently, it has been said that it is necessary to take a well-balanced intake with ω-6 highly unsaturated fatty acids such as arachidonic acid and linoleic acid which are known as components of health foods. To balance this, ethyl esters of omega-3 highly unsaturated fatty acids have been used. However, the ethyl ester of this fatty acid has a lower degree of digestion and absorption as compared with the raw material fat and oil, that is, fatty acid triglyceride [eg Carol M. et al. Bioc of Wojenshi et al.
hem. Biophys. Acta 1081 (199
1) 33-38]. On the other hand, as a means for hydrolyzing raw material fats such as fish oil to concentrate and produce ω-3 highly unsaturated fatty acids, a method using an enzyme is considered, and the decomposition rate of unsaturated fatty acids and saturated fatty acids when lipase is used. Method using the difference of [T. Hoshino, T .; Yamane
And S. Shimizu et al., Agric. Bio
l. Chem. 54 (1990), 1459-146.
7] Alternatively, after selectively hydrolyzing ω-3 highly unsaturated fatty acid oil and fat using a lipase derived from Candida, the fatty acid and glycerin are treated by the action of lipase derived from a bacterium of the genus Chromobacterium. Method of concentrating ω-3 highly unsaturated fatty acid by reacting to synthesize triglyceride ["Practical bioreactor" by Tanaka, Imamura and Kosuge et al., Food industry bioreactor system technology research association research papers (1990) 391st to 41st
1 page] is known, but in these methods, ω-3
All of the processes for concentrating polyunsaturated fatty acids are placed only in the step of selectively hydrolyzing the raw material fats and oils by the action of lipase, and it cannot be said that the method as a whole is efficient. Furthermore, as a method using lipase, hydrolyzing sardine oil,
A method of concentrating ω-3 highly unsaturated fatty acids, and then using glycerin to synthesize triglycerides of these fatty acids by the action of lipases also derived from Chromobacter bacteria. Water magazine 57 (1991), pp. 119-125] is known, but the yield of triglyceride by this method is 5
It is less than 0% and cannot be said to be an efficient method. As described above, ω-3 highly unsaturated fatty acid-containing fats and oils are desired to be ingested as health foods, but these are easily oxidized at the unsaturated bond portion in the molecule, and double bond transfer and isomerization Is likely to occur and is very expensive in price. Therefore, it is desired to obtain fats and oils containing a higher proportion of ω-3 highly unsaturated fatty acid.
【０００３】[0003]
【発明が解決しようとする課題】本発明は、高度不飽和
脂肪酸含有油脂、特に、ω−３高度不飽和脂肪酸含有油
脂を、この脂肪酸の変性を起すことなく効率的に加水分
解し、さらに加水分解生成物からこの高度不飽和脂肪酸
を濃縮し、次いでグリセリンを作用させて高度に濃縮さ
れた高度不飽和脂肪酸のトリグリセリドを製造する方法
を提供することをその課題とする。SUMMARY OF THE INVENTION The present invention efficiently hydrolyzes highly unsaturated fatty acid-containing fats and oils, particularly ω-3 highly unsaturated fatty acid-containing fats and oils without causing modification of the fatty acid, and further hydrolyzes the fatty acid. It is an object of the present invention to provide a method for concentrating this highly unsaturated fatty acid from a decomposition product and then allowing glycerin to act thereon to produce a highly concentrated triglyceride of highly unsaturated fatty acid.
【０００４】[0004]
【課題を解決するための手段】本発明者らは前記課題を
解決するため鋭意検究を重ねた結果、本発明を完成し
た。The present inventors have completed the present invention as a result of intensive investigations to solve the above-mentioned problems.
【０００５】本発明によれば、高度不飽和脂肪酸含有油
脂または高度不飽和脂肪酸のアルコールエステルを固定
化されたリパーゼを用いて加水分解する工程、得られた
加水分解生成物に含まれる高度不飽和脂肪酸を濃縮する
工程及び高度不飽和脂肪酸濃縮物を固定化されたリパー
ゼを用いてトリグリセリドに変換させる工程からなる濃
縮された高度不飽和脂肪酸含有油脂の製造方法が提供さ
れる。高度不飽和脂肪酸は化学的に不安定なために前記
の各工程は不活性気体の雰囲気中で実施するのが好まし
い。According to the present invention, a step of hydrolyzing a fat or oil containing a polyunsaturated fatty acid or an alcohol ester of a polyunsaturated fatty acid using an immobilized lipase, a polyunsaturation contained in the obtained hydrolysis product, Provided is a method for producing a concentrated highly unsaturated fatty acid-containing fat or oil, which comprises a step of concentrating a fatty acid and a step of converting a highly unsaturated fatty acid concentrate into a triglyceride using immobilized lipase. Since the polyunsaturated fatty acids are chemically unstable, it is preferable to carry out the above steps in an atmosphere of an inert gas.
【０００６】本発明は、高度不飽和脂肪酸含有油脂を微
生物由来のリパーゼの作用により選択的に加水分解し、
得られたω−３高度不飽和脂肪酸を濃縮し、次いで微生
物由来のリパーゼの作用によりグリセリンと反応させて
トリグリセリドとすることにより濃縮されたω−３高度
不飽和脂肪酸含有油脂を得ることを意図するものであ
る。The present invention selectively hydrolyzes oils and fats containing highly unsaturated fatty acids by the action of lipase derived from microorganisms,
It is intended to obtain a concentrated ω-3 highly unsaturated fatty acid-containing oil or fat by concentrating the obtained ω-3 highly unsaturated fatty acid and then reacting it with glycerin by the action of a lipase derived from a microorganism to form a triglyceride. It is a thing.
【０００７】本発明において原料として使用される油脂
は、高度不飽和脂肪酸トリグリセリド、特に、ω−３高
度不飽和脂肪酸トリグリセリドを高割合例えば１０重量
％以上含有する油脂であり、このような油脂は海産性ま
たは淡水産性のプランクトンを捕食している生物、例え
ば魚類の油脂、一般的に魚油と呼ばれるもので、大量に
捕獲できる鰯、さば等の魚油、またはこれら魚類の肝油
である。また鰯油のようなω−３高度不飽和脂肪酸を含
有する油脂をエタノールのようなアルコールでアルコー
ル分解したアルコールエステルも本発明の原料として使
用できる。The fats and oils used as a raw material in the present invention are highly unsaturated fatty acid triglycerides, particularly fats and oils containing a high proportion of ω-3 highly unsaturated fatty acid triglycerides, for example, 10% by weight or more. Organisms that eat plankton that are natural or freshwater, for example, fish oils and fats, generally called fish oil, which are fish oils such as sardines, mackerel, and the like, or liver oils of these fishes. Further, an alcohol ester obtained by alcoholyzing an oil or fat containing ω-3 highly unsaturated fatty acid such as sardine oil with an alcohol such as ethanol can also be used as the raw material of the present invention.
