US20170326090A1 - Production of ultrapure epa and polar lipids from largely heterotrophic culture - Google Patents
Production of ultrapure epa and polar lipids from largely heterotrophic culture Download PDFInfo
- Publication number
- US20170326090A1 US20170326090A1 US15/444,027 US201715444027A US2017326090A1 US 20170326090 A1 US20170326090 A1 US 20170326090A1 US 201715444027 A US201715444027 A US 201715444027A US 2017326090 A1 US2017326090 A1 US 2017326090A1
- Authority
- US
- United States
- Prior art keywords
- epa
- polar
- culture
- rich
- fatty acids
- 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.)
- Abandoned
Links
- 150000002632 lipids Chemical class 0.000 title claims abstract description 154
- 238000004519 manufacturing process Methods 0.000 title abstract description 60
- 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 claims abstract description 257
- 235000020673 eicosapentaenoic acid Nutrition 0.000 claims abstract description 256
- 229960005135 eicosapentaenoic acid Drugs 0.000 claims abstract description 256
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 claims abstract description 256
- 244000005700 microbiome Species 0.000 claims abstract description 29
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 114
- 239000000194 fatty acid Substances 0.000 claims description 113
- 229930195729 fatty acid Natural products 0.000 claims description 113
- 150000004665 fatty acids Chemical class 0.000 claims description 112
- 239000002028 Biomass Substances 0.000 claims description 22
- 239000000203 mixture Substances 0.000 abstract description 116
- 238000000034 method Methods 0.000 abstract description 76
- 230000008569 process Effects 0.000 abstract description 32
- 230000001225 therapeutic effect Effects 0.000 abstract description 27
- 241001104939 Nitzschia laevis Species 0.000 abstract description 14
- 235000021323 fish oil Nutrition 0.000 abstract description 13
- 230000000069 prophylactic effect Effects 0.000 abstract description 12
- 150000001875 compounds Chemical class 0.000 abstract description 6
- 241000251468 Actinopterygii Species 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- 235000019626 lipase activity Nutrition 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 238000001311 chemical methods and process Methods 0.000 abstract 1
- 230000003467 diminishing effect Effects 0.000 abstract 1
- 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 62
- 210000004027 cell Anatomy 0.000 description 40
- 102000004190 Enzymes Human genes 0.000 description 33
- 108090000790 Enzymes Proteins 0.000 description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 33
- 235000021342 arachidonic acid Nutrition 0.000 description 30
- 229940114079 arachidonic acid Drugs 0.000 description 30
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 27
- 239000000463 material Substances 0.000 description 24
- 235000015872 dietary supplement Nutrition 0.000 description 20
- 238000000746 purification Methods 0.000 description 20
- 230000000243 photosynthetic effect Effects 0.000 description 19
- 108090001060 Lipase Proteins 0.000 description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 18
- 230000008901 benefit Effects 0.000 description 17
- 239000000047 product Substances 0.000 description 17
- 230000012010 growth Effects 0.000 description 16
- 235000015097 nutrients Nutrition 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 102000004882 Lipase Human genes 0.000 description 14
- 239000004367 Lipase Substances 0.000 description 14
- 238000000605 extraction Methods 0.000 description 14
- 235000019421 lipase Nutrition 0.000 description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 13
- 235000013361 beverage Nutrition 0.000 description 13
- 235000013305 food Nutrition 0.000 description 13
- 239000012528 membrane Substances 0.000 description 13
- 235000020660 omega-3 fatty acid Nutrition 0.000 description 13
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 13
- 238000007792 addition Methods 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 12
- 150000003904 phospholipids Chemical class 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- -1 sulfoquinovosyl diacylglycerol Chemical class 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 compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 10
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 10
- 229910052799 carbon Inorganic materials 0.000 description 10
- 125000004432 carbon atom Chemical group C* 0.000 description 10
- 230000007935 neutral effect Effects 0.000 description 10
- 238000000926 separation method Methods 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 229930182830 galactose Natural products 0.000 description 9
- 230000001965 increasing effect Effects 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- 241000894007 species Species 0.000 description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 150000003626 triacylglycerols Chemical class 0.000 description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 241000124008 Mammalia Species 0.000 description 7
- 102000015439 Phospholipases Human genes 0.000 description 7
- 108010064785 Phospholipases Proteins 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- CYQFCXCEBYINGO-IAGOWNOFSA-N delta1-THC Chemical compound C1=C(C)CC[C@H]2C(C)(C)OC3=CC(CCCCC)=CC(O)=C3[C@@H]21 CYQFCXCEBYINGO-IAGOWNOFSA-N 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 238000003306 harvesting Methods 0.000 description 7
- 230000036541 health Effects 0.000 description 7
- 229940012843 omega-3 fatty acid Drugs 0.000 description 7
- 235000020665 omega-6 fatty acid Nutrition 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 239000011550 stock solution Substances 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 210000003763 chloroplast Anatomy 0.000 description 6
- 238000002425 crystallisation Methods 0.000 description 6
- 235000005911 diet Nutrition 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 150000002148 esters Chemical class 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 235000013336 milk Nutrition 0.000 description 6
- 239000008267 milk Substances 0.000 description 6
- 210000004080 milk Anatomy 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 5
- 241000206745 Nitzschia alba Species 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 230000000378 dietary effect Effects 0.000 description 5
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 5
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 5
- 230000001976 improved effect Effects 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000003921 oil Substances 0.000 description 5
- 235000019198 oils Nutrition 0.000 description 5
- 229940033080 omega-6 fatty acid Drugs 0.000 description 5
- 230000002572 peristaltic effect Effects 0.000 description 5
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- KDYAPQVYJXUQNY-OPHDRXFHSA-N 1,2-di-(alpha-linolenoyl)-3-[alpha-D-galactosyl-(1->6)-beta-D-galactosyl]-sn-glycerol Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](OC[C@@H](COC(=O)CCCCCCC\C=C/C\C=C/C\C=C/CC)OC(=O)CCCCCCC\C=C/C\C=C/C\C=C/CC)O[C@@H]1CO[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KDYAPQVYJXUQNY-OPHDRXFHSA-N 0.000 description 4
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 4
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 4
- 102100033357 Pancreatic lipase-related protein 2 Human genes 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 230000003042 antagnostic effect Effects 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 230000008821 health effect Effects 0.000 description 4
- 230000009569 heterotrophic growth Effects 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000000693 micelle Substances 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 3
- 241000206761 Bacillariophyta Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 241000195493 Cryptophyta Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 150000001298 alcohols Chemical class 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 239000012620 biological material Substances 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 235000014171 carbonated beverage Nutrition 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000037149 energy metabolism Effects 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 238000005886 esterification reaction Methods 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 230000001747 exhibiting effect Effects 0.000 description 3
- 230000037406 food intake Effects 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 3
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- OPGOLNDOMSBSCW-CLNHMMGSSA-N Fursultiamine hydrochloride Chemical compound Cl.C1CCOC1CSSC(\CCO)=C(/C)N(C=O)CC1=CN=C(C)N=C1N OPGOLNDOMSBSCW-CLNHMMGSSA-N 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 2
- 102100022119 Lipoprotein lipase Human genes 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 208000001132 Osteoporosis Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920006362 Teflon® Polymers 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000000370 acceptor Substances 0.000 description 2
- 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 2
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 2
- 235000019789 appetite Nutrition 0.000 description 2
- 230000036528 appetite Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010960 commercial process Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 150000001982 diacylglycerols Chemical class 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 238000004508 fractional distillation Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 235000013376 functional food Nutrition 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 125000002519 galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 description 2
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 2
- 229960002733 gamolenic acid Drugs 0.000 description 2
- 239000003365 glass fiber Substances 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 208000000509 infertility Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 208000021267 infertility disease Diseases 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000005657 iodolactonization reaction Methods 0.000 description 2
- 208000017169 kidney disease Diseases 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
- 229960004488 linolenic acid Drugs 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 208000024714 major depressive disease Diseases 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 239000002417 nutraceutical Substances 0.000 description 2
- 235000021436 nutraceutical agent Nutrition 0.000 description 2
- 201000008482 osteoarthritis Diseases 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000010525 oxidative degradation reaction Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000001850 reproductive effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 235000019795 sodium metasilicate Nutrition 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 229910052911 sodium silicate Inorganic materials 0.000 description 2
- JIWBIWFOSCKQMA-UHFFFAOYSA-N stearidonic acid Natural products CCC=CCC=CCC=CCC=CCCCCC(O)=O JIWBIWFOSCKQMA-UHFFFAOYSA-N 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000004808 supercritical fluid chromatography Methods 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 238000005809 transesterification reaction Methods 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 229910019626 (NH4)6Mo7O24 Inorganic materials 0.000 description 1
- 0 *OC1CC*(CC*2CCCCC2)CC1 Chemical compound *OC1CC*(CC*2CCCCC2)CC1 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000000103 Anorexia Nervosa Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 208000036864 Attention deficit/hyperactivity disease Diseases 0.000 description 1
- 206010004446 Benign prostatic hyperplasia Diseases 0.000 description 1
- 208000020925 Bipolar disease Diseases 0.000 description 1
- 206010006298 Breast pain Diseases 0.000 description 1
- HQGPWBVRBMCYKE-UHFFFAOYSA-N C1CCCCC1.CCC=CCCCC(=O)OCC(O)COC1CCCCC1.[SnH2].[SnH][SnH].[SnH][Sn][SnH] Chemical compound C1CCCCC1.CCC=CCCCC(=O)OCC(O)COC1CCCCC1.[SnH2].[SnH][SnH].[SnH][Sn][SnH] HQGPWBVRBMCYKE-UHFFFAOYSA-N 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 208000020401 Depressive disease Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 241001501873 Isochrysis galbana Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 208000007466 Male Infertility Diseases 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 208000006662 Mastodynia Diseases 0.000 description 1
- 208000021642 Muscular disease Diseases 0.000 description 1
- 240000001307 Myosotis scorpioides Species 0.000 description 1
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 208000021384 Obsessive-Compulsive disease Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000206744 Phaeodactylum tricornutum Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 201000009916 Postpartum depression Diseases 0.000 description 1
- 208000004403 Prostatic Hyperplasia Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 240000005384 Rhizopus oryzae Species 0.000 description 1
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 239000004115 Sodium Silicate Substances 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 208000030886 Traumatic Brain injury Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 208000026723 Urinary tract disease Diseases 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 1
- GBBUBIKYAQLESK-UHFFFAOYSA-N [3-(2-methylprop-2-enoylamino)phenyl]boronic acid Chemical compound CC(=C)C(=O)NC1=CC=CC(B(O)O)=C1 GBBUBIKYAQLESK-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 206010068168 androgenetic alopecia Diseases 0.000 description 1
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 229940053200 antiepileptics fatty acid derivative Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 230000037007 arousal Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 208000015802 attention deficit-hyperactivity disease Diseases 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 238000011138 biotechnological process Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 208000028683 bipolar I disease Diseases 0.000 description 1
- 208000025307 bipolar depression Diseases 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 208000030963 borderline personality disease Diseases 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000006931 brain damage Effects 0.000 description 1
- 231100000874 brain damage Toxicity 0.000 description 1
- 230000036995 brain health Effects 0.000 description 1
- 208000029028 brain injury Diseases 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000007073 chemical hydrolysis Effects 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 238000010961 commercial manufacture process Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 229940117173 croton oil Drugs 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000018823 dietary intake Nutrition 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 150000002016 disaccharides Chemical group 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 206010013932 dyslexia Diseases 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 230000002996 emotional effect Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 201000003104 endogenous depression Diseases 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229940013317 fish oils Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 150000002256 galaktoses Chemical class 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002641 glycemic effect Effects 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000010224 hepatic metabolism Effects 0.000 description 1
- 244000059217 heterotrophic organism Species 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 235000015243 ice cream Nutrition 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 229960004592 isopropanol Drugs 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 201000003723 learning disability Diseases 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 235000013310 margarine Nutrition 0.000 description 1
- 239000003264 margarine Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- NBTOZLQBSIZIKS-UHFFFAOYSA-N methoxide Chemical compound [O-]C NBTOZLQBSIZIKS-UHFFFAOYSA-N 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002772 monosaccharides Chemical group 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 239000012454 non-polar solvent Substances 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 210000003254 palate Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 230000009564 phototrophic growth Effects 0.000 description 1
- 230000007180 physiological regulation Effects 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 235000020991 processed meat Nutrition 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 230000000272 proprioceptive effect Effects 0.000 description 1
- 201000004240 prostatic hypertrophy Diseases 0.000 description 1
- 201000007094 prostatitis Diseases 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 150000003214 pyranose derivatives Chemical group 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000007127 saponification reaction Methods 0.000 description 1
- 238000010963 scalable process Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 235000021055 solid food Nutrition 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 235000015096 spirit Nutrition 0.000 description 1
- 235000011496 sports drink Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000000707 stereoselective effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009529 traumatic brain injury Effects 0.000 description 1
- 208000014001 urinary system disease Diseases 0.000 description 1
- 208000037911 visceral disease Diseases 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 235000014101 wine Nutrition 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
- A23L33/12—Fatty acids or derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P7/6432—Eicosapentaenoic acids [EPA]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- This invention relates to lipid compositions synthesized by single-cell organisms, to the manufacture and some applications of omega-3 highly unsaturated fatty acids and to pharmaceutical substances, and in particular to novel therapeutic, dietary and health-food compositions.
- Alpha linolenic acid is an omega-3 fatty acid with eighteen carbon atoms and three double bonds.
- Arachidonic acid is an omega-6 fatty acid with twenty carbon atoms and four double bonds.
- Diacyl galactolipids are galactolipids with both Sn1 and Sn2 occupied by fatty acid molecules.
- Digalactosyl galactolipids are those where two galactose molecules are attached.
- Docosopentaenoic acid is an omega-3 fatty acid with twenty-two carbon atoms and five double bonds.
- Docosahexanoic acid is an omega-3 fatty acid with twenty-two carbon atoms and six double bonds.
- Eicosapentaenoic acid is an omega-3 fatty acid with twenty carbon atoms and five double bonds.
- Effective amount means: an amount sufficient to cause a desired result when administered.
- Esterification of n-3 HUFAs is understood, so that a term such as “99% pure EPA” is understood to take no account of the ethyl or other moiety used to form an ester with the free fatty acid.
- Nutraceutical is any substance that is a food or a part of a food and provides medical or health benefits, including the prevention and treatment of disease.
- the EPA composition is substantially free of those materials herein defined as “undesired”.
- An EPA composition according to this definition of purity may include only 1 to 10% EPA (although more preferably 60-90% EPA; the remainder may include galactolipids or components thereof, or added pharmaceutically acceptable excipients, anti-oxidants, adsorbents, flavours, and the like.
- compositions comprising around 90% of EPA or more by weight and simultaneously a ratio of EPA to any individual undesired molecule of at least around 90 to 1.
- Undesired molecules or materials are defined as those that may diminish the desired beneficial health effect of EPA-rich compositions when co-consumed. These include molecules that may diminish the desired effect through actions which may be antagonistic, competitive, block, reverse, mediate, synergise or otherwise alter the desired beneficial health effect of EPA when consumed in an effective (or clinically relevant) amount.
- undesired molecules are to include structurally or functionally similar molecules including fatty acids, docosohexaerjoic acid (DHA), arachidonic acid (AA), 18:4 n-3, 18:3 n-3, 18:2 n-6 and other omega-3 and 6 fatty acids in general.
- DHA docosohexaerjoic acid
- AA arachidonic acid
- AA is a precursor of EPA.
- EPA-rich means that the composition includes more than 1% of EPA as dry weight.
- EPA productivity means the amount of EPA which can be produced per unit cost.
- a proxy measure for productivity in largely heterotrophic cultures of microalgae culture is often taken as the combined effect of yield, growth rate, cell density, nutrient utilisation efficiency, and dilution rate.
- Fatty acid compositions include—ethyl esters, salts, free fatty acids, methyl esters, and other alcohol esters of fatty acids, and combinatorial lipids.
- Galactolipids are comprised of a glycerol backbone with at least two separate molecules attached; at least one of which is a fatty acid and the other is. either one or two molecules of galactose (in mono or disaccharide form) bonded by, in the case of the fatty acid or fatty acids, an ester link or rarely an ether link and in the case of the galactose or galactoses an ether link in all cases. At least one galactose is present and normally attached to the Sn3 position of glycerol. The galactose is always in pyranose form.
- a second and rarely a third galactose molecule is attached by beta-d-3-pyranosyl bonds to the preceding galactose molecule.
- Positions are labeled according to a convention based on an original stereospecific structure (see table 1 showing the 1.3 isomer of the DGMG galactolipid class containing an EPA molecule′ acylated in the Sn1. Normally when only one fatty acid molecule is attached it occupies the Sn1 position. However, occasionally one fatty acid may be attached in the Sn2 position while the Sn1 position remains unoccupied. This may be referred to as a lyso derivative.
- Gamma linolenic acid is an omega-6 fatty acid with eighteen carbon atoms and four double bonds.
- Heterotrophic culture means a culture of organisms for which the sole energy source is derived from supplied nutrients (the major nutrient group for energy metabolism) which is usually a form or forms of organic carbon (e.g. glucose, acetate).
- supplied nutrients the major nutrient group for energy metabolism
- organic carbon e.g. glucose, acetate
- Largely (or partially) heterotrophic culture means a mixotrophic culture of organisms for which the major energy source is derived from supplied nutrients (the major nutrient group for energy metabolism) which is usually a form or forms of organic carbon (e.g. glucose, acetate) and the minor energy source for energy metabolism is light.
- supplied nutrients the major nutrient group for energy metabolism
- organic carbon e.g. glucose, acetate
- Linoleic acid is an omega-6 fatty acid with eighteen carbon atoms and two double bonds.
- Monoacyl galactolipids are galactolipids with only one position occupied by a fatty acid. In a monoacyl galactolipid the fatty acid molecule is attached either at position Sn1 or Sn2.
- Monogalactosyl galactolipids are those with only one galactose molecule attached.
- Neutral lipids are those lipids contained in an organism which can be isolated through the use of non-polar solvents and include mono-di- and triacylglycerols
- Photosynthetic lipids are those polar lipids whose production can be significantly altered via the addition of light to a largely heterotrphic culture of microorganisms. These may occur within the chloroplast as is predominantly the case with galactolipids or may also be associated with other cell organelles as is predominantly the case with phospholipids.
- Polar lipids are those lipids contained in an organism which can be isolated through the use of polar solvents and include phospholipids and galactolipids
- Photosynthetically active average irradiance inside a culture is the amount of electromagnetic radiation between wavelengths 400 nm and 700 nm incident on a culture averaged over all positions in the culture vessel and over time.
- Stearidonic acid is an omega-3 fatty acid with eighteen carbon atoms and four double bonds.
- Sub-photosynthetic exposure means an exposure to light whereby the combination of intensity and exposure time is equal or less than around the equivalent of a continuous illumination of 1 to about 10 micromol photons per square meter per second.
- Omega-3 fatty acid is a fatty acid with the first double bond three carbon atoms from the n-methyl end of the molecule.
- Omega-6 fatty acid is a fatty acid with the first double bond six carbon atoms from the n-methyl end of the molecule.
- n-3 omega-3 n-6: omega-6 n-3 HUFA: omega-3 highly unsaturated fatty acid
- MGMG monogalatosylmonoacylgalactolipid
- MGDG monogalactosyldiacylgalactolipid
- n-3 HUFA highly unsaturated fatty acids
- human dietary supplementation studies incorporating relatively pure forms of the n-3 HUFA eicosapentaenoic acid (EPA) have suggested this nutrient may promote health and ameliorate or even reverse the effects of a range of common diseases, including but not limited to certain forms of cardiovascular disease and depression (Yokoyama et al., Lancet 369:1062-1063. 2007; Peet & Horrobin Arch. Gen. Psych. 59(10) 913-9 2002).