【０００８】本発明における前記油脂の加水分解は、ア
ルカリ等の化学的手段を用いることなく、リパーゼ等の
酵素を用いて生化学的手段で実施する。リパーゼは、油
脂のアシル基を加水分解し、脂肪酸とグリセリンを生成
させる。本発明で使用するリパーゼは微生物由来のも
の、例えば、シュウドモナス属菌、キャンディダ属菌、
クロモバクテリゥム属菌等のリパーゼで、特に、シュウ
ドモナス属菌のリパーゼが好適に使用できる。リパーゼ
は通常担体に固定して使用するのが好適であり、この担
体としては陰イオン交換樹脂が好適であり、リパーゼを
陰イオン交換樹脂に固定化した固定化リパーゼは市販品
も入手できる。中でもシュウドモナス属の菌に由来する
リパーゼを陰イオン交換樹脂に固定したリパーゼは加水
分解の反応物質が流動性を持つ５０〜６０℃の温度にお
いても長時間安定であり、魚油等の油脂の分解性もよ
く、後記する実施例１に記載したように分解率６６％以
上に加水分解を行うとω−３高度不飽和脂肪酸を効率よ
く生成するので望ましい分解酵素である。魚油分解の際
の化学組成は未分解のトリグリセリド、一部分解のジグ
リセリドおよび完全分解した脂肪酸であり、モノグリセ
リドの割合は極めて少ない。このモノグリセリドは界面
活性作用が大きいので生成した脂肪酸の分離を困難にす
るので好ましくないが、このモノグリセリドの量が少な
いことは分離効率を高めることになる。また未分解のグ
リセリドにはエイコサンペンタエン酸が濃縮される傾向
を示す。油脂の加水分解率は、脂肪酸にまで分解された
油脂の重量％で表わして、７０％以上、好ましくは８０
〜９９重量％である。The hydrolysis of the fat or oil in the present invention is carried out by a biochemical means using an enzyme such as lipase without using a chemical means such as an alkali. Lipase hydrolyzes the acyl groups of fats and oils to produce fatty acids and glycerin. The lipase used in the present invention is of microbial origin, for example, Pseudomonas spp, Candida spp,
Lipases of the genus Chromobacterium and the like, particularly lipases of the genus Pseudomonas, can be preferably used. Usually, it is preferable to use the lipase immobilized on a carrier, and an anion exchange resin is suitable for this carrier. An immobilized lipase obtained by immobilizing the lipase on the anion exchange resin is also commercially available. Among them, lipase obtained by fixing lipase derived from Pseudomonas spp. To anion exchange resin is stable for a long time even at a temperature of 50 to 60 ° C at which the reaction product of hydrolysis has fluidity and is degradable to oils and fats such as fish oil. In addition, as described in Example 1 described later, hydrolysis is performed at a decomposition rate of 66% or more to efficiently produce ω-3 highly unsaturated fatty acid, which is a desirable degrading enzyme. The chemical composition during decomposition of fish oil is undecomposed triglyceride, partially decomposed diglyceride and completely decomposed fatty acid, and the ratio of monoglyceride is extremely small. This monoglyceride is not preferable because it has a large surface-active effect and thus makes it difficult to separate the produced fatty acid, but a small amount of this monoglyceride increases the separation efficiency. Further, eicosampentapentanoic acid tends to be concentrated in undegraded glyceride. The degree of hydrolysis of fats and oils is 70% or more, preferably 80% or less, expressed by weight% of fats and oils decomposed to fatty acids.
~ 99% by weight.
【０００９】この加水分解を実施するには、反応器に固
定したリパーゼを満たし軽質の原料油脂と水とを向流的
に接触させる。この際生成した脂肪酸を含む流れとグリ
セリンおよび水を含む流れとがよく分離するように容器
は直立型のものとし、重力によってこれらの流れが向流
的に流れるようにするとよい。この際の反応温度は一般
に４０〜７０℃であり、５０〜６０℃の温度に保持する
のが好ましい。この加水分解反応を実施する際、反応器
を不活性気体の雰囲気に保持することが好ましく、不活
性気体としては窒素、アルゴン、炭酸ガス、ネオン等を
使用することができ、反応器の空気をこれら不活性気体
のいずれかで置換する。炭酸ガスおよびアルゴンは比重
が大きいので置換処理には便利な気体である。反応帯域
を不活性気体の雰囲気にすることにより、高度不飽和脂
肪酸中の二重結合が酸素の作用により過酸化結合を生じ
過酸化物価が上昇するのが防止できる。また、この反応
の際、水より軽質成分の脂肪酸、未反応グリセリド等を
分離するのを助長するために水非混和性軽質溶剤を用い
ることができる。本明細書において「軽質」とは比重的
に水より小さい、すなわち水より軽いことを意味する。
この水非混合性軽質溶剤には、例えば、インオクタン、
オクタン、ペンタン、ヘプタン、ヘキサン等の炭化水素
及びその他の疎水性溶剤が挙げられる。この溶剤は反応
前に油脂に添加してもよく、また反応の任意の時点で反
応系中に加えることができる。この溶剤を添加すること
により、低比重生成物として水より分離される生成物の
分離が容易になる。反応は連続的にまたは非連続的に実
施することができ、回分式で加水分解反応を実施した場
合、一般に１５〜２０時間を必要とする。高度不飽和脂
肪酸を含む生成物は低比重生産物として、水およびグリ
セリンを含む高比重生成物から分離される。To carry out this hydrolysis, the lipase fixed to the reactor is filled and the light raw material oil and fat is brought into countercurrent contact with water. It is advisable that the container is of an upright type so that the flow containing the fatty acid and the flow containing glycerin and water produced at this time are well separated, and these flows flow countercurrently by gravity. The reaction temperature at this time is generally 40 to 70 ° C, and it is preferable to maintain the temperature at 50 to 60 ° C. When carrying out this hydrolysis reaction, it is preferable to maintain the reactor in an atmosphere of an inert gas, and nitrogen, argon, carbon dioxide, neon or the like can be used as the inert gas, and the air in the reactor is Substitute with one of these inert gases. Since carbon dioxide and argon have a large specific gravity, they are gases convenient for the substitution process. By making the reaction zone an inert gas atmosphere, it is possible to prevent the double bond in the polyunsaturated fatty acid from forming a peroxide bond due to the action of oxygen and increasing the peroxide value. Further, in this reaction, a water-immiscible light solvent can be used in order to promote separation of light components such as fatty acid and unreacted glyceride from water. As used herein, “light” means smaller in specific gravity than water, that is, lighter than water.
This water-immiscible light solvent includes, for example, inoctane,
Hydrocarbons such as octane, pentane, heptane, hexane and other hydrophobic solvents are mentioned. This solvent may be added to the fat or oil before the reaction, or may be added to the reaction system at any point in the reaction. The addition of this solvent facilitates the separation of products that are separated from water as low specific gravity products. The reaction can be carried out continuously or discontinuously, and when the hydrolysis reaction is carried out batchwise, it generally requires 15 to 20 hours. The product containing polyunsaturated fatty acids is separated from the high density product containing water and glycerin as a low density product.
【００１０】加水分解反応によって得られ、高比重生成
物から分離された高度不飽和脂肪酸を含有する低比重生
成物は、これを軽質溶剤に溶解してその脂肪酸の濃縮を
行うことができる。軽質溶剤の例としては、脂肪酸エス
テルを生成する恐れのあるアルコール性ＯＨ基を分子内
に持たない溶剤、例えば、アセトン、メチルエチルケト
ンのようなケトン、ペンタン、ヘキサン、ペプタン、オ
クタン、イソオクタンのような炭化水素、その他エーテ
ル類がある。加水分解生成物中に高度不飽和脂肪酸のア
ルコールエステルが存在すると、最終工程で高度不飽和
脂肪酸をトリグリセリドに変換する際の収率が低下する
ので、軽質溶剤としてはアルコール性ＯＨ基を分子内に
持たない前記のような溶剤を使用する。The low specific gravity product containing the highly unsaturated fatty acid obtained by the hydrolysis reaction and separated from the high specific gravity product can be dissolved in a light solvent to concentrate the fatty acid. Examples of the light solvent include a solvent having no alcoholic OH group in the molecule which may form a fatty acid ester, such as acetone, ketone such as methyl ethyl ketone, pentane, hexane, peptane, octane and isooctane. There are hydrogen and other ethers. The presence of alcohol ester of highly unsaturated fatty acid in the hydrolysis product lowers the yield when converting the highly unsaturated fatty acid to triglyceride in the final step. Therefore, as a light solvent, an alcoholic OH group is incorporated in the molecule. Use a solvent as described above that does not have.
【００１１】高度不飽和脂肪酸の濃縮はそれ自体周知の
方法で実施され、例えば低温分別結晶法、塩形成法、尿
素付加法、吸着分離法などが使用される。この濃縮工程
も不活性気体の雰囲気で実施して高度不飽和脂肪酸の酸
化による変成を防止することが望ましい。Concentration of the polyunsaturated fatty acid is carried out by a method known per se, and for example, a low temperature fractional crystallization method, a salt formation method, a urea addition method, an adsorption separation method and the like are used. It is desirable to carry out this concentration step also in an atmosphere of an inert gas to prevent denaturation of highly unsaturated fatty acids due to oxidation.
【００１２】加水分解時に水非混合性軽質溶剤を用いた
場合には、溶剤の回収を簡単にするために、濃縮時に用
いる溶剤は、それと同一の溶剤であることが望ましい
が、加水分解時と濃縮時とで異った溶剤を用いることも
可能である。この濃縮工程において高度不飽和脂肪酸の
濃度は濃縮前の濃度の約２〜６倍に濃縮することができ
る。When a water-immiscible light solvent is used at the time of hydrolysis, the solvent used at the time of concentration is preferably the same solvent as the solvent used at the time of concentration in order to simplify the recovery of the solvent. It is also possible to use a different solvent for the concentration. In this concentration step, the concentration of highly unsaturated fatty acid can be concentrated to about 2 to 6 times the concentration before concentration.
【００１３】溶剤を蒸発させることによって得られた高
度不飽和脂肪酸濃縮物は次にグリセリンと反応（エステ
ル化）させてトリグリセリドに変換させる。このトリグ
リセリド合成において生成した水はエステル化と同時に
脱水処理によって除去する。この合成反応は固定化され
たリパーゼとの接触反応によって実施される。この固定
化されたリパーゼは水分含有量が１００ｐｐｍ以下のほ
ぼ無水状態でも活性を維持でき、高度不飽和脂肪酸トリ
グリセリドの合成に好適なものである。このような固定
化されたリパーゼには、例えばキャンデイダ属菌に由来
するリパーゼをアクリル樹脂に固定化したもの、ムコー
ル属菌に由来するリパーゼをマクロポーラスな陰イオン
交換樹脂に固定化したもの等が挙げられる。またほぼ無
水の状態でも活性が維持できるようにレシチンやポリオ
ールの存在下に固定化したシュウドモナス属菌やクロモ
バクテリウム属菌由来のリパーゼも使用することができ
る。ムコール属菌に由来するリパーゼも使用することが
できるが、ドコサヘキサエン酸トリグリセリドの合成は
良くない。なお高度不飽和脂肪酸濃縮物中にアルコール
エステルが存在する場合にはリパーゼの基質特異性によ
り、遊離脂肪酸を用いた場合と比較してトリグリセリド
の収率が悪くなる。したがって本発明における加水分解
工程、濃縮工程で用いる溶剤の選択には注意を要する。The highly unsaturated fatty acid concentrate obtained by evaporating the solvent is then reacted (esterified) with glycerin to convert it into triglycerides. The water generated in this triglyceride synthesis is removed by dehydration at the same time as esterification. This synthetic reaction is carried out by a catalytic reaction with immobilized lipase. This immobilized lipase can maintain the activity even in a substantially anhydrous state having a water content of 100 ppm or less, and is suitable for the synthesis of highly unsaturated fatty acid triglyceride. Such immobilized lipases include, for example, lipase derived from Candida spp. Immobilized on an acrylic resin, lipase derived from Mucor spp. Immobilized on a macroporous anion exchange resin, and the like. Can be mentioned. In addition, lipases derived from Pseudomonas spp. Or Chromobacterium spp. Immobilized in the presence of lecithin or polyol so that the activity can be maintained even in an almost anhydrous state can also be used. Lipases derived from Mucor can be used, but the synthesis of docosahexaenoic acid triglyceride is not good. When alcohol esters are present in the polyunsaturated fatty acid concentrate, the substrate specificity of the lipase results in a poor yield of triglycerides as compared with the case where free fatty acids are used. Therefore, care must be taken in selecting the solvent used in the hydrolysis step and the concentration step in the present invention.
【００１４】このトリグリセリドの合成に際して使用さ
れるグリセリンは、この反応の化学量論量付近である。
高度不飽和脂肪酸が反応中に変成するのを考慮して脂肪
酸の分子量から計算すると、使用する高度不飽和脂肪酸
濃縮物の８〜１０重量％の量のグリセリンが使用され
る。The glycerin used in the synthesis of this triglyceride is near the stoichiometric amount of this reaction.
Glycerin is used in an amount of 8 to 10% by weight of the polyunsaturated fatty acid concentrate used, calculated from the molecular weight of the fatty acids, taking into account that the polyunsaturated fatty acids are modified during the reaction.
【００１５】本発明における固定化リパーゼの接触反応
と同時に行われる脱水処理は真空脱水方式または乾燥不
活性気体の通気方式が採用される。この際反応系に存在
する水分および固化されたリパ−ゼの作用でグリセリン
と高度不飽和脂肪酸濃縮物との反応によって生成した水
分が共に脱水され、反応系内の水分濃度をほぼ無水の１
００ｐｐｍ以下にする。このトリグリセリド合成反応に
おいてアルコ−ルエステルが存在すると前述したように
リパ−ゼの基質特異性によりトリグリセリドの収率が低
下し、また副生するアルコールを活性炭充填カラムに通
して脱アルコ−ル処理することが必要となる。The dehydration treatment carried out at the same time as the catalytic reaction of the immobilized lipase in the present invention employs a vacuum dehydration method or a dry inert gas aeration method. At this time, the water present in the reaction system and the water produced by the reaction between the glycerin and the polyunsaturated fatty acid concentrate are dehydrated by the action of the solidified lipase, so that the water concentration in the reaction system becomes almost anhydrous.
It should be below 00 ppm. If alcohol ester is present in this triglyceride synthesis reaction, the yield of triglyceride is lowered due to the substrate specificity of lipase as described above, and by-product alcohol is subjected to de-alcohol treatment by passing through a column packed with activated carbon. Is required.
【００１６】前記のトリグリセリド合成反応に際して、
反応温度は高度不飽和脂肪酸中の二重結合の移動等を防
止するため低い方が好ましいが、酵素反応であることを
考慮し、その効率性から３０〜６０℃、望ましくは４０
〜６０℃である。反応に要する時間は温度その他の条件
にもよるが一般に２４〜４８時間である。In the above triglyceride synthesis reaction,
The reaction temperature is preferably low in order to prevent migration of double bonds in the highly unsaturated fatty acid, but considering that it is an enzymatic reaction, its temperature is 30 to 60 ° C., preferably 40 ° C.