- the therapeutic effect of dietary supplementation with concentrated forms of EPA are dependent to some extent on purity.
- High purity dose forms have an advantage in terms of increased bioavailability.
- desired effects of EPA are limited or even reversed by the co-consumption of undesired molecules; (as herein defined) in particular docosohexaenoic acid (DHA); also AA and other omega-3 and 6 fatty acids in general. Therefore to enable effective pharmaceutical or therapeutic use of EPA, high purity dose forms, free of the undesired molecules, are required.
- DHA docosohexaenoic acid
- Purification processes are also rendered less efficient by the relatively complex mixture of fatty acids, and a high degree of natural variability contained in fish oil.
- Mixotrophic production systems have been proposed for production of EPA-rich microorganisms. These provide a proportion of the energy for growth in the form of organic carbon supplied to the culture medium.
- An advantage of mixotrophy includes higher productivities than are achievable with solely photosynthetic production and potentially also lower the overall requirement for light.
- a disadvantage of the addition of organic carbon sources to outdoor photobioreactor cultures is the creation of an additional contamination risk by presenting a substrate for growth of non-photosynthetic contaminating organisms.
- a number of solely heterotrophic systems for producing EPA-rich microorganisms have been disclosed. These overcome many of the limitations of photosynthetic systems due to their ability to achieve growth of EPA rich species in the absence of light. By eliminating the requirement for light it is possible to significantly reduced the surface-to-volume ratio of reactors and consequently also reduce capital expenditure and sterilisation costs.
- An additional advantage of heterotrophic production systems is that culture parameters can be tightly controlled leading to production of a product of a consistent quality.
- the fatty acid composition of certain EPA-rich microalgae contain low proportions of fatty acids with structural similarity to EPA. Together with the generally less complex fatty acid composition of microalgae this may offer advantages in terms of purification over fish oil.
- lipids may be accumulated in the form of triglyceride, a lipid class not utilized extensively in lipid membrane structure.
- EPA-rich triglycerides are of potential therapeutic value. EPA may be recovered from triglcerides and further purified via an array of conventional and emerging techniques. Processes designed to extract, concentrate or purify EPA-rich lipid or fatty acid compositions from triglycerides however may be disadvantaged by the presence of a relatively high level and wide range of undesirable fatty acid molecules, and a low level of stereospecificity in terms of the location of EPA within the triglycerides.
- Certain polar lipid classes produced in cultures of microalgae are relatively rich in EPA. At the same time some of these lipid classes may exhibit a high degree of stereospecificty in terms of the location of EPA within the class and its isomers. This concentration of EPA in a predictable manner in particular lipid classes provides an additional opportunity to sequester undesirable molecules in unused fractions during a purification process. In addition certain lipid classes produced by cultures of microalgae may also have therapeutic value in their own right.
- Microalgae produce two major types of polar lipids;—phospholipids and glycolipids. All these major polar lipid classes comprise a glycerol backbone with three positions conventionally labeled Sn 1-3. Phospho and galacto lipid classes are categorised respectively according to phosphate- and galactose-containing functional groups which are attached to the glycerol backbone usually at the Sn-3 position. Fatty acids are acylated at one or more positions 1-2. Isometric forms of these lipid classes arise from acylation patterns where not all available positions are occupied by fatty acids or where a functional group is attached at an alternative position.
- Galactolipids are produced predominantly in the chloroplast and are a structural component of the photosynthetic membrane. Galactolipids are one of the most polar of all the lipid classes; there is a substantial difference in charge distribution over the molecule because of the polar nature of the one or more galactose moieties that are attached to the glycerol backbone, providing spacially separated centres of positive and negative charge. Hence galactolipids have found application as emulsifying agents and have been proposed as drug delivery conjugates.
- galactolipids leads to a number of useful opportunities, including but without limitation to potential advantageous routes for extraction and purification of galactolipids and galactolipid fatty acids, formulation of galactolipids and galactolipid fatty acids into foods, functional foods, beverages, pharmaceutical and industrial compositions, delivery of galactolipids and galactolipid fatty acid nutritional and therapeutic products in a bioavailable form, as well as advantageous therapeutic effects and mechanisms of action their use may promote.
- Phospholipids are major structural components of cellular membranes.
- the highly polar ‘head’ of the molecules coupled with their hydrophobic fatty acid ‘tails’ lead the phospholipids to spontaneously form micelles and bilayers in aqueous media.
- Phosopholipids both within and external to the chloroplast are expected to play a number of important roles in relaton to the physiological response of microorganisms to light. Or example it has been proposed that fatty acids located in cytoplasmic phospholipids classes are a reservoir for incorporation into chloroplastic lipids during production of photosynthetic membranes.
- the polar nature of phospholipids among other physiochemical properties presents a number of useful opportunities similar to those stated above for galactolipids.
- Certain phosopholipids including PC are known be absorbed differentially in mammals which could be turned to a therapeutic advantage. Work on absorption of galactolipids in particular MGDG in mammals is limited.
- Kyle et al in U.S. Pat. No. 5,567,732 disclose a method for producing EPA-rich oils from cells of the diatom Nitzschia alba in the dark and teach that it is possible to induce this organism in heterotrophic culture to enter an oleogenic phase by allowing nitrogen depletion to occur and after 12-24 hours allowing a silicate depletion state to also occur, while continuing to supply other nutrients to the culture.
- the colourless species of diatoms are preferred.
- Coldless species in general and in particular the microorganism preferred by Kyle et al are colourless because they do not exhibit the phenotype of photosynthetic pigments.
- N. alba for example is believed to be an obligate heterotroph which means that it does not have any active photosynthetic capacity.
- fatty acid DHA may be produced from the phosphatidylethanolamine (PE) lipid fraction of the photosynthetic organism Isochrysis galbana by extracting total lipids and subsequently producing DHA rich compositions by employing the well known technique of urea crystallisation.
- PE phosphatidylethanolamine
- Vali et al U.S. Pat. No. 6,953,849 disclose a process involving dewaxing of rice bran and hexane extraction and includes HPLC with a silicic acid column.
- Colarow U.S. Pat. No. 5,284,941 discloses a method involving solvent boric acid gel separation. Buchholz et al U.S. Pat.
- No. 5,440,028 discloses a method isolation through membrane separation, with pH adjustment.
- Bergqvist et al 1995 report after their work on oat kernels that galactolipids may be commercially extracted from a range of biological materials using a solid phase extraction using the known differences in solubility in acetone between phospholipids and glycolipids. They started with a hot ethanol extraction then used hexane then hexane/acetone then acetone.
- a number of prior art publications disclose the use of enzymes to liberate lipids and arrive at concentrated and purified lipid and fatty acid containing compositions from fish oil and other starting materials.
- the inventors appreciate that various lipases and phospholipases are capable of dis-assembling lipids.
- a variety of solvent-based extraction systems and crystallisation techniques have been disclosed that favour extraction of lipids of a particular class or fatty acids of a particular chain length or degree of unsaturation.
- These enzymes which may include lipases and proteases are known to act preferentially on different substrates.
- lipases for example, enzymes are expected to some degree to be specific for lipid class, fatty acid, and the position of the fatty acid within the lipid class.
- the activity and preference of enzymes can be altered by altering environmental conditions such as temperature, and via the addition of cofactors and techniques such as immobilization.
- a common analytical technique used to estimate the localisation of fatty acids at different positions in the lipid structure is to expose a fatty acid class to a lipase capable of selectively hydrolysing fatty acids located in a particular position. It follows that the common general knowledge of those skilled in the art includes the recognition that both the proportion of lipids and the localisation of target fatty acids as well as co-localisation or lack thereof of undesired acids within lipid reservoirs of a biological material constitute critical aspects in a purification process at an analytical scale. To our knowledge however no previous authors have disclosed methods of producing therapeutic or prophylactic compositions via the selective enzymatic hydrolysis of algal polar lipids at least not from polar lipids produced in largely heterotrophic cultures.
- Winget teaches use of topically applied MGDG-EPA compositions in the prevention and treatment of inflammation, but does not disclose application of lipase-type or indeed any enzymes.
- Bruno et al disclose that the galactolipid classes MGDG, DGDG and SQDG obtained from thermophilic blue-green algae have in-vivo anti-inflammatory activities in a croton-oil induced mouse ear inflammatory response.
- any of the n-3 HUFAs were present.
- the present invention provides novel methods for obtaining EPA-rich compositions that provide the public with a useful choice.
- the present invention provides compositions including EPA-rich galactolipids and highly purified EPA-rich fatty acid compositions that provide the public with a useful amount of therapeutic, prophylactic, or dietary EPA, or at least provides the public with a useful choice.
- the invention provides a process for obtaining an eicosapentaenoic acid (EPA)-rich composition for therapeutic or prophylactic use, wherein the process employs a culture of micro-organisms of a type selected for a capability of largely heterotrophic growth, and a capability of production of EPA, and a capability of photosynthetic lipid production; the process including a culture phase in which cells are grown under conditions in which organic carbon is used as an energy source; the conditions including use of controlled illumination at a level corresponding to an average photosynthetically active irradiance inside the culture of less than 40 ⁇ mol photons m ⁇ 2 s ⁇ 1 and including imposition of limitation of nutrients selected from a range including phosphorus and silicon; said procedures being undertaken in order to maximise the amount of recoverable polar lipids including at least one EPA side chain, and a harvesting process that creates a composition rich in EPA.
- EPA eicosapentaenoic acid
- the invention provides a process as previously described in this section wherein the culture of micro-organisms comprises identified microalgae, funghi or bacteria.
- micro-organisms are comprised of the marine single-celled diatom known as Nitzschia laevis , University of Texas microalgal collection UTEX 2047.
- the micro-organisms comprises a strain of micro-organism selected, when under culture conditions, for an improved yield of recoverable polar lipids having molecules which include at least one side chain bearing EPA.
- the micro-organisms accumulate galactolipids rich in EPA concentrated at the Sn1 position within the galactolipid or like classes.
- micro-organisms are capable of accumulating commercially useful quantities of polar lipids, including galactolipids rich in EPA at the same time as exhibiting high EPA productivity in general, including the EPA found in triglycerides.
- the EPA-rich lipid classes contained in the total lipid fraction include without limitation one or more of the following: MGDG; MGMG; DGDG; DGMG; non-galactosyl polar lipids including PC and PG; neutral lipids including monacylglycerol, diacylglycerol, triacylglycerol.
- the culture is capable under managed conditions of producing a proportion of its total dry weight as fatty acids; the proportion lying in the range of between 5 and 80%.
- the culture is capable under managed conditions of producing a proportion of its total fatty acids as EPA; the proportion (by dry weights) lying in the range of between 1 and 80%.
- the culture is capable under controlled conditions of producing 25% to 60% of total fatty acids as fatty acids contained in polar lipids; more preferably the proportion is more than 30%, more preferably over 40% and even more preferably over 50%.
- the organic carbon component is fed incrementally over time, according to the future predicted growth of the culture in a period of 4 to 24 hours.
- the culture is capable under controlled conditions of producing 5 to 40% of total fatty acids as fatty acids contained in galactolipids; more preferably the proportion is more than 10%, more preferably over 20% and even more preferably over 30%.
- the culture is capable under controlled conditions of producing 40 to 70% of EPA as EPA contained in polar lipids (as distinct from neutral lipids; more preferably the proportion is more than 50%, more preferably over 60%.
- the invention provides an EPA-rich composition derived from a culture as previously described in this section, wherein the EPA-rich compositions are obtained by a harvesting process including the steps of:
- the invention provides an EPA-rich composition derived from a process as previously described in this section, wherein the method includes a procedure in which EPA is hydrolysed from a polar EPA-rich lipid class obtained from the polar EPA-rich lipid fraction using the steps of:
- the enzymic process uses at least one enzyme selected from the range of lipases, phospholipases and galactolipases.
- the enzyme is affixed to a surface.
- the addition of materials including (without limitation), an alcohol, hexane or calcium chloride may assist enzymic release by removing products of the enzyme-catalysed reactions from proximity of the enzyme.
- physico-chemical fractionation of the polar lipid fraction may precede the application of one or more enzymes.
- galactolipids rich in EPA will be contained in preparations of the biomass of organisms.
- galactolipids rich in EPA will be contained in the total lipid fraction extracted from the biomass of organisms from which fraction they may be subsequently separated from the total lipid fraction.
- the invention provides a first method for isolating useful products comprising the steps of: (a) taking a preparation of the biomass of the cultured organisms,
- lipid classes contained in the total lipid fraction including but not limited to one or more of the following classes: MGDG; MGMG; DGDG; DGMG; SQDG; non-galactosyl polar lipids including PI, PE, LPC, PC and PG; neutral lipids including monacylglycerol, diacylglycerol, triacylglycerol;
- the EPA is concentrated in galactolipid classes which are the preferred substrate of and/or able to be made accessible to an enzyme capable of hydrolyzing one or more acyl bonds and thereby liberating the EPA contained within the galactolipid classes.
- EPA is concentrated within the diacyl galactolipid classes.
- EPA is concentrated within the monogalactosyldiacylgalactolipid (MGDG) class.
- MGDG monogalactosyldiacylgalactolipid
- the EPA-rich composition is processed after separation in order to further purify the composition, using known techniques though usefully not having to contend with DHA.
- the undesired molecules are not cleaved from the polar lipid, so that (for example) the EPA composition is substantially free of DHA which remains acylated to the glycerol backbone.
- Preferred known techniques for separation include low temperature crystallisations, purification processes taking advantage of differential solubility of fatty acid esters or salts in various solvents including ionic solvents, precipitation using metal salts, the use of selectively permeable membranes, column chromatography of fatty acids or their esters, supercritical fluid chromatography, urea addition crystallisation, fractional distillation, preparative HPLC, iodolactonization, and selective re-esterification by enzymes.
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises 50-60% EPA, less than 5.5% arachidonic acid and substantially no DHA.
- the composition comprises 50-60% EPA, less than 4.5% arachidonic acid and substantially no DHA.
- the composition comprises 50-60% EPA, less than 3.5% arachidonic acid and substantially no DHA.
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises 50-60% EPA, less than 5.5% arachidonic acid and less than about 2% DHA.
- the composition comprises 50-60% EPA, less than 4.5% arachidonic acid and less than about 2% DHA. More preferably the composition comprises 50-60% EPA, less than 3.5% arachidonic acid and less than about 2% DHA. Even more preferably the composition comprises 50-60% EPA, less than 2.5% arachidonic acid and less than about 2% DHA
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises 60-70% EPA, less than 4.5% arachidonic acid and substantially no DHA.
- the composition comprises 60-70% EPA, less than 3.5% arachidonic acid and substantially no DHA.
- the composition comprises 60-70% EPA, less than 2.5% arachidonic acid and substantially no DHA.
- the composition comprises 50-60% EPA, less than 1.5% arachidonic acid and substantially no DHA
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises 60-70% EPA, less than 4.5% arachidonic acid and less than about 1.5% DHA.
- the composition comprises 60-70% EPA, less than 3.5% arachidonic acid and less than about 1.5% DHA. More preferably the composition comprises 60-70% EPA, less than 2.5% arachidonic acid and less than about 1.5% DHA. Even more preferably the composition comprises 50-60% EPA, less than 1.5% arachidonic acid and less than about 1.5% DHA.
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 95 and 99% EPA, less than 1% of arachidonic acid and less than about 0.5% of DHA.
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 95 and 99% EPA, less than 0.5% of arachidonic acid and less than about 0.5% of DHA.
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 95 and 99% EPA, less than 1% of arachidonic acid and less than about 0.1% of DHA.
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 95 and 99% EPA, less than 0.5% of arachidonic acid and less than about 0.1% of DHA.
- the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 99.6 and 99.9% EPA, less than 0.1% of arachidonic acid and less than 0.1% of DHA.
- the invention provides for use of a composition, as previously described in this section, in the manufacture of a medicament for treatment of a person affected by certain medical conditions or disorders including but not limited to those selected from diabetes (type I, and type II), glycaemic disorders diabetes-associated hypertension, cancer, osteoarthritis, autoimmune diseases, rheumatoid arthritis, inflammatory and auto-immune diseases other than arthritis, respiratory diseases, neurological disorders, neurodegenerative disorders (including Huntington's disease, Parkinson's disease, Alzheimer's disease, schizophrenia, major depression, unipolar depression, bipolar depression, obsessive compulsive disorder, borderline personality disorder, post natal depression, organic brain damage, and traumatic brain injury), renal and urinary tract disorders, cardiovascular disorders, cerebrovascular disorders, degenerative diseases of the eye, psychiatric disorders, reproductive disorders, visceral disorders, muscular disorders, metabolic disorders, prostatic hypertrophy and prostatitis, impotence and male infertility, mastalgia, male pattern baldness
- diabetes type I
- the invention provides a composition prepared as previously described in this section, wherein the composition is prepared in the form of a human dietary supplement for therapeutic or prophylactic use.
- the invention provides for use of a polar lipid, prepared as previously described in this section and including an effective amount of EPA in the manufacture of a medicament for use in treating the medical conditions or disorders as previously listed in this section.
- the polar lipid is formulated in order to provide a prophylactic, health or dietary daily health supplement including an amount in the range of from 0.1 to 50 grams of EPA.
- the amount is in the range of from 0.5 to 5 grams of EPA.
- the content of EPA in the relatively low purity composition is less than about 20% and the content of undesired molecules remains low even if the purity of the EPA is below 20%.
- the invention provides one or more relatively low purity EPA compositions which are nevertheless pharmaceutically effective owing to a substantially low level of undesired molecules in one or more lipid fractions.
- compositions having a high ratio of EPA to undesirable molecules which is currently restricted to highly purified EPA from fish oil, which is less accessible to the general population.
- the amount is formulated so as to be suitable for repeated ingestion as a prophylactic, health or dietary daily health supplement.
- the products when consumed are capable of promoting brain and mental health, cognition and behaviour.
- the products when consumed are capable of eliciting health promoting effects on any of the following non limiting list of body systems and tissues; auditory, appetite, arousal, balance, blood, bone, bowel, cardiovascular, digestive, endocrine, enteric, emotional, gastric, hair, hepatic, immune, lymphatic, kineaesthetic, marrow, memory, metabolic, musculoskeletal, neurotransmitter, nasopharyngeal, pancreatic, musculoskeletal, reproductive, respiratory, ocular, oesophagal, olfactory, palate, pulmonary, proprioceptive, renal, skin, sleep, stomach, sensorimotor, skin, urinogenital, wound healing.
- the prophylactic, health or dietary supplement is formulated as a solid substance compatible with direct ingestion by humans; the range of formulations including: a cake, a powder, granules, tablets, boluses, pills, capsules, lozenges or beads.
- the EPA is re-esterified or combined with polar lipids, or alternatively the EPA moiety is cleaved from the galactolipids and re-esterified into phospholipid fractions.
- More preferably intact or semi-intact polar lipids rich in EPA are capable of efficiently passing through endothelial and other peripheral cell membranes in mammals.
- polar lipid fatty acids are incorporated into cell membranes within the mammal.
- the EPA-rich extracted polar lipids are encapsulated such that n-3 HUFAs and other PUFAs are protected from light including ultraviolet light in order to assist long-term stability.
- the EPA-rich extracted polar lipids are encapsulated such that n-3 HUFAs and other PUFAs are protected from oxidative degradation.
- the EPA-rich extracted polar lipids (including galactolipids) will be released into the beverage from a temporary encapsulation shortly prior to consumption.