~ 60 ° C. The time required for the reaction depends on the temperature and other conditions, but it is generally 24 to 48 hours.
【００１７】前記の反応により得られた高度不飽和脂肪
酸トリグリセリドは種々の方法、例えば、カラム処理に
より精製できる。溶剤としてエーテルやヘキサンを用
い、塩基性アルミナ充填カラムまたはマグネシウムで活
性化したシリカゲル充填カラムにより精製を行うと過酸
化物も除去されたトリグリセリドが得られるので好都合
である。前記のトリグリセリド合成反応によって得られ
る生成物の８０％以上が高度不飽和脂肪酸トリグリセリ
ドである。この生成物をエ−テルを溶剤として塩基性ア
ルミナ充填カラムで処理し、吸着物を除去したエーテル
溶出液として処理し、この溶出液を蒸発させると約９９
％以上の高純度の高度不飽和脂肪酸トリグリセリドが得
られる。高度不飽和脂肪酸中にアルコ−ルエステルが混
在するとトリグリセリドの収量が悪くなり、また、トリ
グリセリド合成も脱アルコール処理を同時に行う必要が
生じるようになり、反応操作が複雑になる。この工程も
不活性気体の雰囲気中で実施するのが望ましい。The polyunsaturated fatty acid triglyceride obtained by the above reaction can be purified by various methods such as column treatment. It is convenient to use ether or hexane as a solvent and perform purification with a column packed with basic alumina or a column packed with silica gel activated with magnesium to obtain triglyceride from which the peroxide is removed. 80% or more of the products obtained by the above triglyceride synthesis reaction are highly unsaturated fatty acid triglycerides. This product was treated with a basic alumina packed column using ether as a solvent, treated with an ether eluate from which the adsorbate was removed, and the eluate was evaporated to about 99%.
% Or more of highly pure highly unsaturated fatty acid triglyceride can be obtained. When alcohol ester is mixed in the polyunsaturated fatty acid, the yield of triglyceride is deteriorated, and it becomes necessary to carry out dealcoholization treatment for triglyceride synthesis at the same time, which complicates the reaction operation. It is desirable to carry out this step also in an atmosphere of an inert gas.
【００１８】本発明方法の好適な一例によれば、シュウ
ドモナス属の細菌に由来するリパ−ゼを陰イオン交換樹
脂に固定化して製造した固定化リパ−ゼの作用により、
鰯油のようなω−３高度不飽和脂肪酸含有油脂を分解率
８０％以上まで加水分解することにより遊離のω−３高
度不飽和脂肪酸を８０％以上含有する低比重分解産物が
得られる。この際未分解のグリセリドには界面活性の大
きいモノグリセリドが含まれずしかもエイコサペンタエ
ン酸が濃縮されていた。また原料のω−３高度不飽和脂
肪酸含有油脂を水非混和性軽質溶剤としてイソオクタン
に溶解して加水分解を行うと、酵素的油脂加水分解に悪
影響を与えずに油分と水分との分離が促進され、工程を
連続的に実施した場合、生成物である低比重分解産物の
回収過程が容易になる。こうして得られたω−３高度不
飽和脂肪酸を含む生成物をアルコ−ル性ＯＨ基を持たな
い溶剤に溶解し、濃縮を行うことによりその脂肪酸のエ
ステル化を防止することができる。次にキャンデイタ属
菌の生産したリパ−ゼを例えばアクリル樹脂に固定化し
て製造した固定化リパ−ゼを用いて濃縮されたω−３高
度不飽和脂肪酸とグリセリンを反応させるとドコサヘキ
サエン酸のようなω−３高度不飽和脂肪酸もトリグリセ
ライドに効率よく合成できる。According to a preferred example of the method of the present invention, the lipase derived from a bacterium of the genus Pseudomonas is immobilized on an anion exchange resin by the action of the immobilized lipase,
By hydrolyzing an oil / fat containing ω-3 highly unsaturated fatty acid such as sardine oil to a decomposition rate of 80% or more, a low specific gravity decomposition product containing 80% or more of free ω-3 highly unsaturated fatty acid can be obtained. At this time, undegraded glyceride did not contain monoglyceride having large surface activity and eicosapentaenoic acid was concentrated. When the raw material ω-3 highly unsaturated fatty acid-containing fats and oils are dissolved in isooctane as a water-immiscible light solvent and hydrolyzed, the separation of oil and water is promoted without adversely affecting the enzymatic fat and oil hydrolysis. In the case where the steps are continuously performed, the recovery process of the low specific gravity decomposition product, which is a product, becomes easy. Esterification of the fatty acid can be prevented by dissolving the product containing the ω-3 highly unsaturated fatty acid thus obtained in a solvent having no alcoholic OH group and concentrating the product. Then, the immobilized lipase produced by immobilizing the lipase produced by the genus Candida on an acrylic resin is reacted with concentrated ω-3 highly unsaturated fatty acid and glycerin to produce docosahexaenoic acid. ω-3 highly unsaturated fatty acids can also be efficiently synthesized into triglycerides.
【００１９】[0019]
【実施例】次に本発明の方法を実施例を挙げてさらに詳
しく説明する。EXAMPLES The method of the present invention will now be described in more detail with reference to examples.
【００２０】実施例１ シュウドモナス フルオレセンス（Pseudomonus fluore
scence)バイオタイプΙにより生産されたリパ−ゼを陰
イオン交換樹脂に固定化したもの（エンチロンPF、洛東
化成社製)1g、鰯油1gおよび水1gを50mlの三角フラスコ
にとり、シリコン栓をつけて50℃で振とうした。各反応
時間で脂肪酸画分をガスクロマトグラフィ−（WCOT CP-
Sil88 Chrompack社製）にかけ脂肪酸分析した結果を表
１に示す。分解率は酸価と鹸化価との比より求めた。表
１をみると、分解率６６％以上分解すればメタノール分
解と同じ程度の炭素数２０で、二重結合を５個有するエ
イコサペンタエン酸（Ｃ２０：５）および炭素数２２
で、二重結合を６個有するドコサヘキサエン酸（Ｃ２
２：６）が得られることが解った。Example 1 Pseudomonus fluore
Scence) Lipase produced by biotype Ι was immobilized on anion exchange resin (Enchiron PF, manufactured by Rakuto Kasei Co., Ltd.) 1 g, sardine oil 1 g and water 1 g were placed in a 50 ml Erlenmeyer flask and a silicon stopper was placed on it. Shake it at 50 ° C. Gas chromatography- (WCOT CP-
The results of fatty acid analysis by Sil88 Chrompack) are shown in Table 1. The decomposition rate was obtained from the ratio of the acid value and the saponification value. Table 1 shows that if the decomposition rate is 66% or more, eicosapentaenoic acid (C20: 5) having 20 carbon atoms, which has the same carbon number as methanol decomposition, and has 5 double bonds, and 22 carbon atoms.
And docosahexaenoic acid (C2 having 6 double bonds)
2: 6) was obtained.
【００２１】[0021]
【表１】 [Table 1]
【００２２】実施例２ 図１に示す反応器を用意した。この反応器は向流接触型
の二相系反応器である。仕切り板５及びエマルジョン破
壊装置１０は７５ミクロンの編目を持つステンレススチ
ールメッシュの付切り板である。図面には示していない
が反応槽はマントルにより囲まれていて恒温の液体を導
入、排出させることにより、温度が一定に保たれるよう
になっている。上端と下端に円錐状の静置槽２，８を設
け、上端の静置槽２の上端部に低比重生成物排出管１が
あり、下端の静置槽８には高比重生産物排出管９があ
る。中間の６個の撹拌槽のうち上端の撹拌槽には高比重
基質供給管３があり、下端の撹拌槽には低比重基質供給
管７がある。各槽の直径が５０ｍｍ、高さは２６〜３０
ｍｍで上下の静置槽まで含めた反応器内の体積は４２１
ｍｌであった。６個の撹拌槽４に加えた固定化酵素（エ
ンチロンＰＦ）量は、合計で６４．６ｇであった。高比
重生成物排出管９には１２０度の方向に三方に分かれた
分岐管を有する継ぎ手がその分岐管の１つを介してつな
いであり、残りの一方の分岐管１１はパルス発生機６に
つながれ、残りの他方の分岐管１２には送液ポンプが接
続されている。パルス発生機６は１分間に１５回ずつ脈
流が流れるようになっている。１回の脈流の大きさは約
１５ｍｌである。この脈流は撹拌槽内容物を混合させる
作用を示す。高比重生成物排出管９を閉じて高比重基質
供給管３より水、低比重基質供給管７より鰯油を両者が
約１：１になるように反応器内に基質を満たし、送液ポ
ンプは止めたままパルス発生機６のみを一晩動かすと二
相分離が達成される。なお図示していないが基質は窒素
ガスで飽和にしたのち窒素ガス加圧下の容器に保存す
る。パルス発生機６を作動しながら低比重基質供給管７
より鰯油を５ｍｌ／時間、高比重基質供給管３より水を
２．５ｍｌ／時間で供給し、高比重生成物排出管９より
１２０〜１９０ｍｇ／ｍｌのグリセリンを含む高比重生
成物を１〜３ｍｌ／時間で回収してやると、低比重生成
物が３〜５ｍｌ／時間で回収された。低比重生成物は窒
素で容器中の大気を置換した−９０℃の容器に保存し
た。上記のような条件で反応器を８４０時間運転したの
ち、条件を変えて鰯油に水非混和性軽質溶剤として３０
％のヘキサンを加えて２９６時間、３０％のイソオクタ
ンを加えて２４０時間および最初と同じ条件であるが反
応時間を９６時間にして運転した。その結果を表２に示
す。Example 2 The reactor shown in FIG. 1 was prepared. This reactor is a countercurrent contact type two-phase system reactor. The partition plate 5 and the emulsion breaking device 10 are stainless steel mesh partition plates having 75 micron stitches. Although not shown in the drawing, the reaction tank is surrounded by a mantle, and the temperature is kept constant by introducing and discharging a constant temperature liquid. Conical stationary tanks 2 and 8 are provided at the upper and lower ends, a low specific gravity product discharge pipe 1 is provided at the upper end of the upper stationary tank 2, and a high specific gravity product discharge pipe is provided at the lower stationary tank 8. There is 9. Among the six stirring tanks in the middle, a high specific gravity substrate supply pipe 3 is provided at the upper end stirring tank, and a low specific gravity substrate supply pipe 7 is provided at the lower end stirring tank. Each tank has a diameter of 50 mm and a height of 26-30
The volume in the reactor including the upper and lower stationary tanks in mm is 421.
It was ml. The total amount of immobilized enzyme (ENTILON PF) added to the six stirring tanks 4 was 64.6 g. The high specific gravity product discharge pipe 9 is connected to a joint having branch pipes divided in three directions in the direction of 120 degrees through one of the branch pipes, and the other branch pipe 11 is connected to the pulse generator 6. A liquid feed pump is connected to the other branch pipe 12 connected to the other. The pulse generator 6 has a pulsating flow of 15 times per minute. The size of one pulsating flow is about 15 ml. This pulsating flow has the function of mixing the contents of the stirring tank. The high specific gravity product discharge pipe 9 is closed, water is supplied from the high specific gravity substrate supply pipe 3, and sardine oil is supplied from the low specific gravity substrate supply pipe 7 in the reactor so that the ratio of both is about 1: 1. Two-phase separation is achieved by moving only the pulse generator 6 overnight while it is stopped. Although not shown, the substrate is saturated with nitrogen gas and then stored in a container under pressure of nitrogen gas. Low specific gravity substrate supply pipe 7 while operating the pulse generator 6
More sardine oil is supplied at 5 ml / hour, water is supplied at 2.5 ml / hour from the high specific gravity substrate supply pipe 3, and high specific gravity products containing 120 to 190 mg / ml glycerin are supplied from the high specific gravity product discharge pipe 9 to When it was collected at 3 ml / hour, low specific gravity products were collected at 3 to 5 ml / hour. The low specific gravity product was stored in a container at −90 ° C. in which the atmosphere in the container was replaced with nitrogen. After operating the reactor under the above conditions for 840 hours, the conditions were changed to 30% as a water-immiscible light solvent in sardine oil.
% Hexane was added for 296 hours, 30% isooctane was added for 240 hours, and the same conditions as at the beginning but with a reaction time of 96 hours. The results are shown in Table 2.
【００２３】[0023]
【表２】 [Table 2]
【００２４】表２のＴＧは未反応トリグリセリド、ＤＧ
はジグリセリド、ＭＧはモノグリセリド、ＦＡは脂肪酸
を表し、ＨＰＬＣカラム（ＳＨＯＤＥＸ ＧＰＣＫｆ−
８０２ 昭和電工社製 ３００ｘ３）で求めた。イソオ
クタンを添加すると無溶媒に比較すると６〜７％分解率
も上昇し油水分離も良好であった。また前記の固定化リ
パーゼによると油水分離に悪影響を及ぼす界面活性作用
の強いモノグリセリドの生成も少なかった。TG in Table 2 is unreacted triglyceride, DG
Is a diglyceride, MG is a monoglyceride, FA is a fatty acid, and an HPLC column (SHODEX GPCKf-
802, Showa Denko KK 300x3). When isooctane was added, the decomposition rate was increased by 6 to 7% as compared with the case where no solvent was used, and oil-water separation was also good. Further, the immobilized lipase produced less monoglyceride having a strong surface-active effect, which adversely affects oil-water separation.
【００２５】また以下の表３には鰯油及び無溶媒の分解
産物（分解率８１％）中の脂肪酸組成を示す。グリセリ
ド及び脂肪酸画分は、分解産物を薄層クロマトグラフィ
ーにより分画した。表２および表３をみると未分解のジ
グリセリド部分（ＤＧ）にはω−３のエイコサペンタン
酸（Ｃ２０：５）、ドコサペンタエン酸（Ｃ２２：
５）、およびドコサヘキサエン酸（Ｃ２２：６）が濃縮
されているが、これは低比重生産物中に回収される。な
お表３中、下線を付した数値はω−３高度不飽和脂肪酸
についてのものである。Table 3 below shows the fatty acid composition in sardine oil and solvent-free decomposition products (decomposition rate 81%). For the glyceride and fatty acid fractions, the degradation products were fractionated by thin layer chromatography. Looking at Tables 2 and 3, the undegraded diglyceride moiety (DG) was found to be ω-3 eicosapentaenoic acid (C20: 5) and docosapentaenoic acid (C22:
5), and docosahexaenoic acid (C22: 6) are concentrated but are recovered in the low specific gravity product. In Table 3, the underlined numerical values are for ω-3 highly unsaturated fatty acids.