- a prophylactic, health or dietary supplement as previously described in this section wherein the health supplement is formulated as a substance selected from the range including: a solution, a suspension, a solid mass, a powder, granules, or the like; the substance including an effective amount of an extracted galactolipid class rich in EPA and, when in use, is compatible with incorporation into a manufactured foodstuff.
- the residual whole cell content is utilized as a product in particular suitable for use as a food for mammals including monogastric and ruminant animals and/or aquaculture species including finfish and crustaceans; there being valuable residual compounds e.g.—sulpho galactolipids, carotenoids, other pigments, amino acids, and other fatty acids capable of serving as foods.
- residual compounds e.g.—sulpho galactolipids, carotenoids, other pigments, amino acids, and other fatty acids capable of serving as foods.
- a method for extracting EPA from a biomass derived from procedures whereby certain biomasses produced via the use of in vitro techniques accumulate commercially useful quantities of galactolipids rich in EPA at the same time as exhibiting high EPA productivity when grown under conditions according to those previously described in this section, in relation to the first broad aspect, may commence by first removing undesired fatty acids or lipids with a relatively low EPA content, and later extracting the EPA.
- the present invention discloses a pharmaceutically pure composition of EPA, wherein the fatty acid composition of the composition preferably contains (a) about 80 to 100% EPA and (b) little or no other omega 3 or omega-6 fatty acids; such compounds being useful in the treatment of those medical conditions that respond to medication with EPA.
- the pharmaceutically pure composition of EPA is derived from galactolipids.
- compositions will contain EPA as at least 90% of total fatty acids in the composition and may be substantially pure EPA.
- the content of any one of the fatty acids selected from the non-limiting range of undesirable compounds including: DHA, AA, DPA, 18:4 n-3, 18:3 n-3, 18:2 n-6 is less than 2% of total fatty acids in the composition and more preferably approaches zero.
- these EPA compositions will contain very low or undetectable levels of undesirable molecules which are either structurally similar to, or biologically related to, or antagonistic to the desired effects of EPA when administered to a mammal.
- the present invention includes a novel culture-related aspect, wherein polar lipids rich in EPA are co-produced together with neutral lipids in cultures that are largely heterotrophic in that they are mixotrophic cultures producing using significantly lower photosynthetically active average irradiances inside the culture. These will optimally be subphotosynthetic (i.e., less than 10 ⁇ mol photons m ⁇ 2 s ⁇ 1 ) but may be as high as 40 ⁇ mol photons m ⁇ 2 s ⁇ 1 .
- compositions that facilitate subsequent purification of EPA-rich lipid classes, or may be directly incorporated into novel supplements rich in EPA.
- Galactolipids are incorporated as a major structural component of the membranes of chloroplasts.
- Other polar lipids including phosphatidyl choline (PC) are also involved in transfer of EPA to the chloroplasts.
- PC phosphatidyl choline
- the invention takes advantage of the non-linear relationship between provision of light and subsequent production of photosynthetic lipids.
- the inventors are aware that the neutral fraction of lipids will also contain significant amounts of EPA. This material is likely to be of commercial value in itself and fatty acid derivatives thereof could be purified by traditional or emerging methods mentioned below to give an EPA product. Thus the present invention should be seen as an adjunct to neutral lipid production rather than a replacement.
- Low light levels lie in a range wherein organic carbon consumption is reduced or the efficiency of its use is improved, yet without the technical complications of providing high levels of light either by use of outdoors culture with technical and environment-related complications or by the use of large amounts of artificial light with associated energy costs to be met.
- a further option is the induction of nutrient depletion in cultures in order to encourage particular types of polar lipid production.
- induced deficiencies include phosphate deficiency which are expected to shift lipid production from phospho- to galacto-lipids.
- Silicate depletion in diatoms is also contemplated by the invention as a means of causing photosynthetic lipids to accumulate.
- the invention makes use of the specific molecular structures of certain polar lipids in certain organisms under certain conditions in order to facilitate purification of EPA.
- the invention contemplates the use of particular enzymes having specific appetites such as stereospecificity in order to facilitate purification.
- the unique physiochemical properties of photosynthetic polar lipid classes produced including the polar nature of the galactolipid molecules, provides useful methods of administering EPA.
- formulations including the EPA and galactolipid-rich compositions and applications for the formulations.
- An “ideal enzyme” for use in the invention would be able to excise side chains from the glycerol backbone of any polar lipid class if the side chain comprises a EPA. Whilst several lipases have been isolated that show selectivity for chain length we know of no cases where absolute specificity based on chain length has been demonstrated. None of these enzymes is currently available for industrial processes.
- lipases have the restricted ability of being able to act at the Sn1 position only, which suggests the production and isolation of polar lipids having a desired n-3 HUFA predominantly at the Sn1 position would be a route to enrichment. Selection of a particular enzyme for use in a commercial process is also cost-dependent and it may be necessary to rely on those lipase-type enzymes already produced in bulk for use in the dairy industry or the baking industry, which includes 1,3 specific lipase-type enzymes made from fungi—for instance the lipase/phospholipase derived from Aspergillus spp, “Bakezyme PH 800 BG” (DSM Food Specialities), or the lipase derived from Rhizopus oryzae , “Piccantase R8000”.
- the engineered enzyme “Lecitase Ultra” (Novozymes) has 1,3 specific lipase activity but at elevated temperatures demonstrates phospholipase A1 activity. Both activities are likely to be of use in the isolation of fatty acids from the Sn1 position of polar lipids.
- the current specification discloses a method to produce a relatively pure EPA composition by exposing a microalgal biomass rich in galactolipid EPA to an enzyme.
- any enzymes used will be adsorbed on to a surface or otherwise retained within the process, in order to conserve supplies, by the use of techniques for handling enzymes that are well known in the art. It is also likely that optimisation of working conditions for a selected enzyme will provide a significantly improved rate of attack and a more specific type of attack, as a result of exploitation of working conditions well known to those skilled in the art, such as concentration, pH, temperature, presence of salts, or the presence of competing compounds that inhibit undesired modes of action.
- the polar lipids can be either (A) isolated as particular types, or (B) used as one, collective group. For either A or B, they may then be (i) used directly, (ii) further processed into EPA-rich fatty acid compositions by cleaving the fatty acids from the lipid species. In the case of further processing, a polar lipid fraction or fractions may be hydrolysed with a specific phospholipase (or other lipase), the released EPA captured by a suitable acceptor molecule. Examples of acceptors include: glycerol and alcohols including ethanol, propanol, iso-propanol, or long-chain alcohols (which will yield waxes).
- the EPA may be transferred by a phospholipase, galactolipases, or other lipase on to a suitable carrier type molecule (such as phosphatidylcholine (PC)).
- a suitable carrier type molecule such as phosphatidylcholine (PC)
- PC phosphatidylcholine
- Typical applications include, for (i): production and use of ultra pure EPA and an active pharmaceutical ingredient (ii) production and use of non-pharmaceutical EPA-only therapeutic compositions (iii) production and use of EPA-rich polar lipid containing foods, functional foods and food supplements, and (iv) production and use of whole-cell products for foods or food supplements.
- Actively growing cells of the species N. laevis obtained as above are produced in 200 mL of media in stoppered 500 mL Erlenmeyer flasks. Multiple flasks are used to produce large volumes of material. An inoculum of 0.2 g L ⁇ 1 of exponential or early stationary phase cells is used. Flasks are incubated in temperature- and light-controlled growth chambers by placing them on orbital shakers at around 200 rpm to maintain the cells in suspension and aid in gas transfer between atmosphere and media. Temperature is maintained at 20° C.
- Light is provided at an average irradiance of photosynthetically active light in the culture of 40 ⁇ mol photons m ⁇ 2 s ⁇ 1 as measured by an Apogee quantum sensor digital pyranometer and calculated from conditions such as culture depth and cell density. Aliquots of culture are taken during growth to determine the dry weight of the culture at that time point. Cultures are fed a heat sterilised glucose stock solution (400 g L ⁇ 1 ) daily at a level that is projected to provide organic carbon requirements for the predicted biomass production over the subsequent 24 hours. Total glucose added to culture over the entire culture period amounted to 3 grams per litre.
- Biomass dry weight is measured, using the pre-weighed glass fibre filter method as follows.
- a 10 ml sample is removed from a larger representative sample taken whilst stirring to achieve a broadly homogenous dispersion of cells and cell aggregates; culture flasks are generally sterilised with a Teflon-coated magnetic stir bar in place to aid with this.
- the 10 ml sample is placed in a centrifuge tube and spun at 3000 rpm in a Heraeus Sepatech Megafuge 1.0 with swing-out rotor for 4 min and the liquid decanted leaving a cell pellet.
- the cell pellet is washed with phosphate-buffered saline and re-centrifuged.
- a Sartorius glass fibre filter is washed by passing 1 litre of deionised water through the filter then dried overnight in a vacuum oven at 30° C. prior to being weighed.
- the 10 ml sample is passed through the preweighed filter in a vacuum filter apparatus and is then placed in an oven at 60 deg C. for two hours prior to being reweighed.
- the difference in grams between the pre and post weights times 100 is taken as a measure of the dry weight per litre.
- Cells are harvested after 3 days of growth since at this point the culture(s) are still in exponential phase.
- Cellular extract containing the lipids can be obtained by Folch extraction following the method of Bligh and Dyer (1959). Cells from several flasks are combined to allow production of sufficient material for further use.
- Total fatty acid analyses of samples of cellular extract are obtained to identify the composition of the cultured material. Addition of an internal standard such as C23:0 to the reaction allows measurement of the total fatty acid content of the cells.
- the method of fatty acid production entails a basic transesterification with 0.5M methoxide in methanol followed by an acidic transesterification using dry HCl in methanol. Fatty acid methyl esters are recovered by extracting with hexane and drying with sodium sulphate before analysis using gas chromatography.
- the sample is run on a 30 m ⁇ 0.25 mm ID Famewax (crossbond polyethylene glycol) glass capillary column contained within a Shimadzu 2010 GC by autoinjection. By ramping the column temperature from 145 to 240° C. over the course of 50 minutes and then leaving the column at 240° C. for a further 10 minutes fatty acids is identified by co-chromatography with known standards supplied by Restek.
- Cellular extract is separated into polar and non-polar fractions using column chromatography. 0.5-2 g of the cellular extract is dissolved in a small volume of diethyl ether and loaded onto a column containing 40 g silica gel (with a mesh size of 230-350) in diethyl ether. 10 mL of diethyl ether and 80 mL of chloroform is used to elute non-polar material including the triglycerides. Further addition of 10 mL chloroform:methanol (1:1 v/v) and 80 mL methanol to the column elutes the polar material including galactolipids and phospholipids.
- the 1,3 specific lipase and phospholipase A1 “Lecitase Ultra®” from Novozymes is used to cleave the fatty acids from the Sn-1 position of MGDG isolated in the manner described above.
- 5 mg of MGDG is dissolved in 3 mL of methanol whilst 12 u of enzyme is dissolved in 3 mL 10 mM citric acid buffer at pH6.0. These are incubated together at 60° C. for 5-15 minutes and after incubation the reaction could is washed 3 times with 2 mL hexane to collect the free fatty acids produced.
- the hexane washes are collected in a fresh tube with 3 mL methanol and the mixture incubated at 50° C. for 2 hours to produce Fatty acid methyl esters. At the end of this period the hexane layer is removed and concentrated before being analysed on a GC.
- Table 1 shows in the left hand column the total fatty acid profile of MGDG isolated using the method described in the present example and in the right hand column the total fatty acid profile of fatty acids recovered from the hydrosylate.
- the method allows significant increases in the polar lipid production of the organism to take place over that which is measured in a purely heterotrophic culture (where 75% or more of the fatty acids are seen in the non-polar fraction).
- Application of the enzyme in the present example provides for enrichment of EPA and the exclusion of DHA from the fatty acids recovered from the hydrosylate as compared to the total fatty acids of the MGDG fraction.
- Flask cultures of N. laevis is produced according to the method of example one except that: 5-10 grams of glucose is added over the course of the culture run. Light is provided at an average irradiance of photosynthetically active light in the culture of 10 ⁇ mol photons m ⁇ 2 Harvesting, separation and analysis techniques are all according to the method of example one.
- a biomass dry weight reaches between 3 and 5 grams per litre in flask culture.
- Fatty acids form at least 10% of the dry material grown in the culture.
- EPA reaches at least 20% of total fatty acids.
- fatty acids were recovered in the non-polar fraction than the polar fraction but the amounts of fatty acids in the polar fraction are still substantially higher (50-200% greater) than those from a similar heterotrophic culture grown in the absence of light.
- EPA is found as a higher proportion of fatty acids in the polar fraction both in comparison to the non-polar fraction and in comparison to a polar fraction from a totally heterotrophic culture.
- EXAMPLE 3 “PERFUSION CULTURE” MODE OF CULTURING N. LAEVIS INCLUDING VARIATION OF NUTRIENT(S) AND/OR EXPOSURE TO SUB-PHOTOSYNTHETIC LIGHT INTENSITIES
- the vessel is internally lined with “Teflon®” and comprise a stirred, jacketed tank.
- the jacket is provided with hot or cold water as required in order to maintain an internal temperature of 20° C., as sensed by internal probes and controlled with a SCADA device controlling water valves.
- a mechanical seal admits an impeller shaft of 19 mm diameter, having a 6-blade Rushton impeller at one end, placed near an air sparger, and a marine impellor 250 mm from the end.
- a 0.25 kW 3-phase 6-pole motor drives the shaft at between 100 and 900 revolutions per minute.
- Motor speed is controlled with a variable speed drive capable of receiving an analogue signal from the supervisory control device.
- Pressurised air (1.5 bar) is injected through a sterilizing filter at a rate of between 2 to 10 litres per minute to the air sparger.
- the air flow is measured with a Dwyer flow meter model TF 2110 and the flow rate is controlled either manually through the use of a regulator or by a “Festo” proportional solenoid controller.
- gas outflow from the vessel is measured and regulated.
- Dissolved oxygen is measured by an oxygen sensor (Broadley-James Corporation, Irvine, Calif.) and the dissolved oxygen maintained at around 50% of saturation via supervisory control feedback loops controlling motor speed and, to a lesser degree, air flow.
- an immersed pH sensor Broadley-James Corporation, Irvine, Calif.
- a peristaltic pump for the addition of either alkali (as NaOH or KOH) or for the addition of acid (as HCl or Acetic acid) as required.
- Concentrations of nutrients including feed stocks that are sources of nitrogen, silicate, phosphorus, glucose, and organic carbon (e.g. glucose) are separately controlled by feeding sterile stock solutions of desired concentrations aseptically through corresponding peristaltic pumps.
- Sterile basal media is also supplied to the vessel through an aseptic pump.
- Culture volume is monitored using a Kubler level sensor and input of basal media or nutrient controlled by the SCADA device.
- Precautions related to sterility include operations being conducted in a production environment equipped with an air lock and supplied with filtered sterile air to create a positive pressure directing air away from culture vessels.
- the vessel is inoculated by introducing a freshly growing culture through a previously steam-sterilised manifold in order to achieve an initial concentration of from 0.1 to 1 g per litre of cells in the vessel.
- Motive pressure for the transfer is provided through displacement of the inoculating culture with sterile air.
- An optical density probe is also be immersed in the tank in order to indicate the amount of biomass present in a culture.
- the culture vessel is also be provided with one or more settling devices, which are external separating funnels into which cells within their medium may be pumped from time to time aseptically via the operation of a peristaltic pump. These devices function by allowing cell-containing media to be pumped into them to settle whilst at the same time permitting spent media, substantially free of cells to be removed in a sterile manner. The suspension rich in settled cells located at the bottom of the settling device is then pumped back into the culture vessel via the alternate operation of a second peristaltic pump.
- the reduction in total medium volume in the main culture vessel is made up with fresh media, so that excretory products can be taken out of the vessel and fresh nutrients added.
- the dimensions and volume of the settlers is optimised via deign methodologies well know to those skilled in the art to allow the total volume of the tank to be changed over a 24 hour period via the operation of one or more settlers whilst minimizing the residence time of cells in the settlers.
- Means for providing light to the micro-algal cells during culture include one or more of: use of an optically translucent or transparent section of the vessel with a surface illuminated externally, insertion of light guides through ports from the exterior into the culture, insertion of a sterilisable light emitting device (e.g. fibre optics, conventional bulbs or LEDs).
- Cells can be pumped of cells, using a peristaltic pump or the like, from within the culture medium outside and along tubes that are exposed to a light source. For instance a transparent flat panel vessel illuminated using artificial light is used. Relative amounts of time spent in the main vessel and the flat panel system determine the amount of light that the cells are exposed to.
- a low continuous level of exposure using, for example, light guides may be preferable to a high intermittent exposure in an external system.
- cells may be pulsed intermittently with high intensity light so as to achieve an average low intensity over the period of the culture.
- a preferred method for the production of Nitzschia laevis is the use of low intensity light providing a photosynthetically active average irradiance in the culture of less than 40 micromol photons per square meter per second,
- Nitzschia laevis An even more preferred method for the production of Nitzschia laevis is the use of low intensity light providing a photosynthetically active average irradiance in the culture of 1-10 micromol photons per square meter per second.
- the biomass is harvested as a batch after 5 to 9 days of growth and subjected to extraction with supercritical dimethyl ether (DME).
- DME supercritical dimethyl ether
- cells are first collected and heat killed at 70° C. for 15 minutes to denature endogenous enzymes.
- the cells are then spray dried to form a powder with less than 10% water content.
- Supercritical DME at 60° C. and 40 bar pressure
- Removal of the DME leaves a tar-like extract which contains the lipids as well as pigments and other cellular material. Around 50% of the dry weight is extracted using this method.
- Subsequent extraction of the complex lipid mixture with supercritical CO 2 may also be performed which has the effect of separating polar material (left as a residue in the CO 2 process) from non-polar (dissolved in the supercritical CO 2 ).
- a variety of other methods of isolating lipids are known to those skilled in the art.
- the total extract or isolated neutral or polar lipid fraction may be used in its own right or fatty acids recovered by direct saponification via methods well known to those skilled in the art. Further chromatographic processes as described in example one can then be utilised as necessary to further purify lipid classes or fatty acid fractions.
- Isolation of fatty acids from the Sn1 position of specific polar lipid classes is carried out by dissolving the lipid material in methanol and passing it through a column of immobilised Lecitase Ultra. Addition of hexane to the material flowing from the column isolates the fatty acids hydrolysed by the enzyme. These are then purified further as desired. Productivity of this system is between 5 and 50 mg EPA per litre per hour.
- the invention may rely on use of higher plants cells normal or transgenic organisms including but not limited to algae, fungi, and bacteria.
- a possible improvement option involves growing micro-organisms for a period under conditions in which the media is depleted of either silicate or phosphate or both in order to cause the organisms to produce more polar galactolipids in their lipid membranes, which are subsequently extracted.
- the nutrient limitation is imposed on the culture over the last phase of growth prior to harvesting.
- the culture may be maintained under temperatures below the previously stated 20 degrees Celsius, and above the freezing point of the culture medium.
- the cooling is applied to the culture over the last phase before harvesting.
- a therapeutic composition containing less than (for example) 20% EPA yet having substantial absence of other potentially antagonistic molecules such as DHA AA etc. should not be just as effective as a 100% pure EPA oil (not counting esters).
- the drive to get substantially 100% purity could be re-expressed as a desire to have substantially none of the “undesired molecules” such as DHA. Therefore, acceptance of (for example a 10 to 95% pure EPA) becomes a matter of satisfying the relevant regulatory authorities.
- the role of the inventors becomes a matter of exclusion of certain impurities.
- a therapeutic composition that delivers EPA in a relatively water-soluble form (or a stable emulsion in water) has substantial formulation advantages.