【００２６】[0026]
【表３】 [Table 3]
【００２７】実施例３ 実施例２の無溶媒の低比重生成物（分解率８１％）にヘ
キサンを加え無水硫酸ナトリウムで脱水し、ヘキサンを
蒸散させた分解産物３５ｇに、低温分別結晶法による濃
縮のため軽質溶剤として７倍量のアセトンを加え、−５
０℃で一晩放置後濾過し、−５０℃にしたアセトンで洗
浄し濾液及び洗液のアセトンを蒸散させ１次濃縮物１
８．７２ｇを得た。再び７倍量のアセトンを加え−８０
℃で一晩放置後同様にして２次濃縮物１０．３１ｇを得
た。２次濃縮物１ｇにグリセリン０．０７１４ｇをリボ
ザイムＩＭ６０を０．１ｇ加え、１０〜２０トル（Ｔｏ
ｒｒ）まで脱気し真空乾燥しながら、６０℃で振とう反
応した。リポザイムＩＭ６０（Ｎｏｖｏ社製）はムコー
ル・ミーヘイの生産したリパーゼをマクロ細孔の陰イオ
ン交換樹脂に固定化したものである。４８時間後の反応
物の組成は、トリグリセリド６１．２％、ジグリセリド
２２．０％、モノグリセリド２．８％および脂肪酸１
３．９％であった。この反応物をエチルエーテルに溶か
し固定化酵素を濾別した後、塩基性アルミナカラム（ア
ルミニウムオキシド９０（メルク社製製品番号１０７
６）およびアルカリ脱酸法により精製した。各段階の組
成および脂肪酸組成を、表４に示す。表中、下線を付し
た数値はいずれもω−３高度不飽和脂肪酸についてのも
のであり、特にエイコサペンタエン酸およびドコサペン
タエン酸の場合にはアセトンによる低温分別結晶法によ
る濃縮の結果、第２次濃縮物にいずれも２倍濃縮されて
いることが判る。またこれらの脂肪酸はグリセリンとの
反応によりドコサヘキサエン酸（Ｃ22：６）の取込みが
若干悪いがトリグリセリドに変換されることを示してい
る。Example 3 Hexane was added to the solventless low specific gravity product (decomposition rate 81%) of Example 2 and dehydrated with anhydrous sodium sulfate, and 35 g of the decomposition product obtained by evaporating hexane was concentrated by the low temperature fractional crystallization method. Therefore, add 7 times the amount of acetone as a light solvent, and
The mixture was left at 0 ° C overnight, filtered, washed with acetone at -50 ° C, and the acetone in the filtrate and washings was evaporated to obtain a primary concentrate 1
8.72 g was obtained. Add 7 times more acetone again -80
After standing overnight at 0 ° C., 10.31 g of a secondary concentrate was obtained in the same manner. Glycerin (0.0714 g) and ribozyme IM60 (0.1 g) were added to the secondary concentrate (1 g) to obtain 10 to 20 Torr (To
The mixture was degassed to rr) and vacuum-dried while reacting with shaking at 60 ° C. Lipozyme IM60 (manufactured by Novo) is obtained by immobilizing lipase produced by Mucor Mihay on an anion exchange resin having macropores. The composition of the reaction product after 48 hours was as follows: triglyceride 61.2%, diglyceride 22.0%, monoglyceride 2.8% and fatty acid 1
It was 3.9%. This reaction product was dissolved in ethyl ether and the immobilized enzyme was filtered off, followed by a basic alumina column (aluminum oxide 90 (product number 107 manufactured by Merck).
6) and the alkaline deoxidation method. Table 4 shows the composition of each stage and the fatty acid composition. In the table, all underlined numbers are for ω-3 highly unsaturated fatty acids, and particularly in the case of eicosapentaenoic acid and docosapentaenoic acid, the result of concentration by the low temperature fractional crystallization method with acetone was It can be seen that each of the subsequent concentrates was twice concentrated. The docosahexaenoic acid by reaction with these fatty acids Glycerin: indicates that (C 22 6) Although uptake of slightly poor are converted into triglycerides.
【００２８】[0028]
【表４】 [Table 4]
【００２９】実施例４ 実施例２のイソオクタン添加の低比重生成物（分解率８
１％）にイソオクタンを蒸散させた加水分解物１９０ｇ
に塩形成法による濃縮のために軽質溶剤として７倍量の
アセトン１３３０ｇおよび４規定の水酸化カリウムメタ
ノール溶液２０２ｍｌを加え３０分間室温で撹拌し−２
０℃に１６時間放置した。次いでカリウム塩として沈殿
する部分を除き濾液５８．２ｇに対し、５８２ｇのアセ
トンを加えて、−２０℃に１６時間放置した。沈殿する
部分を除き濾液を４規定の塩酸で酸性にしアセトンを蒸
散させたのちへキサンを加え洗液がｐＨ７なるまで水洗
した。ヘキサンを蒸散させて２４．４ｇのω−３高度不
飽和脂肪酸濃縮物を得た。この濃縮物１ｇにグリセリン
０．０９ｇおよび固定化リパーゼｓｐ３８２を０．１ｇ
を加え、６０℃で２４時間１０〜２０トル（Ｔｏｒｒ）
まで脱気し、真空乾燥しながら６０℃で振とう反応し
た。固定化リパーゼｓｐ３８２（Ｎｏｖｏ社製）はキャ
ンデダ・アンタクチカの生産するリパーゼをアクリル樹
脂に固定化したものである。２４時間後のこの反応物の
組成は、トリグリセリド６８．３％、ジグリセリド２
４．７％、モノグリセリド３．７％および脂肪酸３．３
％であった。この反応物をエチルエーテルに溶かし固定
化酵素を濾別した後、塩基性アルミナカラムにより精製
した。各段階の組成および脂肪酸組成を、表５に示す。Example 4 A low specific gravity product (decomposition rate: 8) obtained by adding isooctane in Example 2
190 g of hydrolyzate obtained by evaporating isooctane to 1%)
For concentration by the salt-forming method, 1330 g of 7 times amount of acetone as a light solvent and 202 ml of 4N potassium hydroxide methanol solution were added and stirred at room temperature for 30 minutes.
It was left at 0 ° C. for 16 hours. Then, 582 g of acetone was added to 58.2 g of the filtrate except the portion precipitated as a potassium salt, and the mixture was allowed to stand at -20 ° C for 16 hours. After removing the precipitated portion, the filtrate was acidified with 4N hydrochloric acid to evaporate acetone, and then hexane was added to the solution and the solution was washed with water until the pH became 7. Hexane was evaporated to obtain 24.4 g of ω-3 highly unsaturated fatty acid concentrate. To 1 g of this concentrate, 0.09 g of glycerin and 0.1 g of immobilized lipase sp382
10 to 20 Torr at 60 ° C. for 24 hours
The mixture was degassed, and the mixture was shaken at 60 ° C. while being dried under vacuum. Immobilized lipase sp382 (manufactured by Novo) is obtained by immobilizing a lipase produced by Candeda antactica on an acrylic resin. After 24 hours, the composition of this reaction product was: triglyceride 68.3%, diglyceride 2
4.7%, monoglyceride 3.7% and fatty acid 3.3
%Met. This reaction product was dissolved in ethyl ether, the immobilized enzyme was filtered off, and then purified by a basic alumina column. Table 5 shows the composition of each stage and the fatty acid composition.
【００３０】[0030]
【表５】 [Table 5]
【００３１】表中、下線を付した数値はω−３高度不飽
和脂肪酸についてのものである。本方法によると、トリ
グリセリド合成の際の酵素としてキャンデイダ属の菌由
来のリパーゼを用いているので、ドコサヘキサエン酸
（Ｃ22：６）のトリグリセリドへの取り込みが良くトリ
グリセリド合成量も６８．３％と良いものであった。In the table, the underlined numbers are for ω-3 highly unsaturated fatty acids. According to this method, since lipase derived from a bacterium belonging to the genus Candida is used as an enzyme in the synthesis of triglyceride, the incorporation of docosahexaenoic acid (C 22 : 6) into triglyceride is good and the amount of triglyceride synthesis is as good as 68.3%. It was a thing.
【００３２】[0032]
【発明の効果】ω−３高度不飽和脂肪酸は、循環器系疾
病の予防効果、ラットの学習能力の向上効果等重要な生
理的意義がある。本発明によれば、ω−３高度不飽和脂
肪酸を豊富に含み多量に供給入手が可能な魚油等の油脂
を原料として、酵素反応を利用してω−３高度不飽和脂
肪酸含有油脂を一旦加水分解して脂肪酸とし、これを低
温分別結晶法などの濃縮方法により他の脂肪酸などから
分離濃縮し、再度酵素反応を利用してグリセリンと反応
させることにより、ω−３高度不飽和脂肪酸トリグリセ
リド油脂を効率良く得ることができる。この際の原料油
脂は安価であり、反応はすべて緩和な条件下で実施で
き、しかもω−３高度不飽和脂肪酸を実質的な変成を起
すことなく高度に濃縮できる。従って本発明は単に機能
性食品としての利用ばかりでなく医学的および薬学的な
応用にたいしても寄与するものである。EFFECT OF THE INVENTION ω-3 highly unsaturated fatty acids have important physiological significance such as a preventive effect on cardiovascular diseases and an effect of improving learning ability of rats. According to the present invention, an oil or fat such as fish oil, which is rich in ω-3 highly unsaturated fatty acids and can be supplied and supplied in a large amount, is used as a raw material to hydrolyze an ω-3 highly unsaturated fatty acid-containing oil or fat by utilizing an enzymatic reaction. By decomposing it into fatty acid, separating and concentrating it from other fatty acids by a concentrating method such as low temperature fractional crystallization, and reacting again with glycerin using an enzymatic reaction, ω-3 highly unsaturated fatty acid triglyceride It can be obtained efficiently. In this case, the raw material fats and oils are inexpensive, all the reactions can be carried out under mild conditions, and the ω-3 highly unsaturated fatty acid can be highly concentrated without causing substantial alteration. Therefore, the present invention not only serves as a functional food but also contributes to medical and pharmaceutical applications.
【図面の簡単な説明】[Brief description of drawings]
【図１】向流接触型の二相系固定化酵素充填反応器の説
明断面図である。FIG. 1 is an explanatory cross-sectional view of a countercurrent contact type two-phase system immobilized enzyme packed reactor.
【符号の説明】 １．低比重生産物排出管 ２．上端の静置層 ３．高比重基質供給管 ４．撹拌槽 ５．仕切り板 ６．パルス発生機 ７．低比重基質供給管 ８．下端の静置層 ９．高比重生産物排出管 １０．エマルジョン破壊装置 １１．パルス発生機につながれた分岐管 １２．定量ポンプにつながれた分岐管[Explanation of symbols] 1. Low specific gravity product discharge pipe 2. Top stationary layer 3. High specific gravity substrate supply pipe 4. Stirring tank 5. Partition plate 6. Pulse generator 7. Low specific gravity substrate supply tube 8. Bottom static layer 9. High specific gravity product discharge pipe 10. Emulsion breaker 11. Branch pipe connected to pulse generator 12. Branch pipe connected to metering pump
───────────────────────────────────────────────────── フロントページの続き (72)発明者 冨塚 登 茨城県つくば市東１丁目１番３号 工業技 術院微生物工業技術研究所内 (72)発明者 東 直輝 千葉県船橋市日の出２丁目17番１号 ボー ソー油脂株式会社内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Noboru Tomizuka, Inventor No. 1-3, Higashi 1-3, Tsukuba-shi, Ibaraki Inside Institute for Microbial Technology, Industrial Technology Institute (72) Naoki Higashi, 2-17-1 Hinode, Funabashi, Chiba No. Boso Oil & Fat Co., Ltd.
Claims (8)
飽和脂肪酸のアルコールエステルを固定化されたリパー
ゼを用いて加水分解する工程、得られた加水分解生成物
に含まれる高度不飽和脂肪酸を濃縮する工程及び高度不
飽和脂肪酸濃縮物を固定化されたリパーゼを用いてトリ
グリセリドに変換させる工程からなる濃縮された高度不
飽和脂肪酸含有油脂の製造方法。1. A step of hydrolyzing a polyunsaturated fatty acid-containing oil or fat or an alcohol ester of a polyunsaturated fatty acid using immobilized lipase, and concentrating the polyunsaturated fatty acid contained in the obtained hydrolysis product. A process for producing a concentrated highly unsaturated fatty acid-containing oil or fat, which comprises the steps of converting a highly unsaturated fatty acid concentrate to triglyceride using immobilized lipase.
る請求項１の方法。2. The method of claim 1 wherein all reactions are carried out in an atmosphere of inert gas.
存在下に実施する請求項１又は２の方法。3. The method according to claim 1, wherein the hydrolysis is carried out in the presence of a water-immiscible light solvent.
の生産したリパーゼを含む固定化されたリパーゼを用い
て実施する請求項１〜３のいずれかの方法。4. The method according to claim 1, wherein the hydrolysis is carried out using an immobilized lipase containing a lipase produced by Pseudomonas bacteria.
の変換をキャンデイダ属菌の生産したリパーゼを含む固
化されたリパーゼを用いて実施する請求項５の方法。5. The method according to claim 5, wherein the conversion to the highly unsaturated concentrated triglyceride is carried out by using a solidified lipase containing a lipase produced by Candida.
重量％からなる混合液を、固定化されたリパーゼと接触
反応させると同時に副生水を脱水させることを特徴とす
る高度不飽和脂肪酸濃縮物トリグリセリドの製造方法。6. A polyunsaturated fatty acid concentrate and its 8-10.
A method for producing a highly unsaturated fatty acid concentrate triglyceride, which comprises subjecting a mixed solution of wt% to a catalytic reaction with immobilized lipase and simultaneously dehydrating by-product water.
イダ属菌の生産したリパーゼを含む請求項６の方法。7. The method of claim 6, wherein the immobilized lipase comprises a Candida sp. Produced lipase.