- a basis for making foods, food supplements, nutraceuticals, or therapeutic preparations that rely on the EPA held in polar lipid molecules is that on oral administration of polar lipids rich in EPA to a mammal leads to a significant proportion of their fatty acids being absorbed into the blood stream via enteric lymph vessels thereby bypassing first-pass liver metabolism and thus providing greater bioavailability of the EPA.
- polar nature of the lipids renders this form of EPA, which does not behave as in the same manner as a fatty acid or ester or as a neutral lipid, easier to formulate and to administer.
- Polar lipids derived from microorganisms would not carry a fishy flavour of the type usually present in fish oil extracts.
- Galactolipids and certain phospholipids according to the invention are recognised to be of particular utility due to a combination of their high EPA content, low content of other potentially antagonistic molecules and undesirable fatty acids.
- An EPA-rich galactolipid that has been manufactured from a culture according to the invention may be prepared for storage, shipping and sale as a substance having one of a variety of physical forms such as a solution a suspension (feasible with water), or in a cake, a powder, granules, tablets, boluses, pills, capsules, or beads.
- a powder the galactolipid may be bound to inert particles (such as of starch) or encapsulated by means well known to those skilled in the relevant arts.
- Means to restrict oxidation of the EPA may be included in packaging such as by sparging with nitrogen.
- microencapsulation for foods may be assisted with phospholipids (such as crude or purified lecithins) so that the EPA is protected
- the composition includes an effective amount of an extracted galactolipid rich in EPA and is suitable for oral ingestion either directly or after a technical process of food preparation.
- a beverage may be a most preferred route since beverages are consumed by most people. Due to the vulnerability of n-3 HUFA and other PUFAs to oxidative degradation it may be preferable to encapsulate the galactolipid such that it is protected from light especially sunlight, and released into the beverage shortly prior to consumption. Alternatively it may be desirable to sparge the liquid containing the galactolipid with an inert gas to prevent exposure of the EPA to oxygen. Carbon dioxide in carbonated beverages may assist dispersion of the galactolipid. Micelles may also hold dissolved carbon dioxide in the case of carbonated beverages and the composition of the micelle may be altered in order to enhance this property.
- glycerol in certain cases it may be preferable to add glycerol to these beverage preparations to optimise the dispersion of the galactolipids and also to provide a more desirable mouth feel for the beverage.
- a water-free concentrate which would be made up by the user with water at or near the time of consumption may be distributed. Steps to minimise oxidation of the n-3 HUFA within the concentrate would have to be taken including the use of light-tight packaging, and microencapsulation of the galactolipid.
- Milk is a fairly universally consumed beverage and there are many processed variants of “plain” milk on sale.
- EPA-supplemented milk is made by adding an EPA-rich galactolipid during processing. The usual practice is to homogenize and pasteurize milk in a single process. In order to minimise exposure of the EPA to heat, the EPA-rich galactolipid is preferably added during or after cooling of the milk.
- Spreads may have EPA-rich materials included. Such spreads include the protein-rich type such as yeast extracts, or fat-based spreads such as aioli, butter and margarine, in which water is the dispersed phase. In manufacture, the galactolipid is added either to the fatty component or to the water component and the polar nature of the molecule assists in solubility. Spreads also include jams and jellies. The EPA-rich galactolipid may be added to a jam in the form of an emulsion. More solid preparations include sweets and chocolates. The EPA-rich galactolipid may be added as a fat-soluble component during manufacture preferably after heating in order that the galactolipid is not exposed to heat.
- EPA-rich galactolipids may be added to ice cream, for example, along with selected long-chain fatty acid molecules and optionally a phosphatidyl choline (PC) molecule as a carrier.
- PC phosphatidyl choline
- An alternative is to transfer the EPA on to a selected PC molecule using a selective galactolipase/phospholipase.
- Long-chain lyso-phosphatidylcholine is a possible suitable receptor having advantages in processing and in products.
- the product formed may be insoluble within the enzyme system which tends to drive the equation towards its formation.
- EPA-rich phosphatidylcholine isolated from the culture may be used.
- Whole-cell preparations which may be intact, partially hydrolysed, or lysed, may be incorporated directly into spread-type foods, baking products, processed meats or other food supplements either (i) as is, or (ii) after homogenisation (by shear or pressure) or (iii) controlled enzymatic or chemical hydrolysis to aid proteolysis.
- the whole cell will offer useful protein and generally high HUFA levels, even if some stripping of 1000 EPA has taken place.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Diabetes (AREA)
- Nutrition Science (AREA)
- Endocrinology (AREA)
- Dermatology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Cardiology (AREA)
Abstract
Eicosapentaenoic acid (EPA) compositions and EPA-rich polar lipids for prophylactic or therapeutic applications are described. Production from certain cultured micro-organisms (like Nitzschia laevis) promotes synthesis of EPA, including polar lipids including EPA. The EPA-rich polar lipids themselves may be used as polar compounds. EPA can be selectively hydrolyzed from particular positions in isolated polar lipids by lipase activity, then optionally further purified. The process bypasses reliance on diminishing fish stocks and on physico-chemical processes that may not adequately separate desirable n-3 HUFAs from unwanted products like DHA also found in fish oil and cultured organisms.
Description
- The present application is a continuation of U.S. patent application Ser. No. 14/531,783, filed Nov. 3, 2014, which is a continuation of U.S. patent application Ser. No. 12/307,532, filed Nov. 5, 2009, which is a US National Phase of International Patent Application No. PCT/NZ2007/000172, filed Jul. 5, 2007, which claims the benefit of New Zealand Patent Application No. 548339, filed Jul. 5, 2006, all of which are incorporated by reference herein in their entireties.
- This invention relates to lipid compositions synthesized by single-cell organisms, to the manufacture and some applications of omega-3 highly unsaturated fatty acids and to pharmaceutical substances, and in particular to novel therapeutic, dietary and health-food compositions.
- Alpha linolenic acid is an omega-3 fatty acid with eighteen carbon atoms and three double bonds.
- Arachidonic acid is an omega-6 fatty acid with twenty carbon atoms and four double bonds.
- Diacyl galactolipids are galactolipids with both Sn1 and Sn2 occupied by fatty acid molecules.
- Digalactosyl galactolipids are those where two galactose molecules are attached.
- Docosopentaenoic acid is an omega-3 fatty acid with twenty-two carbon atoms and five double bonds.
- Docosahexanoic acid is an omega-3 fatty acid with twenty-two carbon atoms and six double bonds.
- Eicosapentaenoic acid is an omega-3 fatty acid with twenty carbon atoms and five double bonds.
- Effective amount means: an amount sufficient to cause a desired result when administered.
- EPA-only means: a composition containing in its highly unsaturated omega-3 fatty acid component, substantially only EPA.
- Esterification of n-3 HUFAs is understood, so that a term such as “99% pure EPA” is understood to take no account of the ethyl or other moiety used to form an ester with the free fatty acid.
- Nutraceutical is any substance that is a food or a part of a food and provides medical or health benefits, including the prevention and treatment of disease.
- Purity: 1. Chemical purity: The EPA composition is substantially free of all molecules other than EPA.
- 2. Functional purity: The EPA composition is substantially free of those materials herein defined as “undesired”. An EPA composition according to this definition of purity may include only 1 to 10% EPA (although more preferably 60-90% EPA; the remainder may include galactolipids or components thereof, or added pharmaceutically acceptable excipients, anti-oxidants, adsorbents, flavours, and the like.
- 3. Pharmaceutical purity: refers to EPA-rich compositions comprising around 90% of EPA or more by weight and simultaneously a ratio of EPA to any individual undesired molecule of at least around 90 to 1. Undesired molecules or materials are defined as those that may diminish the desired beneficial health effect of EPA-rich compositions when co-consumed. These include molecules that may diminish the desired effect through actions which may be antagonistic, competitive, block, reverse, mediate, synergise or otherwise alter the desired beneficial health effect of EPA when consumed in an effective (or clinically relevant) amount. For the purposes of the present specification undesired molecules are to include structurally or functionally similar molecules including fatty acids, docosohexaerjoic acid (DHA), arachidonic acid (AA), 18:4 n-3, 18:3 n-3, 18:2 n-6 and other omega-3 and 6 fatty acids in general. AA is a precursor of EPA.
- EPA-rich means that the composition includes more than 1% of EPA as dry weight.
- EPA productivity means the amount of EPA which can be produced per unit cost. A proxy measure for productivity in largely heterotrophic cultures of microalgae culture is often taken as the combined effect of yield, growth rate, cell density, nutrient utilisation efficiency, and dilution rate.
- Fatty acid compositions include—ethyl esters, salts, free fatty acids, methyl esters, and other alcohol esters of fatty acids, and combinatorial lipids.
- Galactolipids are comprised of a glycerol backbone with at least two separate molecules attached; at least one of which is a fatty acid and the other is. either one or two molecules of galactose (in mono or disaccharide form) bonded by, in the case of the fatty acid or fatty acids, an ester link or rarely an ether link and in the case of the galactose or galactoses an ether link in all cases. At least one galactose is present and normally attached to the Sn3 position of glycerol. The galactose is always in pyranose form. A second and rarely a third galactose molecule is attached by beta-d-3-pyranosyl bonds to the preceding galactose molecule. Positions are labeled according to a convention based on an original stereospecific structure (see table 1 showing the 1.3 isomer of the DGMG galactolipid class containing an EPA molecule′ acylated in the Sn1. Normally when only one fatty acid molecule is attached it occupies the Sn1 position. However, occasionally one fatty acid may be attached in the Sn2 position while the Sn1 position remains unoccupied. This may be referred to as a lyso derivative.
- The 1,3 Isomer of DGMG, with an EPA Molecule Acylated to the Sn1 Position.
- Gamma linolenic acid is an omega-6 fatty acid with eighteen carbon atoms and four double bonds.
- Heterotrophic culture means a culture of organisms for which the sole energy source is derived from supplied nutrients (the major nutrient group for energy metabolism) which is usually a form or forms of organic carbon (e.g. glucose, acetate).
- Largely (or partially) heterotrophic culture means a mixotrophic culture of organisms for which the major energy source is derived from supplied nutrients (the major nutrient group for energy metabolism) which is usually a form or forms of organic carbon (e.g. glucose, acetate) and the minor energy source for energy metabolism is light.
- Linoleic acid is an omega-6 fatty acid with eighteen carbon atoms and two double bonds.
- Monoacyl galactolipids are galactolipids with only one position occupied by a fatty acid. In a monoacyl galactolipid the fatty acid molecule is attached either at position Sn1 or Sn2.
- Monogalactosyl galactolipids are those with only one galactose molecule attached.
- Neutral lipids are those lipids contained in an organism which can be isolated through the use of non-polar solvents and include mono-di- and triacylglycerols
- Photosynthetic lipids are those polar lipids whose production can be significantly altered via the addition of light to a largely heterotrphic culture of microorganisms. These may occur within the chloroplast as is predominantly the case with galactolipids or may also be associated with other cell organelles as is predominantly the case with phospholipids.
- Polar lipids are those lipids contained in an organism which can be isolated through the use of polar solvents and include phospholipids and galactolipids
- Photosynthetically active average irradiance inside a culture is the amount of electromagnetic radiation between wavelengths 400 nm and 700 nm incident on a culture averaged over all positions in the culture vessel and over time.
- Stearidonic acid is an omega-3 fatty acid with eighteen carbon atoms and four double bonds.
- Sub-photosynthetic exposure means an exposure to light whereby the combination of intensity and exposure time is equal or less than around the equivalent of a continuous illumination of 1 to about 10 micromol photons per square meter per second.
- Omega-3 fatty acid is a fatty acid with the first double bond three carbon atoms from the n-methyl end of the molecule.
- Omega-6 fatty acid is a fatty acid with the first double bond six carbon atoms from the n-methyl end of the molecule.
- 18:2 n-6 or LA: linoleic acid
18:3 n-3 or ALA: alpha linolenic acid
18:4 n-3 or SDA: stearidonic acid
18:4 n-6 or GLA: gamma linolenic acid
20:4 n-6 or AA: arachidonic acid
20:5 n-3 or EPA: eicosapentaenoic acid
22:5 n-3 or DPA: docosopentaenoic acid
22:6 n-3 or DHA: docosohexaenoic acid
DGDG digalactosyldiacylgalactolipid
DGMG: digalactosylmonoacylgalactolipid - n-3: omega-3
n-6: omega-6
n-3 HUFA: omega-3 highly unsaturated fatty acid
MGMG: monogalatosylmonoacylgalactolipid
MGDG monogalactosyldiacylgalactolipid
N. laevis: Nitzschia laevis (UTEX 2047)
N. alba: Nitzschia alba - SQDG: sulfoquinovosyl diacylglycerol
- It was discovered in the late 1920s that certain “essential” dietary fatty acids must be present in effective quantities for normal growth and health in rats to ensue (Burr & Burr J. Biol. Chem., 82: 345-367 1929). Epidemiological data collected from human populations beginning in the 1940s then suggested relatively high dietary intakes of n-3 HUFA may be protective against the development of a number of medical conditions and that low n-3 intake may increase risk (Sinclair. Lancet 1:381-3 1956; Bang et al., Lancet 1:1143-5.1971; Hirai et al., Lancet 2:1132-3. 1980; Kromhout et al Am J. Clin. Nutr., 85:1142-1147).
- In recent decades supplementation studies incorporating individual omega-3 highly unsaturated fatty acids (n-3 HUFA) in the diet of humans have demonstrated beneficial health effects of individual dietary n-3 HUFA. In particular, human dietary supplementation studies incorporating relatively pure forms of the n-3 HUFA eicosapentaenoic acid (EPA) have suggested this nutrient may promote health and ameliorate or even reverse the effects of a range of common diseases, including but not limited to certain forms of cardiovascular disease and depression (Yokoyama et al., Lancet 369:1062-1063. 2007; Peet & Horrobin Arch. Gen. Psych. 59(10) 913-9 2002).
- The therapeutic effect of dietary supplementation with concentrated forms of EPA are dependent to some extent on purity. High purity dose forms have an advantage in terms of increased bioavailability. Furthermore the desired effects of EPA are limited or even reversed by the co-consumption of undesired molecules; (as herein defined) in particular docosohexaenoic acid (DHA); also AA and other omega-3 and 6 fatty acids in general. Therefore to enable effective pharmaceutical or therapeutic use of EPA, high purity dose forms, free of the undesired molecules, are required.
- Should the demand for high purity EPA increase, which seems likely, large numbers of clinically or subclinically diseased persons may come to depend on continuity of supply long term to maintain quality of life. To date, however, commercial manufacturers have not been capable of economically producing EPA-only compositions with relatively high EPA purities which are at the same time devoid of undesired molecules.
- Reasons include: (1) The raw material for commercial production is exclusively limited to particular fish oils containing high levels of undesired molecules. (2) The undesired molecules contained in fish oil are structurally or physico-chemically similar to EPA and cannot be easily removed during purification (3) The cost of further purification rises in a non-linear fashion with increasing purity.
- Consequently even at EPA purities up to around the high 90th percentile up to 1% or more of these undesired molecules may remain.
- Purification processes are also rendered less efficient by the relatively complex mixture of fatty acids, and a high degree of natural variability contained in fish oil.
- The practical effect of the abovementioned factors is that commercial products currently available that contain high purity EPA may also contain unacceptably high concentrations of the abovementioned undesired molecules for therapeutic use. Furthermore the high cost of purifying fish oil to an extent where only small amounts of undesired molecules remain constrain the use of these ultrapure compositions.
- Up to 15 kgs of high EPA fish oil are required to produce 1 kg of highly purified EPA in current purification processes. Because the efficiency of such manufacturers is sensitive to the initial concentration of EPA these are based on fish caught with a high percentage of EPA in their lipids. The fish oil must also be carefully handled and stored during processing to protect against damage which can result in the formation of unacceptable molecular species such as trans EPA which is an unacceptable contaminant in therapeutic formulations and virtually impossible to remove during purification. The complex structure of the fishing industry, the careful handling requirements and the dwindling and finite resource of high EPA fish species means that production of high purity EPA from sea fish is difficult to scale up in order to meet increasing demands and is likely to be unsustainable.
- Many publications have reported the potential of alternative sources of EPA-rich compositions or EPA produced from cultured microbes including (micro)algae, fungi, and bacteria. Some of these sources contain low levels of undesired fatty acids. Additionally, the generally less complex fatty acid composition of microbes as compared to fish oil may offer advantages in purification. Variation in fatty acid composition in cultured microorganisms is minimal as compared to fish oil conferring an additional advantage for purification. Production of EPA-rich compositions in biotechnological processes is likely to be rapidly scalable and provide EPA-rich compositions suitable for both nutritional and therapeutic use that are of consistent quality.
- The majority of the publications relating to production of EPA from microalgae concern the development of outdoor production systems. The advantage of these systems is the main source of energy for growth,—sunlight, is free. Outdoor production systems however suffer from several key defficiencies. Firstly contamination from competing microorganisms limits the applicability of open pond or raceway cultures to species which are able to withstand environmental conditions that limit the growth of other competing microorganisms. Secondly “photobioreactor” production systems designed to restrict contamination require very large surface to volume ratios to facilitate penetration of light into the culture createing a requirement for large upfront capital expenditure in the establishment of these systems and an ongoing technical challenge and cost with regard to maintaining sterility.
- A further weakness of largely photosynthetic cultures developed to date is that species have not yet been isolated that accumulate significant quantities of intracellular lipid in the form of triglycerides when produced photosynthetically. This limits EPA production to that accumulated in polar lipids, the upper limit of which appears to be under tight physiological regulation.
- Mixotrophic production systems have been proposed for production of EPA-rich microorganisms. These provide a proportion of the energy for growth in the form of organic carbon supplied to the culture medium. An advantage of mixotrophy includes higher productivities than are achievable with solely photosynthetic production and potentially also lower the overall requirement for light. A disadvantage of the addition of organic carbon sources to outdoor photobioreactor cultures however is the creation of an additional contamination risk by presenting a substrate for growth of non-photosynthetic contaminating organisms.
- A number of solely heterotrophic systems for producing EPA-rich microorganisms have been disclosed. These overcome many of the limitations of photosynthetic systems due to their ability to achieve growth of EPA rich species in the absence of light. By eliminating the requirement for light it is possible to significantly reduced the surface-to-volume ratio of reactors and consequently also reduce capital expenditure and sterilisation costs. An additional advantage of heterotrophic production systems is that culture parameters can be tightly controlled leading to production of a product of a consistent quality.
- The fatty acid composition of certain EPA-rich microalgae contain low proportions of fatty acids with structural similarity to EPA. Together with the generally less complex fatty acid composition of microalgae this may offer advantages in terms of purification over fish oil.
- In addition to achieving a favorable overall fatty acid composition in cultured EPA-rich microalgae the selective production of EPA in particular lipid classes is also possible.
- One particular strategy for enhancement of lipid and overall EPA production in EPA-rich microalgal species is the timed imposition of a nitrogen limitation in microbial culture medium in heterotrophic cultures of microalgae. When microorganisms are deprived of key nutrients required for synthesis of membranes, lipids may be accumulated in the form of triglyceride, a lipid class not utilized extensively in lipid membrane structure.
- EPA-rich triglycerides are of potential therapeutic value. EPA may be recovered from triglcerides and further purified via an array of conventional and emerging techniques. Processes designed to extract, concentrate or purify EPA-rich lipid or fatty acid compositions from triglycerides however may be disadvantaged by the presence of a relatively high level and wide range of undesirable fatty acid molecules, and a low level of stereospecificity in terms of the location of EPA within the triglycerides.