飽和脂肪酸である請求項１〜７のいずれかの方法。8. The method according to claim 1, wherein the polyunsaturated fatty acid is an ω-3 polyunsaturated fatty acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3283560A JP2516860B2 (en) | 1991-10-03 | 1991-10-03 | Method for producing concentrated highly unsaturated fatty acid-containing fats and oils |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3283560A JP2516860B2 (en) | 1991-10-03 | 1991-10-03 | Method for producing concentrated highly unsaturated fatty acid-containing fats and oils |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0595792A true JPH0595792A (en) | 1993-04-20 |
| JP2516860B2 JP2516860B2 (en) | 1996-07-24 |
Family
ID=17667115
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3283560A Expired - Fee Related JP2516860B2 (en) | 1991-10-03 | 1991-10-03 | Method for producing concentrated highly unsaturated fatty acid-containing fats and oils |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2516860B2 (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996026287A1 (en) * | 1995-02-24 | 1996-08-29 | Goemar S.A. | Enzymatic methods for polyunsaturated fatty acid enrichment |
| EP0739589A1 (en) * | 1995-04-28 | 1996-10-30 | Loders Croklaan B.V. | Triglyceridus, rich in Polyunsaturated fatty acids |
| EP0739591A1 (en) * | 1995-04-28 | 1996-10-30 | Loders Croklaan B.V. | Triglycerides rich in polyunsaturated fatty acids |
| EP0739590A3 (en) * | 1995-04-28 | 1996-11-06 | Loders Croklaan B.V. | Triglycerides, rich in polyunsaturated fatty acids |
| WO1996037587A1 (en) * | 1995-05-24 | 1996-11-28 | Loders Croklaan B.V. | Production of materials high in long chain polyunsaturated fatty acids |
| WO1996037586A1 (en) * | 1995-05-24 | 1996-11-28 | Loders Croklaan B.V. | Production method for fats with long chain polyunsaturated fatty acids |
| WO1997019601A1 (en) * | 1995-11-24 | 1997-06-05 | Loders Croklaan B.V. | Composition based on fish oil |
| WO1999009119A1 (en) * | 1997-08-18 | 1999-02-25 | Kao Corporation | Process for producing diglycerides |
| WO2003094625A1 (en) * | 2002-05-08 | 2003-11-20 | Danmarks Tekniske Uni Technica | A facile two-step enzyme process for increasing the content of polyunsaturated fatty acids in fish oil |
| JP2004285182A (en) * | 2003-03-20 | 2004-10-14 | Yuji Shimada | Glyceride and its manufacturing process |
| JP2010503748A (en) * | 2006-09-14 | 2010-02-04 | イルシン ウェルズ カンパニー リミテッド | Fish oil-derived glyceride oil composition and method for producing the same |
| WO2011074758A1 (en) * | 2009-12-17 | 2011-06-23 | Chemport Inc. | Process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids |
| US8241875B2 (en) | 2005-06-21 | 2012-08-14 | Kao Corporation | Method for producing fatty acids with an immobilized enzyme packed column |
| CN103025880A (en) * | 2009-04-06 | 2013-04-03 | 诺维信公司 | Triglycerides with high unsaturated fatty acid content |
| CN119709395A (en) * | 2024-12-20 | 2025-03-28 | 中国农业科学院油料作物研究所 | A gas-liquid-solid three-phase fluidized bed enzyme reactor and a method for simultaneously preparing diglyceride by enzymatic deacidification of oil and fat using the same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6291188A (en) * | 1985-10-17 | 1987-04-25 | Nisshin Oil Mills Ltd:The | Production of highly unsaturated fatty acid glyceride |
| JPS62278991A (en) * | 1986-05-28 | 1987-12-03 | Japanese Res & Dev Assoc Bio Reactor Syst Food Ind | Production of highly unsaturated fatty acid |
| JPH0225447A (en) * | 1988-07-13 | 1990-01-26 | Nippon Oil & Fats Co Ltd | Production of highly unsaturated fatty acids |
-
1991
- 1991-10-03 JP JP3283560A patent/JP2516860B2/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6291188A (en) * | 1985-10-17 | 1987-04-25 | Nisshin Oil Mills Ltd:The | Production of highly unsaturated fatty acid glyceride |
| JPS62278991A (en) * | 1986-05-28 | 1987-12-03 | Japanese Res & Dev Assoc Bio Reactor Syst Food Ind | Production of highly unsaturated fatty acid |
| JPH0225447A (en) * | 1988-07-13 | 1990-01-26 | Nippon Oil & Fats Co Ltd | Production of highly unsaturated fatty acids |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996026287A1 (en) * | 1995-02-24 | 1996-08-29 | Goemar S.A. | Enzymatic methods for polyunsaturated fatty acid enrichment |
| FR2731015A1 (en) * | 1995-02-24 | 1996-08-30 | Sci Sartone | PROCESS FOR THE ENZYMATIC ENRICHMENT OF OILS OF MARINE ORIGIN AND THE TRIGLYCERIDES OF POLYUNSATURATED FATTY ACIDS THUS OBTAINED |
| EP0739589A1 (en) * | 1995-04-28 | 1996-10-30 | Loders Croklaan B.V. | Triglyceridus, rich in Polyunsaturated fatty acids |
| EP0739591A1 (en) * | 1995-04-28 | 1996-10-30 | Loders Croklaan B.V. | Triglycerides rich in polyunsaturated fatty acids |
| EP0739590A3 (en) * | 1995-04-28 | 1996-11-06 | Loders Croklaan B.V. | Triglycerides, rich in polyunsaturated fatty acids |
| WO1996037587A1 (en) * | 1995-05-24 | 1996-11-28 | Loders Croklaan B.V. | Production of materials high in long chain polyunsaturated fatty acids |
| WO1996037586A1 (en) * | 1995-05-24 | 1996-11-28 | Loders Croklaan B.V. | Production method for fats with long chain polyunsaturated fatty acids |
| WO1997019601A1 (en) * | 1995-11-24 | 1997-06-05 | Loders Croklaan B.V. | Composition based on fish oil |
| WO1999009119A1 (en) * | 1997-08-18 | 1999-02-25 | Kao Corporation | Process for producing diglycerides |
| WO2003094625A1 (en) * | 2002-05-08 | 2003-11-20 | Danmarks Tekniske Uni Technica | A facile two-step enzyme process for increasing the content of polyunsaturated fatty acids in fish oil |
| JP2004285182A (en) * | 2003-03-20 | 2004-10-14 | Yuji Shimada | Glyceride and its manufacturing process |
| US8241875B2 (en) | 2005-06-21 | 2012-08-14 | Kao Corporation | Method for producing fatty acids with an immobilized enzyme packed column |
| JP2010503748A (en) * | 2006-09-14 | 2010-02-04 | イルシン ウェルズ カンパニー リミテッド | Fish oil-derived glyceride oil composition and method for producing the same |
| CN103025880A (en) * | 2009-04-06 | 2013-04-03 | 诺维信公司 | Triglycerides with high unsaturated fatty acid content |
| WO2011074758A1 (en) * | 2009-12-17 | 2011-06-23 | Chemport Inc. | Process for preparing triglyceride oil containing high concentration of polyunsaturated fatty acids |
| CN119709395A (en) * | 2024-12-20 | 2025-03-28 | 中国农业科学院油料作物研究所 | A gas-liquid-solid three-phase fluidized bed enzyme reactor and a method for simultaneously preparing diglyceride by enzymatic deacidification of oil and fat using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2516860B2 (en) | 1996-07-24 |
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