- Certain polar lipid classes produced in cultures of microalgae are relatively rich in EPA. At the same time some of these lipid classes may exhibit a high degree of stereospecificty in terms of the location of EPA within the class and its isomers. This concentration of EPA in a predictable manner in particular lipid classes provides an additional opportunity to sequester undesirable molecules in unused fractions during a purification process. In addition certain lipid classes produced by cultures of microalgae may also have therapeutic value in their own right.
- It may seem surprising then that little, if any, attention has been given to the possibility of inducing heterotrophic or largely heterotrophic cultures of microalgae to localise EPA in polar lipid reservoirs in such a way as to enhance the efficiency and applicability of extraction, concentration and purification processes and to provide a source of polar lipid for incorporation into therapeutic products. In fact prior art disclosures appear to teach away from this possibility.
- Unfortunately until now strategies applied to enhancing the productivity of processes providing alternative sources of EPA-rich compositions have led to a reduction in the polar lipid content of EPA-rich microorganisms.
- Microalgae produce two major types of polar lipids;—phospholipids and glycolipids. All these major polar lipid classes comprise a glycerol backbone with three positions conventionally labeled Sn 1-3. Phospho and galacto lipid classes are categorised respectively according to phosphate- and galactose-containing functional groups which are attached to the glycerol backbone usually at the Sn-3 position. Fatty acids are acylated at one or more positions 1-2. Isometric forms of these lipid classes arise from acylation patterns where not all available positions are occupied by fatty acids or where a functional group is attached at an alternative position.
- Galactolipids are produced predominantly in the chloroplast and are a structural component of the photosynthetic membrane. Galactolipids are one of the most polar of all the lipid classes; there is a substantial difference in charge distribution over the molecule because of the polar nature of the one or more galactose moieties that are attached to the glycerol backbone, providing spacially separated centres of positive and negative charge. Hence galactolipids have found application as emulsifying agents and have been proposed as drug delivery conjugates.
- The polar nature (among other physiochemical properties) of galactolipids leads to a number of useful opportunities, including but without limitation to potential advantageous routes for extraction and purification of galactolipids and galactolipid fatty acids, formulation of galactolipids and galactolipid fatty acids into foods, functional foods, beverages, pharmaceutical and industrial compositions, delivery of galactolipids and galactolipid fatty acid nutritional and therapeutic products in a bioavailable form, as well as advantageous therapeutic effects and mechanisms of action their use may promote.
- Phospholipids are major structural components of cellular membranes. The highly polar ‘head’ of the molecules coupled with their hydrophobic fatty acid ‘tails’ lead the phospholipids to spontaneously form micelles and bilayers in aqueous media. Phosopholipids both within and external to the chloroplast are expected to play a number of important roles in relaton to the physiological response of microorganisms to light. Or example it has been proposed that fatty acids located in cytoplasmic phospholipids classes are a reservoir for incorporation into chloroplastic lipids during production of photosynthetic membranes. The polar nature of phospholipids among other physiochemical properties presents a number of useful opportunities similar to those stated above for galactolipids. Certain phosopholipids including PC are known be absorbed differentially in mammals which could be turned to a therapeutic advantage. Work on absorption of galactolipids in particular MGDG in mammals is limited.
- Cohen et al. Journal of Applied Phycology 5: 109-115, 1993 disclose a general scheme for obtaining microalgal galactolipids and producing compositions enriched with the fatty acid GLA. The method disclosed involves extracting the total lipids of the organism and then separating the galactolipids from the total lipid fraction. These authors recognise in the same prior art publication that in order to be industrially useful the content of GLA in the microalgae (and presumably in the galactolipid fraction) would have to be increased. The organisms used in this study were grown under totally photosynthetic conditions. To our knowledge, prior to the present invention neither Cohen and colleagues nor any other previous authors have suggested that the required increases in yield could be accomplished by using largely heterotrophic growth.
- Kyle et al in U.S. Pat. No. 5,567,732 disclose a method for producing EPA-rich oils from cells of the diatom Nitzschia alba in the dark and teach that it is possible to induce this organism in heterotrophic culture to enter an oleogenic phase by allowing nitrogen depletion to occur and after 12-24 hours allowing a silicate depletion state to also occur, while continuing to supply other nutrients to the culture. The colourless species of diatoms are preferred. (Colourless species in general and in particular the microorganism preferred by Kyle et al are colourless because they do not exhibit the phenotype of photosynthetic pigments. N. alba for example is believed to be an obligate heterotroph which means that it does not have any active photosynthetic capacity. Nevertheless, a published lipid class analysis of N. alba reports that a few percent of the lipid composition are comprised of galactolipids.) The authors claim that diatoms can successfully be economically cultivated to produce large quantities of single cell oil and they state “for the purposes of this specification, single cell oil means a triglyceride product of a unicellular microorganism”. To our knowledge prior to the present invention neither Kyle et al nor previous authors to our knowledge have disclosed heterotrophic or largely processes useful in the commercial co-production of EPA-rich polar lipids.
- The mixotrophic production of the EPA-rich microalga Phaeodactylum tricornutum in a tubular photobioreactor is disclosed in Ceron Garcia et al Journal of Applied Phycology 12: 239-248, 2000. This manufacture utilizes 9.2 g L−1 glycerol as an organic carbon source and supplies an external irradiance of 165 μmol photons m−2 s−1 to the photobioreactor surface. These authors claim that by reducing the need for light this form of mixotrophic growth has a number of advantages including the possibility of greatly increasing the algal cell concentration and EPA productivity in outdoor mass culture on a large-scale. Ceron Garcia and colleagues did not however identify the advantages of largely heterotrophic growth in terms of increased polar lipid production. Furthermore neither these authors nor any previous authors have identified the potential for utilizing relatively low levels of irradiance in largely heterotrophic culture for producing EPA-rich microorganisms.
- A number of prior art publications have disclosed that culture conditions including light intensity and wavelength can enhance lipid and overall EPA production in specified EPA-rich microalgal species. However, prior to the present invention it had not been proposed that sub photosynthetic light intensities could be used to alter the relative production of lipid classes in a commercially useful manner. Nor had it been proposed that sub photosynthetic light intensities could be utilized to alter the localization of EPA in lipid reservoirs of microalgae in a commercially useful manufacture.
- Extraction of Polar Lipids from Microalgae
- Several techniques of potential industrial utility have been proposed for extraction and concentration of galactolipids and/or fatty acids from galactolipid fractions of biological material. Winget (U.S. Pat. No. 5,767,095) describes in detail a range of extraction and concentration techniques used to recover particular lipid classes, including relatively pure galactolipids containing EPA, from a number of photosynthetically produced microalga including those of the diatom genus Chlorella.
- Cohen et al (J. Appl. Phycol. 5: 109, 1993.) disclose the fatty acid DHA may be produced from the phosphatidylethanolamine (PE) lipid fraction of the photosynthetic organism Isochrysis galbana by extracting total lipids and subsequently producing DHA rich compositions by employing the well known technique of urea crystallisation. Vali et al U.S. Pat. No. 6,953,849 disclose a process involving dewaxing of rice bran and hexane extraction and includes HPLC with a silicic acid column. Colarow U.S. Pat. No. 5,284,941 discloses a method involving solvent boric acid gel separation. Buchholz et al U.S. Pat. No. 5,440,028 discloses a method isolation through membrane separation, with pH adjustment. Bergqvist et al 1995 report after their work on oat kernels that galactolipids may be commercially extracted from a range of biological materials using a solid phase extraction using the known differences in solubility in acetone between phospholipids and glycolipids. They started with a hot ethanol extraction then used hexane then hexane/acetone then acetone.
- No prior art publications are known to teach that it is possible to selectively isolate EPA-rich compositions in a commercial manufacture from the lipids of organisms that have been cultured using largely heterotrophic culture capable of enhancing EPA productivity and simultaneously increasing the concentration of EPA in specific polar lipid fractions.
- A number of prior art publications disclose the use of enzymes to liberate lipids and arrive at concentrated and purified lipid and fatty acid containing compositions from fish oil and other starting materials. The inventors appreciate that various lipases and phospholipases are capable of dis-assembling lipids. For example a variety of solvent-based extraction systems and crystallisation techniques have been disclosed that favour extraction of lipids of a particular class or fatty acids of a particular chain length or degree of unsaturation. These enzymes which may include lipases and proteases are known to act preferentially on different substrates. In the case of lipases for example, enzymes are expected to some degree to be specific for lipid class, fatty acid, and the position of the fatty acid within the lipid class. The activity and preference of enzymes can be altered by altering environmental conditions such as temperature, and via the addition of cofactors and techniques such as immobilization.
- A common analytical technique used to estimate the localisation of fatty acids at different positions in the lipid structure is to expose a fatty acid class to a lipase capable of selectively hydrolysing fatty acids located in a particular position. It follows that the common general knowledge of those skilled in the art includes the recognition that both the proportion of lipids and the localisation of target fatty acids as well as co-localisation or lack thereof of undesired acids within lipid reservoirs of a biological material constitute critical aspects in a purification process at an analytical scale. To our knowledge however no previous authors have disclosed methods of producing therapeutic or prophylactic compositions via the selective enzymatic hydrolysis of algal polar lipids at least not from polar lipids produced in largely heterotrophic cultures.
- Winget teaches use of topically applied MGDG-EPA compositions in the prevention and treatment of inflammation, but does not disclose application of lipase-type or indeed any enzymes. Later, Bruno et al (Eur J Pharmacol: 524; 159-168 7 Nov. 2005) disclose that the galactolipid classes MGDG, DGDG and SQDG obtained from thermophilic blue-green algae have in-vivo anti-inflammatory activities in a croton-oil induced mouse ear inflammatory response. However there is no indication in the abstract that any of the n-3 HUFAs were present.
- The present invention provides novel methods for obtaining EPA-rich compositions that provide the public with a useful choice. In addition, the present invention provides compositions including EPA-rich galactolipids and highly purified EPA-rich fatty acid compositions that provide the public with a useful amount of therapeutic, prophylactic, or dietary EPA, or at least provides the public with a useful choice.
- In a first broad aspect the invention provides a process for obtaining an eicosapentaenoic acid (EPA)-rich composition for therapeutic or prophylactic use, wherein the process employs a culture of micro-organisms of a type selected for a capability of largely heterotrophic growth, and a capability of production of EPA, and a capability of photosynthetic lipid production; the process including a culture phase in which cells are grown under conditions in which organic carbon is used as an energy source; the conditions including use of controlled illumination at a level corresponding to an average photosynthetically active irradiance inside the culture of less than 40 μmol photons m−2 s−1 and including imposition of limitation of nutrients selected from a range including phosphorus and silicon; said procedures being undertaken in order to maximise the amount of recoverable polar lipids including at least one EPA side chain, and a harvesting process that creates a composition rich in EPA.
- In a related aspect the invention provides a process as previously described in this section wherein the culture of micro-organisms comprises identified microalgae, funghi or bacteria.
- Preferably the micro-organisms are comprised of the marine single-celled diatom known as Nitzschia laevis, University of Texas microalgal collection UTEX 2047.
- In another related aspect, the micro-organisms comprises a strain of micro-organism selected, when under culture conditions, for an improved yield of recoverable polar lipids having molecules which include at least one side chain bearing EPA.
- Preferably the micro-organisms accumulate galactolipids rich in EPA concentrated at the Sn1 position within the galactolipid or like classes.
- More preferably the micro-organisms are capable of accumulating commercially useful quantities of polar lipids, including galactolipids rich in EPA at the same time as exhibiting high EPA productivity in general, including the EPA found in triglycerides.
- In particular, the EPA-rich lipid classes contained in the total lipid fraction include without limitation one or more of the following: MGDG; MGMG; DGDG; DGMG; non-galactosyl polar lipids including PC and PG; neutral lipids including monacylglycerol, diacylglycerol, triacylglycerol.
- Preferably the culture is capable under managed conditions of producing a proportion of its total dry weight as fatty acids; the proportion lying in the range of between 5 and 80%. Preferably the culture is capable under managed conditions of producing a proportion of its total fatty acids as EPA; the proportion (by dry weights) lying in the range of between 1 and 80%. Preferably the culture is capable under controlled conditions of producing 25% to 60% of total fatty acids as fatty acids contained in polar lipids; more preferably the proportion is more than 30%, more preferably over 40% and even more preferably over 50%.
- Preferably the organic carbon component is fed incrementally over time, according to the future predicted growth of the culture in a period of 4 to 24 hours.
- Preferably the culture is capable under controlled conditions of producing 5 to 40% of total fatty acids as fatty acids contained in galactolipids; more preferably the proportion is more than 10%, more preferably over 20% and even more preferably over 30%.
- Preferably the culture is capable under controlled conditions of producing 40 to 70% of EPA as EPA contained in polar lipids (as distinct from neutral lipids; more preferably the proportion is more than 50%, more preferably over 60%.
- In a second broad aspect the invention provides an EPA-rich composition derived from a culture as previously described in this section, wherein the EPA-rich compositions are obtained by a harvesting process including the steps of:
-
- harvesting cells from the culture medium at a selected time;
- heat or otherwise killing the cells to denature endogenous enzymes;
- forming the cells into a cake of biomass;
- extracting the cake of biomass with a first, non-selective lipid solvent and recovering the extracted material from the solvent as a residue; (use of near critical di-methyl ether is one option)
- extracting the residue with a second, selective solvent so that a neutral, EPA-rich lipid composition is separated from the polar, EPA-rich lipid composition and optionally further purifying either or both types of EPA-rich compositions in order to achieve a required standard of purity.
- In a related aspect the invention provides an EPA-rich composition derived from a process as previously described in this section, wherein the method includes a procedure in which EPA is hydrolysed from a polar EPA-rich lipid class obtained from the polar EPA-rich lipid fraction using the steps of:
-
- presenting the polar lipid as a substrate to at least one enzyme capable of cleaving EPA from the Sn1 position and
- after cleavage separating the EPA as a free or esterified fatty acid or metal-salt, thereby obtaining an EPA composition intended for use as a therapeutic composition or for prophylactic use.
- Preferably the enzymic process uses at least one enzyme selected from the range of lipases, phospholipases and galactolipases. Optionally the enzyme is affixed to a surface.
- Alternatively the recovery of fractions having a relatively high EPA purity and an increasing yield is accomplished by matching the enzymic release of fatty acids over time with a series of recovery steps in a continuous or multistage batch extraction process.
- Alternatively, the addition of materials including (without limitation), an alcohol, hexane or calcium chloride may assist enzymic release by removing products of the enzyme-catalysed reactions from proximity of the enzyme.
- Optionally physico-chemical fractionation of the polar lipid fraction, including techniques such as partitioning, chromatographic fractionation and the like, may precede the application of one or more enzymes.
- Preferably said galactolipids rich in EPA will be contained in preparations of the biomass of organisms.
- More preferably said galactolipids rich in EPA will be contained in the total lipid fraction extracted from the biomass of organisms from which fraction they may be subsequently separated from the total lipid fraction.
- In a first related aspect the invention provides a first method for isolating useful products comprising the steps of: (a) taking a preparation of the biomass of the cultured organisms,
- (b) extracting the total lipid fraction from the biomass, and
(c) isolating one or more lipid classes contained in the total lipid fraction including but not limited to one or more of the following classes: MGDG; MGMG; DGDG; DGMG; SQDG; non-galactosyl polar lipids including PI, PE, LPC, PC and PG; neutral lipids including monacylglycerol, diacylglycerol, triacylglycerol; - Alternatively, the EPA is concentrated in galactolipid classes which are the preferred substrate of and/or able to be made accessible to an enzyme capable of hydrolyzing one or more acyl bonds and thereby liberating the EPA contained within the galactolipid classes.
- In another alternative, EPA is concentrated within the diacyl galactolipid classes.
- More preferably EPA is concentrated within the monogalactosyldiacylgalactolipid (MGDG) class.
- Preferably the EPA-rich composition is processed after separation in order to further purify the composition, using known techniques though usefully not having to contend with DHA.
- Preferably the undesired molecules (even if present) are not cleaved from the polar lipid, so that (for example) the EPA composition is substantially free of DHA which remains acylated to the glycerol backbone.
- Preferred known techniques for separation include low temperature crystallisations, purification processes taking advantage of differential solubility of fatty acid esters or salts in various solvents including ionic solvents, precipitation using metal salts, the use of selectively permeable membranes, column chromatography of fatty acids or their esters, supercritical fluid chromatography, urea addition crystallisation, fractional distillation, preparative HPLC, iodolactonization, and selective re-esterification by enzymes.
- In a first alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises 50-60% EPA, less than 5.5% arachidonic acid and substantially no DHA. Preferably the composition comprises 50-60% EPA, less than 4.5% arachidonic acid and substantially no DHA. More preferably the composition comprises 50-60% EPA, less than 3.5% arachidonic acid and substantially no DHA. Even more preferably the composition comprises 50-60% EPA, less than 2.5% arachidonic acid and substantially no DHA In a second alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises 50-60% EPA, less than 5.5% arachidonic acid and less than about 2% DHA. Preferably the composition comprises 50-60% EPA, less than 4.5% arachidonic acid and less than about 2% DHA. More preferably the composition comprises 50-60% EPA, less than 3.5% arachidonic acid and less than about 2% DHA. Even more preferably the composition comprises 50-60% EPA, less than 2.5% arachidonic acid and less than about 2% DHA
- In a third alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises 60-70% EPA, less than 4.5% arachidonic acid and substantially no DHA. Preferably the composition comprises 60-70% EPA, less than 3.5% arachidonic acid and substantially no DHA. More preferably the composition comprises 60-70% EPA, less than 2.5% arachidonic acid and substantially no DHA. Even more preferably the composition comprises 50-60% EPA, less than 1.5% arachidonic acid and substantially no DHA
- In a fourth alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises 60-70% EPA, less than 4.5% arachidonic acid and less than about 1.5% DHA. Preferably the composition comprises 60-70% EPA, less than 3.5% arachidonic acid and less than about 1.5% DHA. More preferably the composition comprises 60-70% EPA, less than 2.5% arachidonic acid and less than about 1.5% DHA. Even more preferably the composition comprises 50-60% EPA, less than 1.5% arachidonic acid and less than about 1.5% DHA.
- In a fifth alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 95 and 99% EPA, less than 1% of arachidonic acid and less than about 0.5% of DHA.
- In a sixth alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 95 and 99% EPA, less than 0.5% of arachidonic acid and less than about 0.5% of DHA.
- In a seventh alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 95 and 99% EPA, less than 1% of arachidonic acid and less than about 0.1% of DHA.
- In an eighth alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 95 and 99% EPA, less than 0.5% of arachidonic acid and less than about 0.1% of DHA.
- In a ninth alternative aspect, the invention provides a composition derived from a process as previously described in this section, wherein the composition comprises between 99.6 and 99.9% EPA, less than 0.1% of arachidonic acid and less than 0.1% of DHA.
- In a further alternative aspect the invention provides for use of a composition, as previously described in this section, in the manufacture of a medicament for treatment of a person affected by certain medical conditions or disorders including but not limited to those selected from diabetes (type I, and type II), glycaemic disorders diabetes-associated hypertension, cancer, osteoarthritis, autoimmune diseases, rheumatoid arthritis, inflammatory and auto-immune diseases other than arthritis, respiratory diseases, neurological disorders, neurodegenerative disorders (including Huntington's disease, Parkinson's disease, Alzheimer's disease, schizophrenia, major depression, unipolar depression, bipolar depression, obsessive compulsive disorder, borderline personality disorder, post natal depression, organic brain damage, and traumatic brain injury), renal and urinary tract disorders, cardiovascular disorders, cerebrovascular disorders, degenerative diseases of the eye, psychiatric disorders, reproductive disorders, visceral disorders, muscular disorders, metabolic disorders, prostatic hypertrophy and prostatitis, impotence and male infertility, mastalgia, male pattern baldness, osteoporosis, dermatological disorders, dyslexia and other learning disabilities, cancer cachexia, obesity, ulcerative colitis, Crohn's disease, anorexia nervosa, burns, osteoarthritis, osteoporosis, attention deficit/hyperactivity disorder, and early stages of colorectal cancer, lung and kidney diseases, and disorders associated with abnormal growth and development.
- In a third broad aspect the invention provides a composition prepared as previously described in this section, wherein the composition is prepared in the form of a human dietary supplement for therapeutic or prophylactic use.
- In a first (therapeutic) alternative, the invention provides for use of a polar lipid, prepared as previously described in this section and including an effective amount of EPA in the manufacture of a medicament for use in treating the medical conditions or disorders as previously listed in this section.
- Preferably the polar lipid is formulated in order to provide a prophylactic, health or dietary daily health supplement including an amount in the range of from 0.1 to 50 grams of EPA.
- Alternatively the amount is in the range of from 0.5 to 5 grams of EPA.
- (Note that even if an EPA-rich polar lipid composition suitable for food and dietary supplement use has a relatively low content of EPA; it is a relatively high ratio of EPA to undesirable molecules that renders the composition fit for purpose, since the undesired molecules exist at a substantially low level in the composition).
- Preferably the content of EPA in the relatively low purity composition is less than about 20% and the content of undesired molecules remains low even if the purity of the EPA is below 20%.
- In a yet further preferred aspect, the invention provides one or more relatively low purity EPA compositions which are nevertheless pharmaceutically effective owing to a substantially low level of undesired molecules in one or more lipid fractions.
- More preferably the content of EPA between about 10% and about 80%.
- Preferably the availability of such polar lipid compositions broadens the choice of compositions having a high ratio of EPA to undesirable molecules which is currently restricted to highly purified EPA from fish oil, which is less accessible to the general population.
- More preferably the amount is formulated so as to be suitable for repeated ingestion as a prophylactic, health or dietary daily health supplement.
- Preferably the products when consumed are capable of promoting brain and mental health, cognition and behaviour.
- Preferably the products when consumed are capable of eliciting health promoting effects on any of the following non limiting list of body systems and tissues; auditory, appetite, arousal, balance, blood, bone, bowel, cardiovascular, digestive, endocrine, enteric, emotional, gastric, hair, hepatic, immune, lymphatic, kineaesthetic, marrow, memory, metabolic, musculoskeletal, neurotransmitter, nasopharyngeal, pancreatic, musculoskeletal, reproductive, respiratory, ocular, oesophagal, olfactory, palate, pulmonary, proprioceptive, renal, skin, sleep, stomach, sensorimotor, skin, urinogenital, wound healing.
- In a further alternative, the prophylactic, health or dietary supplement is formulated as a solid substance compatible with direct ingestion by humans; the range of formulations including: a cake, a powder, granules, tablets, boluses, pills, capsules, lozenges or beads.
- In one option, the EPA is re-esterified or combined with polar lipids, or alternatively the EPA moiety is cleaved from the galactolipids and re-esterified into phospholipid fractions.
- More preferably intact or semi-intact polar lipids rich in EPA are capable of efficiently passing through endothelial and other peripheral cell membranes in mammals.
- Preferably said polar lipid fatty acids are incorporated into cell membranes within the mammal.
- A prophylactic, health or dietary supplement as previously described in this section, wherein the health supplement is provided as a liquid substance carrying finely dispersed EPA-rich extracted polar lipids suitable for consumption as a beverage; the range of beverages including, (without limitation) water, beer, wine, milk, spirits, sports drinks, juices and carbonated drinks, and optionally includes at least one stabilizing substance.
- Preferably the EPA-rich extracted polar lipids are encapsulated such that n-3 HUFAs and other PUFAs are protected from light including ultraviolet light in order to assist long-term stability.
- Preferably the EPA-rich extracted polar lipids are encapsulated such that n-3 HUFAs and other PUFAs are protected from oxidative degradation.
- Optionally, in the case of beverage preparations, the EPA-rich extracted polar lipids (including galactolipids) will be released into the beverage from a temporary encapsulation shortly prior to consumption.
- A prophylactic, health or dietary supplement as previously described in this section, wherein the health supplement is formulated as a substance selected from the range including: a solution, a suspension, a solid mass, a powder, granules, or the like; the substance including an effective amount of an extracted galactolipid class rich in EPA and, when in use, is compatible with incorporation into a manufactured foodstuff.
- An EPA-rich composition suitable for use as an EPA-rich human or animal dietary supplement derived as previously described in this section, wherein the process includes the steps of obtaining the cake of biomass as harvested from the culture, and of preparing substantially the entire biomass for consumption.
- In a yet further alternative, the residual whole cell content is utilized as a product in particular suitable for use as a food for mammals including monogastric and ruminant animals and/or aquaculture species including finfish and crustaceans; there being valuable residual compounds e.g.—sulpho galactolipids, carotenoids, other pigments, amino acids, and other fatty acids capable of serving as foods.
- Optionally a method for extracting EPA from a biomass derived from procedures whereby certain biomasses produced via the use of in vitro techniques accumulate commercially useful quantities of galactolipids rich in EPA at the same time as exhibiting high EPA productivity when grown under conditions according to those previously described in this section, in relation to the first broad aspect, may commence by first removing undesired fatty acids or lipids with a relatively low EPA content, and later extracting the EPA.
- In a fourth broad aspect the present invention discloses a pharmaceutically pure composition of EPA, wherein the fatty acid composition of the composition preferably contains (a) about 80 to 100% EPA and (b) little or no other omega 3 or omega-6 fatty acids; such compounds being useful in the treatment of those medical conditions that respond to medication with EPA.
- Preferably, but not solely, the pharmaceutically pure composition of EPA is derived from galactolipids.
- Preferably said pharmaceutical compositions will contain EPA as at least 90% of total fatty acids in the composition and may be substantially pure EPA.
- Preferably the content of any one of the fatty acids selected from the non-limiting range of undesirable compounds including: DHA, AA, DPA, 18:4 n-3, 18:3 n-3, 18:2 n-6 is less than 2% of total fatty acids in the composition and more preferably approaches zero.
- In a related aspect these EPA compositions will contain very low or undetectable levels of undesirable molecules which are either structurally similar to, or biologically related to, or antagonistic to the desired effects of EPA when administered to a mammal.
- The description of the invention to be provided herein is given purely by way of example and is not to be taken in any way as limiting the scope or extent of the invention. Throughout this specification unless the text requires otherwise, the word “comprise” and variations such as “comprising” or “comprises” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
- Most approaches to culturing single-celled organisms for EPA production have been based on solely heterotrophic culture, or else cultures grown under conditions providing substantial levels of light; substantially daylight or artificial equivalents. The present invention includes a novel culture-related aspect, wherein polar lipids rich in EPA are co-produced together with neutral lipids in cultures that are largely heterotrophic in that they are mixotrophic cultures producing using significantly lower photosynthetically active average irradiances inside the culture. These will optimally be subphotosynthetic (i.e., less than 10 μmol photons m−2 s−1) but may be as high as 40 μmol photons m−2 s−1.
- The invention exploits the tendency for EPA productivity in polar lipids to increase and for EPA to become localised within particular polar lipid classes under these conditions. Compositions are provided that facilitate subsequent purification of EPA-rich lipid classes, or may be directly incorporated into novel supplements rich in EPA.
- Galactolipids are incorporated as a major structural component of the membranes of chloroplasts. Other polar lipids including phosphatidyl choline (PC) are also involved in transfer of EPA to the chloroplasts. Certain types of marine algae, but not all, generate intracellular chloroplasts if illuminated and so would be expected to generate these photosynthetic lipids in response to light. The invention takes advantage of the non-linear relationship between provision of light and subsequent production of photosynthetic lipids.
- The inventors are aware that the neutral fraction of lipids will also contain significant amounts of EPA. This material is likely to be of commercial value in itself and fatty acid derivatives thereof could be purified by traditional or emerging methods mentioned below to give an EPA product. Thus the present invention should be seen as an adjunct to neutral lipid production rather than a replacement.
- Low light levels lie in a range wherein organic carbon consumption is reduced or the efficiency of its use is improved, yet without the technical complications of providing high levels of light either by use of outdoors culture with technical and environment-related complications or by the use of large amounts of artificial light with associated energy costs to be met.
- As well as largely heterotrophic cultures grown under conditions of low light a further option is the induction of nutrient depletion in cultures in order to encourage particular types of polar lipid production. Such induced deficiencies include phosphate deficiency which are expected to shift lipid production from phospho- to galacto-lipids. Silicate depletion in diatoms is also contemplated by the invention as a means of causing photosynthetic lipids to accumulate.
- The invention makes use of the specific molecular structures of certain polar lipids in certain organisms under certain conditions in order to facilitate purification of EPA. In association with the above structural specificity, the invention contemplates the use of particular enzymes having specific appetites such as stereospecificity in order to facilitate purification. In addition, the unique physiochemical properties of photosynthetic polar lipid classes produced, including the polar nature of the galactolipid molecules, provides useful methods of administering EPA.
- Finally the invention discloses formulations including the EPA and galactolipid-rich compositions and applications for the formulations.
- The particular micro-organism that has been used in the procedures described at the time of filing this specification is Nitzschia laevis. Cells were obtained from the University of Texas Microalgal Collection where they are deposited with the call reference UTEX 2047. We expect there to be other micro-algae capable of largely heterotrophic growth of the “coloured” set (those having photosynthetic machinery) to exhibit comparable or improved rates of production. At this time, we have not carried out any strain selection experiments designed to encourage expression of superior metabolic profiles under an imposed set of artificial culture conditions, and which allow cells having those profiles to be isolated. We would, however, expect a measurable gain in process efficiency (with regard to EPA production) to result.
- At this point, trials with (a) other micro-algae, (b) selected micro-algae, (see above) or (c) genetically modified micro-algae (see “variations”) have not been done although all such trials are included in the scope of the invention.
- Large scale commercial cultures of microalgae can be produced according to the methods of this invention under closely monitored and controlled conditions in a vessels with capacities measured in tens or hundreds of thousands of litres. Changes in conditions and requirements associated with the scale of the culture vessel (e.g. cooling/heating, mixing, gas mass transfer) would be anticipated by those skilled in the art.
- Once material with a relatively high level of EPA has been obtained it may be purified further by a number of means. These include low temperature crystallisations, purification processes taking advantage of differential solubility of fatty acid esters or salts in various solvents including ionic solvents, precipitation using metal salts, the use of selectively permeable membranes, column chromatography of fatty acids or their esters, supercritical fluid chromatography, urea addition crystallisation, fractional distillation, preparative HPLC, iodolactonization, and selective re-esterification by enzymes.
- Enzymes and their Provenance.
- An “ideal enzyme” for use in the invention would be able to excise side chains from the glycerol backbone of any polar lipid class if the side chain comprises a EPA. Whilst several lipases have been isolated that show selectivity for chain length we know of no cases where absolute specificity based on chain length has been demonstrated. None of these enzymes is currently available for industrial processes.
- Many known lipases have the restricted ability of being able to act at the Sn1 position only, which suggests the production and isolation of polar lipids having a desired n-3 HUFA predominantly at the Sn1 position would be a route to enrichment. Selection of a particular enzyme for use in a commercial process is also cost-dependent and it may be necessary to rely on those lipase-type enzymes already produced in bulk for use in the dairy industry or the baking industry, which includes 1,3 specific lipase-type enzymes made from fungi—for instance the lipase/phospholipase derived from Aspergillus spp, “Bakezyme PH 800 BG” (DSM Food Specialities), or the lipase derived from Rhizopus oryzae, “Piccantase R8000”. The engineered enzyme “Lecitase Ultra” (Novozymes) has 1,3 specific lipase activity but at elevated temperatures demonstrates phospholipase A1 activity. Both activities are likely to be of use in the isolation of fatty acids from the Sn1 position of polar lipids.
- At this time, attention has been applied in particular to phospholipases. However it will be appreciated that other lipases and galactolipases are also of relevance in the extraction of EPA and of EPA-containing materials from biomass. Clearly there is an opportunity for further exploitation and optimisation of enzyme specificity within the invention, for it is likely that enzyme-based purification will have a number of advantages over physico-chemical separation of fatty acids. Tasks such as separation of DHA from DPA or 18:2 from 18:4 fatty acids are relatively difficult under existing methods many of which are hampered by reliance on physico-chemical factors such as melting points or molecular sizes.
- The current specification discloses a method to produce a relatively pure EPA composition by exposing a microalgal biomass rich in galactolipid EPA to an enzyme.
- In some versions of the commercial process disclosed herein it is likely that any enzymes used will be adsorbed on to a surface or otherwise retained within the process, in order to conserve supplies, by the use of techniques for handling enzymes that are well known in the art. It is also likely that optimisation of working conditions for a selected enzyme will provide a significantly improved rate of attack and a more specific type of attack, as a result of exploitation of working conditions well known to those skilled in the art, such as concentration, pH, temperature, presence of salts, or the presence of competing compounds that inhibit undesired modes of action.
- In general terms the polar lipids can be either (A) isolated as particular types, or (B) used as one, collective group. For either A or B, they may then be (i) used directly, (ii) further processed into EPA-rich fatty acid compositions by cleaving the fatty acids from the lipid species. In the case of further processing, a polar lipid fraction or fractions may be hydrolysed with a specific phospholipase (or other lipase), the released EPA captured by a suitable acceptor molecule. Examples of acceptors include: glycerol and alcohols including ethanol, propanol, iso-propanol, or long-chain alcohols (which will yield waxes). Alternatively, the EPA may be transferred by a phospholipase, galactolipases, or other lipase on to a suitable carrier type molecule (such as phosphatidylcholine (PC)). Metal salts-membrane separation.
- Typical applications include, for (i): production and use of ultra pure EPA and an active pharmaceutical ingredient (ii) production and use of non-pharmaceutical EPA-only therapeutic compositions (iii) production and use of EPA-rich polar lipid containing foods, functional foods and food supplements, and (iv) production and use of whole-cell products for foods or food supplements.
- Actively growing cells of the species N. laevis obtained as above are produced in 200 mL of media in stoppered 500 mL Erlenmeyer flasks. Multiple flasks are used to produce large volumes of material. An inoculum of 0.2 g L−1 of exponential or early stationary phase cells is used. Flasks are incubated in temperature- and light-controlled growth chambers by placing them on orbital shakers at around 200 rpm to maintain the cells in suspension and aid in gas transfer between atmosphere and media. Temperature is maintained at 20° C. Light is provided at an average irradiance of photosynthetically active light in the culture of 40 μmol photons m−2 s−1 as measured by an Apogee quantum sensor digital pyranometer and calculated from conditions such as culture depth and cell density. Aliquots of culture are taken during growth to determine the dry weight of the culture at that time point. Cultures are fed a heat sterilised glucose stock solution (400 g L−1) daily at a level that is projected to provide organic carbon requirements for the predicted biomass production over the subsequent 24 hours. Total glucose added to culture over the entire culture period amounted to 3 grams per litre.
- Initial concentrations of nutrients in standard media are typically, per litre:
- (a) 50 ml Salt stock solution which comprises NaCl; 160.0 g, MgSO4 7H2O; 44.0 g, KCl; 10.8 g, CaCL2; 2.04 g, KH2PO4; 0.8 g per 1 L of distilled water.
(b) 50 ml Nitrogen stock solution, which comprises the following: NaNO3; 17 g, and yeast extract; 16 g per litre of distilled water.
(c) 10 ml Tris buffer stock solution. This stock is made by dissolving 89.2 g Tris buffer in 1 L distilled water.
(d) 5 ml Trace Metal stock solution, which contains the following, per 100 ml; (NH4)6Mo7O24 4H2O; 0.556 g, CoCl2 6H2O_0.046 g, MnCl2 4H2O_0.500 g, Na2MoO4 2H2O_0.048 g, H3BO3_61.120 g, ZnCl2_0.622 g, H2SO4 (concentrated); 18 ml.
(e) 2 ml of vitamin solution which is made by dissolving 6 g 0.1% vitamin B12, 0.01 g Biotin and 0.01 g Thiamine in 100 ml distilled water.
(f) 5 ml Sodium Metasilicate stock solution which is made by dissolving 24 g Na2SiO3 in 1 L distilled water).
(g) 2.7 ml of Chelated Iron stock which is made by dissolving 0.81 g FeCl3 6H2O in 10 ml of 0.1N HCl and dissolving 10 g NaEDTA in 100 ml 0.1N NaOH.
(h) 1 mL Copper sulphate stock which is made by dissolving 9.8 mg CuSO4 5H2O in 1 L distilled water. - Biomass dry weight is measured, using the pre-weighed glass fibre filter method as follows. A 10 ml sample is removed from a larger representative sample taken whilst stirring to achieve a broadly homogenous dispersion of cells and cell aggregates; culture flasks are generally sterilised with a Teflon-coated magnetic stir bar in place to aid with this. The 10 ml sample is placed in a centrifuge tube and spun at 3000 rpm in a Heraeus Sepatech Megafuge 1.0 with swing-out rotor for 4 min and the liquid decanted leaving a cell pellet. The cell pellet is washed with phosphate-buffered saline and re-centrifuged. A Sartorius glass fibre filter is washed by passing 1 litre of deionised water through the filter then dried overnight in a vacuum oven at 30° C. prior to being weighed. The 10 ml sample is passed through the preweighed filter in a vacuum filter apparatus and is then placed in an oven at 60 deg C. for two hours prior to being reweighed. The difference in grams between the pre and post weights times 100 is taken as a measure of the dry weight per litre.
- Cells are harvested after 3 days of growth since at this point the culture(s) are still in exponential phase. Cellular extract containing the lipids can be obtained by Folch extraction following the method of Bligh and Dyer (1959). Cells from several flasks are combined to allow production of sufficient material for further use.
- Total fatty acid analyses of samples of cellular extract are obtained to identify the composition of the cultured material. Addition of an internal standard such as C23:0 to the reaction allows measurement of the total fatty acid content of the cells. The method of fatty acid production entails a basic transesterification with 0.5M methoxide in methanol followed by an acidic transesterification using dry HCl in methanol. Fatty acid methyl esters are recovered by extracting with hexane and drying with sodium sulphate before analysis using gas chromatography. The sample is run on a 30 m×0.25 mm ID Famewax (crossbond polyethylene glycol) glass capillary column contained within a Shimadzu 2010 GC by autoinjection. By ramping the column temperature from 145 to 240° C. over the course of 50 minutes and then leaving the column at 240° C. for a further 10 minutes fatty acids is identified by co-chromatography with known standards supplied by Restek.
- Cellular extract is separated into polar and non-polar fractions using column chromatography. 0.5-2 g of the cellular extract is dissolved in a small volume of diethyl ether and loaded onto a column containing 40 g silica gel (with a mesh size of 230-350) in diethyl ether. 10 mL of diethyl ether and 80 mL of chloroform is used to elute non-polar material including the triglycerides. Further addition of 10 mL chloroform:methanol (1:1 v/v) and 80 mL methanol to the column elutes the polar material including galactolipids and phospholipids. These two classes of material are collected separately and dried down before samples are subjected to fatty acid analysis similar to that described above. The polar fraction is then further separated by placing it on a second chromatographic column. A column is constructed of silica gel in chloroform and is washed with successive washes of 2 column volumes each of 99:1 (v/v), 49:1 (v/v), 29:1 (v/v), 19:1 (v/v), and 9:1 (v/v) chloroform:methanol and 2 column volumes of methanol. Further steps are added as required to separate other galactolipids and phospholipids from one another if so desired.
- Lipase Based Separation of Fatty Acids from Specific Positions within Lipid Molecules.
- The 1,3 specific lipase and phospholipase A1 “Lecitase Ultra®” from Novozymes is used to cleave the fatty acids from the Sn-1 position of MGDG isolated in the manner described above. 5 mg of MGDG is dissolved in 3 mL of methanol whilst 12 u of enzyme is dissolved in 3 mL 10 mM citric acid buffer at pH6.0. These are incubated together at 60° C. for 5-15 minutes and after incubation the reaction could is washed 3 times with 2 mL hexane to collect the free fatty acids produced. The hexane washes are collected in a fresh tube with 3 mL methanol and the mixture incubated at 50° C. for 2 hours to produce Fatty acid methyl esters. At the end of this period the hexane layer is removed and concentrated before being analysed on a GC.
- After 72 hours a biomass dry weight reaches 3 grams per litre in flask culture. Fatty acids form at least 8% of the dry material grown in the culture. EPA reaches 24% of total fatty acids.
- Cultures of Nitzschia laevis grown in this manner demonstrate doubling times as low as 12 hours.
- The analysis of the fractions recovered from the first chromatographic column shows that roughly equal amounts of fatty acids are recovered in polar and non-polar lipid fractions. 67% of the EPA is located in the polar fraction.
- Analysis of the fractions by GC shows that around one third of the polar fatty acids elute in the 9:1 (v/v) fraction, Thin Layer Chromatography of the fractions indicates that the 9:1 (v/v) fraction contains the bulk of the galactolipid MGDG with the remainder of the MGDG and all other lipid classes eluting with methanol.
- Table 1 (below) shows in the left hand column the total fatty acid profile of MGDG isolated using the method described in the present example and in the right hand column the total fatty acid profile of fatty acids recovered from the hydrosylate.
-
TABLE 1 Total and enzyme-hydrolysed fatty acids from MGDG. Figures are percentage of total fatty acids loaded on the GC. Please note that where results are expressed as “ND” (“not detected”) the amount present was beyond the limits of detection of our instrument. Total Fatty acid Enzyme-liberated production from Fatty acids from Fatty acid Name MGDG MGDG C14:0 2.44 3.81 C16:0 3.94 2.17 C16:1 c9 35.79 27.06 C16:2 4.07 1.62 C16:3 14.15 0.49 C16:4 0.47 ND C18:2 c9,12 0.26 0.14 C18:2 t9,12 0.04 ND C18:3 c9,12,15 0.48 0.89 C18:3 c6,9,12 0.22 ND C18:4 n3 0.88 0.43 C20:2 c11,14 0.09 0.76 C20:4 c5,8,11,14-AA 2.99 5.32 C20:4 c8,11,14,17 0.19 0.23 C20:3 c8,11,14 0.05 ND C20:5 c5,8,11,14,17 - EPA 30.11 54.93 C22:2 c13,16 0.07 0.64 C22:5 - DPA 0.2 0.21 C22:6 - DHA 0.68 ND Unknown molecules 1.75 0.21 Other saturates 0.47 0.48 Other monounsaturates 0.68 0.61 - The method allows significant increases in the polar lipid production of the organism to take place over that which is measured in a purely heterotrophic culture (where 75% or more of the fatty acids are seen in the non-polar fraction).
- Roughly one third of the polar fatty acid in the example is contained in the MGDG fraction as compared with only around 15% in purely heterotrophic cultures.
- Application of the enzyme in the present example provides for enrichment of EPA and the exclusion of DHA from the fatty acids recovered from the hydrosylate as compared to the total fatty acids of the MGDG fraction.
- Whilst only 30-35% of the total MGDG fatty acids are recovered in the hydrosylate in the present example 57.0% of the EPA in the sample is recovered by the enzymatic process confirming predominance in the Sn1 position of MGDG.
- Flask cultures of N. laevis is produced according to the method of example one except that: 5-10 grams of glucose is added over the course of the culture run. Light is provided at an average irradiance of photosynthetically active light in the culture of 10 μmol photons m−2 Harvesting, separation and analysis techniques are all according to the method of example one.
- After 72 hours a biomass dry weight reaches between 3 and 5 grams per litre in flask culture. Fatty acids form at least 10% of the dry material grown in the culture. EPA reaches at least 20% of total fatty acids.
- Analysis of the fractions obtained from the first chromatographic column shows that 35 to 40% of fatty acids are recovered in the polar fraction and 60-65% in the non-polar lipid fraction. EPA is preferentially located in the polar fraction.
- Analysis of the fractions by GC shows that between 30 and 40% of the polar fatty acids elute in the 9:1 (v/v) fraction. Thin Layer Chromatography of the fractions indicates that the 9:1 (v/v) fraction contains the bulk of the galactolipid MGDG with the remainder of the MGDG and all other lipid classes eluting with methanol.
- Analysis of the MGDG fraction shows that over 30% of the fatty acids are EPA. Of the material recovered from enzymatic hydrolysis, between 50 and 60% is EPA.
- More fatty acids were recovered in the non-polar fraction than the polar fraction but the amounts of fatty acids in the polar fraction are still substantially higher (50-200% greater) than those from a similar heterotrophic culture grown in the absence of light. EPA is found as a higher proportion of fatty acids in the polar fraction both in comparison to the non-polar fraction and in comparison to a polar fraction from a totally heterotrophic culture.
- Significantly, although the amount of lipids recovered in the polar class is lower as a proportion of the whole when compared to mixotrophic or phototrophic growth, the improved growth rates under largely heterotrophic conditions means that polar and galactolipid yields are equivalent to or better than those of example 1.
- Larger volumes of actively growing cells of the species N. laevis are grown under closely monitored and controlled conditions in a 20 litre vessel, having an effective working capacity of 18.5 litres. The vessel is internally lined with “Teflon®” and comprise a stirred, jacketed tank. The jacket is provided with hot or cold water as required in order to maintain an internal temperature of 20° C., as sensed by internal probes and controlled with a SCADA device controlling water valves. A mechanical seal admits an impeller shaft of 19 mm diameter, having a 6-blade Rushton impeller at one end, placed near an air sparger, and a marine impellor 250 mm from the end. A 0.25 kW 3-phase 6-pole motor drives the shaft at between 100 and 900 revolutions per minute. Motor speed is controlled with a variable speed drive capable of receiving an analogue signal from the supervisory control device. Pressurised air (1.5 bar) is injected through a sterilizing filter at a rate of between 2 to 10 litres per minute to the air sparger. The air flow is measured with a Dwyer flow meter model TF 2110 and the flow rate is controlled either manually through the use of a regulator or by a “Festo” proportional solenoid controller. Similarly gas outflow from the vessel is measured and regulated. Dissolved oxygen is measured by an oxygen sensor (Broadley-James Corporation, Irvine, Calif.) and the dissolved oxygen maintained at around 50% of saturation via supervisory control feedback loops controlling motor speed and, to a lesser degree, air flow.
- The pH of the culture is maintained at pH=8.5 (or at another pH if desired) by using an immersed pH sensor (Broadley-James Corporation, Irvine, Calif.) in a closed control loop, driving a peristaltic pump for the addition of either alkali (as NaOH or KOH) or for the addition of acid (as HCl or Acetic acid) as required.
- Concentrations of nutrients, including feed stocks that are sources of nitrogen, silicate, phosphorus, glucose, and organic carbon (e.g. glucose) are separately controlled by feeding sterile stock solutions of desired concentrations aseptically through corresponding peristaltic pumps. Sterile basal media is also supplied to the vessel through an aseptic pump. Culture volume is monitored using a Kubler level sensor and input of basal media or nutrient controlled by the SCADA device.
- Precautions related to sterility include operations being conducted in a production environment equipped with an air lock and supplied with filtered sterile air to create a positive pressure directing air away from culture vessels. Staff follow protocols well known to those skilled in the art designed to minimize any accidental introduction of contaminants into the production environment. All stocks that can withstand heating are autoclaved at 121° C. for 15 minutes or 132° C. for 4 minutes. Remaining media is filtered through 0.2 micron filters. All pumps, lines and vessels are steam sterilized prior to use of the apparatus and all exposure of culture or cells to the external production environment is undertaken in a laminar flow cabinet in accordance with sterile technique.
- The vessel is inoculated by introducing a freshly growing culture through a previously steam-sterilised manifold in order to achieve an initial concentration of from 0.1 to 1 g per litre of cells in the vessel. Motive pressure for the transfer is provided through displacement of the inoculating culture with sterile air.
- An optical density probe is also be immersed in the tank in order to indicate the amount of biomass present in a culture. The culture vessel is also be provided with one or more settling devices, which are external separating funnels into which cells within their medium may be pumped from time to time aseptically via the operation of a peristaltic pump. These devices function by allowing cell-containing media to be pumped into them to settle whilst at the same time permitting spent media, substantially free of cells to be removed in a sterile manner. The suspension rich in settled cells located at the bottom of the settling device is then pumped back into the culture vessel via the alternate operation of a second peristaltic pump. The reduction in total medium volume in the main culture vessel is made up with fresh media, so that excretory products can be taken out of the vessel and fresh nutrients added. The dimensions and volume of the settlers is optimised via deign methodologies well know to those skilled in the art to allow the total volume of the tank to be changed over a 24 hour period via the operation of one or more settlers whilst minimizing the residence time of cells in the settlers.
- Means for providing light to the micro-algal cells during culture include one or more of: use of an optically translucent or transparent section of the vessel with a surface illuminated externally, insertion of light guides through ports from the exterior into the culture, insertion of a sterilisable light emitting device (e.g. fibre optics, conventional bulbs or LEDs). Cells can be pumped of cells, using a peristaltic pump or the like, from within the culture medium outside and along tubes that are exposed to a light source. For instance a transparent flat panel vessel illuminated using artificial light is used. Relative amounts of time spent in the main vessel and the flat panel system determine the amount of light that the cells are exposed to.
- A low continuous level of exposure using, for example, light guides may be preferable to a high intermittent exposure in an external system. Alternatively cells may be pulsed intermittently with high intensity light so as to achieve an average low intensity over the period of the culture.
- A preferred method for the production of Nitzschia laevis is the use of low intensity light providing a photosynthetically active average irradiance in the culture of less than 40 micromol photons per square meter per second,
- An even more preferred method for the production of Nitzschia laevis is the use of low intensity light providing a photosynthetically active average irradiance in the culture of 1-10 micromol photons per square meter per second.
- The biomass is harvested as a batch after 5 to 9 days of growth and subjected to extraction with supercritical dimethyl ether (DME). In this process cells are first collected and heat killed at 70° C. for 15 minutes to denature endogenous enzymes. The cells are then spray dried to form a powder with less than 10% water content. Supercritical DME (at 60° C. and 40 bar pressure) is then used to extract material from the powder and recovered. Removal of the DME leaves a tar-like extract which contains the lipids as well as pigments and other cellular material. Around 50% of the dry weight is extracted using this method. Subsequent extraction of the complex lipid mixture with supercritical CO2 may also be performed which has the effect of separating polar material (left as a residue in the CO2 process) from non-polar (dissolved in the supercritical CO2). A variety of other methods of isolating lipids are known to those skilled in the art. The total extract or isolated neutral or polar lipid fraction may be used in its own right or fatty acids recovered by direct saponification via methods well known to those skilled in the art. Further chromatographic processes as described in example one can then be utilised as necessary to further purify lipid classes or fatty acid fractions.
- Isolation of fatty acids from the Sn1 position of specific polar lipid classes is carried out by dissolving the lipid material in methanol and passing it through a column of immobilised Lecitase Ultra. Addition of hexane to the material flowing from the column isolates the fatty acids hydrolysed by the enzyme. These are then purified further as desired. Productivity of this system is between 5 and 50 mg EPA per litre per hour.
- The invention may rely on use of higher plants cells normal or transgenic organisms including but not limited to algae, fungi, and bacteria.
- A possible improvement option involves growing micro-organisms for a period under conditions in which the media is depleted of either silicate or phosphate or both in order to cause the organisms to produce more polar galactolipids in their lipid membranes, which are subsequently extracted. Preferably the nutrient limitation is imposed on the culture over the last phase of growth prior to harvesting.
- The culture may be maintained under temperatures below the previously stated 20 degrees Celsius, and above the freezing point of the culture medium.
- One option based on the postulate (whether it is adequate or not) that EPA serves to render lipid membranes more fluid involves growing the micro-organisms for a period under conditions in which the micro-organisms are cooled; perhaps as far as the freezing point of sea water (−1.8 deg C.) in order to cause the organisms to include more EPA either as lipids or as galactolipids in their lipid membranes, which are subsequently extracted. Preferably the cooling is applied to the culture over the last phase before harvesting.
- There is no reason why a therapeutic composition containing less than (for example) 20% EPA yet having substantial absence of other potentially antagonistic molecules such as DHA AA etc. should not be just as effective as a 100% pure EPA oil (not counting esters). The drive to get substantially 100% purity could be re-expressed as a desire to have substantially none of the “undesired molecules” such as DHA. Therefore, acceptance of (for example a 10 to 95% pure EPA) becomes a matter of satisfying the relevant regulatory authorities. The role of the inventors becomes a matter of exclusion of certain impurities. Further, a therapeutic composition that delivers EPA in a relatively water-soluble form (or a stable emulsion in water) has substantial formulation advantages.
- A basis for making foods, food supplements, nutraceuticals, or therapeutic preparations that rely on the EPA held in polar lipid molecules is that on oral administration of polar lipids rich in EPA to a mammal leads to a significant proportion of their fatty acids being absorbed into the blood stream via enteric lymph vessels thereby bypassing first-pass liver metabolism and thus providing greater bioavailability of the EPA.
- In addition the polar nature of the lipids renders this form of EPA, which does not behave as in the same manner as a fatty acid or ester or as a neutral lipid, easier to formulate and to administer. Polar lipids derived from microorganisms would not carry a fishy flavour of the type usually present in fish oil extracts. Galactolipids and certain phospholipids according to the invention are recognised to be of particular utility due to a combination of their high EPA content, low content of other potentially antagonistic molecules and undesirable fatty acids.
- The unique physiochemical properties of galactolipids conferred by the hydrophilicity of the polar carbohydrate head group; rendering them excellent surfactants. This latter property will allow the production of a range of EPA-only food and in particular EPA-only beverages due to the ability of the galactolipids to be dispersed as micelles and remain stable over long periods of time in aqueous oil in water solutions. In vitro techniques can accumulate commercially useful quantities of polar lipids and especially galactolipids rich in EPA at the same time as exhibiting high EPA productivity when grown under conditions according to this invention,
- An EPA-rich galactolipid that has been manufactured from a culture according to the invention may be prepared for storage, shipping and sale as a substance having one of a variety of physical forms such as a solution a suspension (feasible with water), or in a cake, a powder, granules, tablets, boluses, pills, capsules, or beads. In, for example, a powder, the galactolipid may be bound to inert particles (such as of starch) or encapsulated by means well known to those skilled in the relevant arts.
- Means to restrict oxidation of the EPA may be included in packaging such as by sparging with nitrogen. In addition microencapsulation for foods may be assisted with phospholipids (such as crude or purified lecithins) so that the EPA is protected The composition includes an effective amount of an extracted galactolipid rich in EPA and is suitable for oral ingestion either directly or after a technical process of food preparation.
- In order to deliver a recommended daily intake to a population, a beverage may be a most preferred route since beverages are consumed by most people. Due to the vulnerability of n-3 HUFA and other PUFAs to oxidative degradation it may be preferable to encapsulate the galactolipid such that it is protected from light especially sunlight, and released into the beverage shortly prior to consumption. Alternatively it may be desirable to sparge the liquid containing the galactolipid with an inert gas to prevent exposure of the EPA to oxygen. Carbon dioxide in carbonated beverages may assist dispersion of the galactolipid. Micelles may also hold dissolved carbon dioxide in the case of carbonated beverages and the composition of the micelle may be altered in order to enhance this property. In certain cases it may be preferable to add glycerol to these beverage preparations to optimise the dispersion of the galactolipids and also to provide a more desirable mouth feel for the beverage. Alternatively a water-free concentrate which would be made up by the user with water at or near the time of consumption may be distributed. Steps to minimise oxidation of the n-3 HUFA within the concentrate would have to be taken including the use of light-tight packaging, and microencapsulation of the galactolipid.
- Milk is a fairly universally consumed beverage and there are many processed variants of “plain” milk on sale. EPA-supplemented milk is made by adding an EPA-rich galactolipid during processing. The usual practice is to homogenize and pasteurize milk in a single process. In order to minimise exposure of the EPA to heat, the EPA-rich galactolipid is preferably added during or after cooling of the milk.
- Spreads may have EPA-rich materials included. Such spreads include the protein-rich type such as yeast extracts, or fat-based spreads such as aioli, butter and margarine, in which water is the dispersed phase. In manufacture, the galactolipid is added either to the fatty component or to the water component and the polar nature of the molecule assists in solubility. Spreads also include jams and jellies. The EPA-rich galactolipid may be added to a jam in the form of an emulsion. More solid preparations include sweets and chocolates. The EPA-rich galactolipid may be added as a fat-soluble component during manufacture preferably after heating in order that the galactolipid is not exposed to heat.
- EPA-rich galactolipids may be added to ice cream, for example, along with selected long-chain fatty acid molecules and optionally a phosphatidyl choline (PC) molecule as a carrier. An alternative is to transfer the EPA on to a selected PC molecule using a selective galactolipase/phospholipase. Long-chain lyso-phosphatidylcholine is a possible suitable receptor having advantages in processing and in products. The product formed may be insoluble within the enzyme system which tends to drive the equation towards its formation. Alternatively EPA-rich phosphatidylcholine isolated from the culture may be used.
- Whole-cell preparations, which may be intact, partially hydrolysed, or lysed, may be incorporated directly into spread-type foods, baking products, processed meats or other food supplements either (i) as is, or (ii) after homogenisation (by shear or pressure) or (iii) controlled enzymatic or chemical hydrolysis to aid proteolysis. The whole cell will offer useful protein and generally high HUFA levels, even if some stripping of 1000 EPA has taken place.
- The invention as described herein offers:
-
- 1. An industrially upwardly scalable process capable of creating a compound (such as EPA) that is difficult to synthesise, and so at the present time is obtained mainly from the limited resource, marine fish oil, yet is in increasing demand on account of rising populations and better awareness of the consequences of inadequate intake.
- 2. The preferred light levels (of up to about 80 μmol photons m−2 s−1 if at a steady rate, or at a higher yet equivalent rate if intermittently provided) are cheaper to provide by artificial means than the level of full sunlight, or to provide if daylight is used
- 3. A process as above for providing EPA in a variety of purities (suitable for therapeutic use) and importantly, the product includes only traces of the undesired molecule DHA.
- 4. A culture process for creating EPA from a largely heterotrophic organism that is relatively economical to manage, nourish and supply with energy in a large-scale manufacturing environment.
- 5. A process as above for co-producing EPA with triglycerides, and within polar lipid compounds (for example, galactolipids) that open up a number of possibilities for therapeutic and prophylactic administration.
- 6. Acceptable and readily ingested formulations containing useful amounts of EPA (in terms of nutritional and/or therapeutic requirements) within polar lipids.
- Finally, it will be understood that the scope of this invention as described by way of example and/or illustrated herein is not limited to the specified embodiments. Where in the foregoing description, reference has been made to specific components or integers of the invention having known equivalents, then such equivalents are included as if individually set forth. Those of skill will appreciate that various modifications, additions, known equivalents, and substitutions are possible without departing from the scope and spirit of the invention as set forth in the following claims.
Claims (4)
1. A micro-organism biomass comprising fatty acids, wherein at least 20% of the total fatty acids is EPA (eicosapentaenoic acid), more than 30% of the total fatty acids are fatty acids contained in polar lipids and more than 50% of the EPA is contained in the polar lipids.
2. The micro-organism biomass of claim 1 , wherein more than 40% of the total fatty acids are fatty acids contained in polar lipids.
3. The micro-organism biomass of claim 1 , wherein more than 50% of the total fatty acids are fatty acids contained in polar lipids.
4. The micro-organism biomass of claim 1 , wherein more than 60% of the EPA is contained in the polar lipids.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/444,027 US20170326090A1 (en) | 2006-07-05 | 2017-02-27 | Production of ultrapure epa and polar lipids from largely heterotrophic culture |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ54833906 | 2006-07-05 | ||
NZ548339 | 2006-07-05 | ||
US12/307,532 US8877465B2 (en) | 2006-07-05 | 2007-07-05 | Production of ultrapure EPA and polar lipids from largely heterotrophic culture |
PCT/NZ2007/000172 WO2008004900A1 (en) | 2006-07-05 | 2007-07-05 | Production of ultrapure epa and polar lipids from largely heterotrophic culture |
US14/531,783 US20150057353A1 (en) | 2006-07-05 | 2014-11-03 | production of ultrapure EPA and polar lipids form largely heterotrophic culture |
US15/444,027 US20170326090A1 (en) | 2006-07-05 | 2017-02-27 | Production of ultrapure epa and polar lipids from largely heterotrophic culture |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/531,783 Continuation US20150057353A1 (en) | 2006-07-05 | 2014-11-03 | production of ultrapure EPA and polar lipids form largely heterotrophic culture |
Publications (1)
Publication Number | Publication Date |
---|---|
US20170326090A1 true US20170326090A1 (en) | 2017-11-16 |
Family
ID=38894787
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/307,532 Active 2031-08-04 US8877465B2 (en) | 2006-07-05 | 2007-07-05 | Production of ultrapure EPA and polar lipids from largely heterotrophic culture |
US14/531,783 Abandoned US20150057353A1 (en) | 2006-07-05 | 2014-11-03 | production of ultrapure EPA and polar lipids form largely heterotrophic culture |
US15/444,027 Abandoned US20170326090A1 (en) | 2006-07-05 | 2017-02-27 | Production of ultrapure epa and polar lipids from largely heterotrophic culture |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/307,532 Active 2031-08-04 US8877465B2 (en) | 2006-07-05 | 2007-07-05 | Production of ultrapure EPA and polar lipids from largely heterotrophic culture |
US14/531,783 Abandoned US20150057353A1 (en) | 2006-07-05 | 2014-11-03 | production of ultrapure EPA and polar lipids form largely heterotrophic culture |
Country Status (8)
Country | Link |
---|---|
US (3) | US8877465B2 (en) |
EP (1) | EP2044208A4 (en) |
JP (1) | JP5658876B2 (en) |
AU (1) | AU2007270135B9 (en) |
CA (1) | CA2656311C (en) |
NO (1) | NO20090531L (en) |
NZ (1) | NZ573719A (en) |
WO (1) | WO2008004900A1 (en) |
Families Citing this family (82)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PL356587A1 (en) | 2000-01-19 | 2004-06-28 | Omegatech, Inc. | Solventless extraction process |
US8343753B2 (en) | 2007-11-01 | 2013-01-01 | Wake Forest University School Of Medicine | Compositions, methods, and kits for polyunsaturated fatty acids from microalgae |
ES2862336T3 (en) | 2008-09-02 | 2021-10-07 | Amarin Pharmaceuticals Ie Ltd | Pharmaceutical composition comprising eicosapentaenoic acid and nicotinic acid and methods of using the same |
LT2596786T (en) | 2009-02-10 | 2020-01-10 | Amarin Pharmaceuticals Ireland Limited | Use of eicosapentaenoic acid ethyl ester for treating hypertriglyceridemia |
DK3278665T3 (en) | 2009-04-29 | 2020-11-30 | Amarin Pharmaceuticals Ie Ltd | STABLE PHARMACEUTICAL COMPOSITION AND PROCEDURES FOR USE |
NZ771180A (en) | 2009-04-29 | 2022-07-29 | Amarin Pharmaceuticals Ie Ltd | Pharmaceutical compositions comprising epa and a cardiovascular agent and methods of using the same |
CN102625847A (en) | 2009-06-15 | 2012-08-01 | 阿马里纳制药公司 | Compositions and methods for lowering triglycerides without raising LDL-C levels in a subject on concomitant statin therapy |
US8637298B2 (en) | 2009-06-16 | 2014-01-28 | E I Du Pont De Nemours And Company | Optimized strains of yarrowia lipolytica for high eicosapentaenoic acid production |
CN101613724B (en) * | 2009-07-28 | 2011-04-06 | 嘉吉烯王生物工程(湖北)有限公司 | Method for preparing arachidonic acid (ARA) by recycling fungal meal of Mortierella alpine |
ES2732493T3 (en) * | 2009-09-18 | 2019-11-22 | Phycoil Biotechnology Int Inc | Fermentation of microalgae using controlled lighting |
MX2012003555A (en) | 2009-09-23 | 2012-07-03 | Amarin Corp Plc | Pharmaceutical composition comprising omega-3 fatty acid and hydroxy-derivative of a statin and methods of using same. |
KR101801011B1 (en) * | 2009-12-30 | 2017-11-23 | 바스프 파마 (칼라니쉬) 리미티드 | Simulated moving bed chromatographic separation process for the purification of polyunsaturated fatty acids |
AU2013204090B2 (en) * | 2009-12-30 | 2015-01-29 | Basf Pharma (Callanish) Limited | Simulated moving bed chromatographic separation process |
CA2787344C (en) | 2010-01-19 | 2018-03-20 | Dsm Ip Assets B.V. | Eicosapentaenoic acid-producing microorganisms, fatty acid compositions, and methods of making and uses thereof |
EP3583849A1 (en) * | 2010-03-04 | 2019-12-25 | Amarin Pharmaceuticals Ireland Limited | Compositions and methods for treating and/or preventing cardiovascular disease |
CN103124791B (en) | 2010-06-01 | 2016-08-24 | 帝斯曼知识产权资产管理有限公司 | Lipid and thus obtained product is extracted from cell |
AU2011262568B2 (en) | 2010-06-09 | 2015-01-29 | Fermentalg | Compositions comprising eicosapentaenoic acid suitable for high purification |
FR2964667B1 (en) * | 2010-09-15 | 2014-08-22 | Fermentalg | PROCESS FOR THE CULTURE OF MIXOTROPHIC UNICELLULAR ALGAE IN THE PRESENCE OF DISCONTINUOUS LIGHT RATIO IN THE FORM OF FLASHS |
AU2011312987A1 (en) * | 2010-10-06 | 2013-05-02 | Photonz Corporation Limited | Heterotrophic microbial production of xanthophyll pigments |
US10479969B2 (en) | 2010-10-11 | 2019-11-19 | Phycoil Biotechnology International. Inc. | Utilization of wastewater for microalgal cultivation |
US11712429B2 (en) | 2010-11-29 | 2023-08-01 | Amarin Pharmaceuticals Ireland Limited | Low eructation composition and methods for treating and/or preventing cardiovascular disease in a subject with fish allergy/hypersensitivity |
AU2011336856A1 (en) | 2010-11-29 | 2013-07-04 | Amarin Pharmaceuticals Ireland Limited | Low eructation composition and methods for treating and/or preventing cardiovascular disease in a subject with fish allergy/hypersensitivity |
CN103327982A (en) * | 2011-02-01 | 2013-09-25 | 日本水产株式会社 | Sexual function improving agent |
WO2012103685A1 (en) * | 2011-02-01 | 2012-08-09 | Nippon Suisan Kaisha, Ltd. | Sexual function improving agent |
CA2825037A1 (en) * | 2011-02-11 | 2012-08-16 | E.I. Du Pont De Nemours And Company | An eicosapentaenoic acid concentrate |
GB201111594D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | New improved process |
GB201111595D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | Improved process |
GB201111591D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | Further new process |
GB201111589D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | New modified process |
GB201111601D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | New process |
CN105410925A (en) | 2011-07-21 | 2016-03-23 | 帝斯曼知识产权资产管理有限公司 | Fatty acid compositions |
US11291643B2 (en) | 2011-11-07 | 2022-04-05 | Amarin Pharmaceuticals Ireland Limited | Methods of treating hypertriglyceridemia |
US20130131170A1 (en) | 2011-11-07 | 2013-05-23 | Amarin Pharmaceuticals Ireland Limited | Methods of treating hypertriglyceridemia |
WO2013103958A1 (en) | 2012-01-06 | 2013-07-11 | Amarin Pharmaceuticals Ireland Limited | Compositions and methods for lowering levels of high-sensitivity (hs-crp) in a subject |
FR2988098A1 (en) * | 2012-03-16 | 2013-09-20 | Fermentalg | PRODUCTION OF DOCOSAHEXAENOIC ACID IN MIXOTROPHE MODE BY NITZSCHIA |
US9719115B2 (en) | 2012-05-10 | 2017-08-01 | Kyoto University | Method for producing oxo fatty acid and rare fatty acid |
CA2916208A1 (en) | 2012-06-17 | 2013-12-27 | Matinas Biopharma, Inc. | Omega-3 pentaenoic acid compositions and methods of use |
HUE053111T2 (en) | 2012-06-29 | 2021-06-28 | Amarin Pharmaceuticals Ie Ltd | Methods of reducing the risk of a cardiovascular event in a subject on statin therapy using eicosapentaenoic acid ethyl ester |
US20150265566A1 (en) | 2012-11-06 | 2015-09-24 | Amarin Pharmaceuticals Ireland Limited | Compositions and Methods for Lowering Triglycerides without Raising LDL-C Levels in a Subject on Concomitant Statin Therapy |
WO2014074772A1 (en) | 2012-11-09 | 2014-05-15 | Heliae Development, Llc | Mixotrophic, phototrophic, and heterotrophic combination methods and systems |
WO2014074770A2 (en) | 2012-11-09 | 2014-05-15 | Heliae Development, Llc | Balanced mixotrophy methods |
KR20210059779A (en) * | 2012-12-06 | 2021-05-25 | 마티나스 바이오파마, 인코포레이티드 | Omega-3 pentaenoic acid compositions and methods of use |
SG10201709538QA (en) * | 2012-12-24 | 2017-12-28 | Qualitas Health Ltd | Eicosapentaenoic acid (epa) formulations |
US10123986B2 (en) | 2012-12-24 | 2018-11-13 | Qualitas Health, Ltd. | Eicosapentaenoic acid (EPA) formulations |
US9629820B2 (en) | 2012-12-24 | 2017-04-25 | Qualitas Health, Ltd. | Eicosapentaenoic acid (EPA) formulations |
US9814733B2 (en) | 2012-12-31 | 2017-11-14 | A,arin Pharmaceuticals Ireland Limited | Compositions comprising EPA and obeticholic acid and methods of use thereof |
US20140187633A1 (en) | 2012-12-31 | 2014-07-03 | Amarin Pharmaceuticals Ireland Limited | Methods of treating or preventing nonalcoholic steatohepatitis and/or primary biliary cirrhosis |
GB201300354D0 (en) | 2013-01-09 | 2013-02-20 | Basf Pharma Callanish Ltd | Multi-step separation process |
US9452151B2 (en) | 2013-02-06 | 2016-09-27 | Amarin Pharmaceuticals Ireland Limited | Methods of reducing apolipoprotein C-III |
US9624492B2 (en) | 2013-02-13 | 2017-04-18 | Amarin Pharmaceuticals Ireland Limited | Compositions comprising eicosapentaenoic acid and mipomersen and methods of use thereof |
US9662307B2 (en) | 2013-02-19 | 2017-05-30 | The Regents Of The University Of Colorado | Compositions comprising eicosapentaenoic acid and a hydroxyl compound and methods of use thereof |
US9283201B2 (en) | 2013-03-14 | 2016-03-15 | Amarin Pharmaceuticals Ireland Limited | Compositions and methods for treating or preventing obesity in a subject in need thereof |
US20140271841A1 (en) | 2013-03-15 | 2014-09-18 | Amarin Pharmaceuticals Ireland Limited | Pharmaceutical composition comprising eicosapentaenoic acid and derivatives thereof and a statin |
US20140275483A1 (en) * | 2013-03-15 | 2014-09-18 | Aurora Algae, Inc. | Algal oil compositions |
AU2014232168A1 (en) * | 2013-03-15 | 2015-08-20 | Aurora Algae, Inc. | Compositions of crude algal oil |
US9428711B2 (en) | 2013-05-07 | 2016-08-30 | Groupe Novasep | Chromatographic process for the production of highly purified polyunsaturated fatty acids |
US8802880B1 (en) | 2013-05-07 | 2014-08-12 | Group Novasep | Chromatographic process for the production of highly purified polyunsaturated fatty acids |
US10966968B2 (en) | 2013-06-06 | 2021-04-06 | Amarin Pharmaceuticals Ireland Limited | Co-administration of rosiglitazone and eicosapentaenoic acid or a derivative thereof |
EP3027732B1 (en) | 2013-08-01 | 2020-07-22 | Fermentalg | Methods for the production of diatom biomass |
US20150065572A1 (en) | 2013-09-04 | 2015-03-05 | Amarin Pharmaceuticals Ireland Limited | Methods of treating or preventing prostate cancer |
NL2011472C2 (en) * | 2013-09-19 | 2015-03-23 | Univ Delft Tech | Storage compound production by phototrophic diatoms. |
US9585859B2 (en) | 2013-10-10 | 2017-03-07 | Amarin Pharmaceuticals Ireland Limited | Compositions and methods for lowering triglycerides without raising LDL-C levels in a subject on concomitant statin therapy |
EP2883860B1 (en) | 2013-12-11 | 2016-08-24 | Novasep Process | Chromatographic method for producing polyunsaturated fatty acids |
KR102435269B1 (en) | 2013-12-20 | 2022-08-22 | 디에스엠 아이피 어셋츠 비.브이. | Processes for obtaining microbial oil from microbial cells |
SG11201605009RA (en) | 2013-12-20 | 2016-07-28 | Dsm Ip Assets Bv | Processes for obtaining microbial oil from microbial cells |
AR098896A1 (en) | 2013-12-20 | 2016-06-22 | Dsm Ip Assets Bv | PROCESS FOR OBTAINING MICROBIAL OIL FROM MICROBIAL CELLS |
BR112016014517B1 (en) | 2013-12-20 | 2022-06-28 | Dsm Ip Assets B.V. | PROCESS FOR OBTAINING A MICROBIAL OIL COMPRISING ONE OR MORE POLYUNSATURATED FATTY ACIDS FROM ONE OR MORE MICROBIAL CELLS |
KR102165406B1 (en) | 2014-01-07 | 2020-10-14 | 노바셉 프로세스 | Process for purifying aromatic amino acids |
JP2015146788A (en) * | 2014-02-07 | 2015-08-20 | 三菱マテリアル株式会社 | Algae culture method and algae culture apparatus |
US10561631B2 (en) | 2014-06-11 | 2020-02-18 | Amarin Pharmaceuticals Ireland Limited | Methods of reducing RLP-C |
WO2015195662A1 (en) | 2014-06-16 | 2015-12-23 | Amarin Pharmaceuticals Ireland Limited | Methods of reducing or preventing oxidation of small dense ldl or membrane polyunsaturated fatty acids |
US10406130B2 (en) | 2016-03-15 | 2019-09-10 | Amarin Pharmaceuticals Ireland Limited | Methods of reducing or preventing oxidation of small dense LDL or membrane polyunsaturated fatty acids |
JP2019518457A (en) * | 2016-06-08 | 2019-07-04 | スウェディッシュ アルガエ ファクトリー アーベー | Flasture extracted from benthic winged diatoms harvested from an industrial biofilm process |
CN105912054B (en) * | 2016-06-24 | 2018-11-20 | 国网山东省电力公司聊城供电公司 | The temperature control system and its working method of a kind of oil chromatography on-line equipment cabinet and application |
TW201900160A (en) | 2017-05-19 | 2019-01-01 | 愛爾蘭商艾瑪琳製藥愛爾蘭有限公司 | Compositions and Methods for Lowering Triglycerides in a Subject Having Reduced Kidney Function |
CN107099379A (en) * | 2017-06-09 | 2017-08-29 | 云南中烟工业有限责任公司 | A kind of Zimbabwe's pure tobacco oil for electronic cigarette |
US11058661B2 (en) | 2018-03-02 | 2021-07-13 | Amarin Pharmaceuticals Ireland Limited | Compositions and methods for lowering triglycerides in a subject on concomitant statin therapy and having hsCRP levels of at least about 2 mg/L |
FR3085962B1 (en) | 2018-09-14 | 2021-06-18 | Fermentalg | PROCESS FOR EXTRACTING AN OIL RICH IN PUFA |
FI4056176T3 (en) | 2018-09-24 | 2024-05-30 | Amarin Pharmaceuticals Ie Ltd | Methods of reducing the risk of cardiovascular events in a subject |
JP7395144B2 (en) * | 2019-04-05 | 2023-12-11 | 三菱重工機械システム株式会社 | Microalgae culture method and microalgae culture device |
US20240100168A1 (en) * | 2020-04-17 | 2024-03-28 | Wake Forest University | Lipid compositions and methods of preparation thereof |
EP4326244A1 (en) | 2021-04-21 | 2024-02-28 | Amarin Pharmaceuticals Ireland Limited | Methods of reducing the risk of heart failure |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE1969433U (en) * | 1967-04-14 | 1967-09-28 | Bernhard Dipl Ing Winkler | SIGNAGE ELEMENT. |
JPS5946225A (en) | 1982-09-09 | 1984-03-15 | Kogyo Kaihatsu Kenkyusho | Preparation of antiarteriosclerotic agent |
JPS6163624A (en) * | 1984-09-05 | 1986-04-01 | Nisshin Oil Mills Ltd:The | Production of glycolipid of high eicosapentaenoic acid content |
JPH0297373A (en) * | 1988-09-30 | 1990-04-09 | Koujiyouen:Kk | Production of beer |
JPH0297393A (en) | 1988-10-03 | 1990-04-09 | Sagami Chem Res Center | Production of phospholipid composition containing eicosapentaenoic acid using marine microorganism |
JPH02145191A (en) * | 1988-11-29 | 1990-06-04 | Tosoh Corp | Method for separating highly unsaturated fatty acid from marine microorganism |
US5244921A (en) * | 1990-03-21 | 1993-09-14 | Martek Corporation | Eicosapentaenoic acids and methods for their production |
WO1994024984A2 (en) * | 1993-04-30 | 1994-11-10 | Winget Rodner R | Anti-inflammatory compositions containing eicosapentaenoic acid bearing monogalactosyldiacylglycerol and methods relating thereto |
GB9404483D0 (en) | 1994-03-08 | 1994-04-20 | Norsk Hydro As | Refining marine oil compositions |
DE19629433A1 (en) | 1996-07-22 | 1998-01-29 | Hoechst Ag | Preparation containing omega-3 fatty acids from microorganisms as a prophylactic or therapeutic agent against parasitic diseases in animals |
JP3836231B2 (en) * | 1997-10-17 | 2006-10-25 | 日本化学飼料株式会社 | Highly unsaturated fatty acid-containing oil obtained from scallop midgut gland and method for producing the same |
AU2004235641B2 (en) * | 1999-01-27 | 2006-09-14 | Amarin Neuroscience Limited | Highly purified ethyl EPA and other EPA derivatives for psychiatric and neurological disorders |
CA2273570A1 (en) * | 1999-05-31 | 2000-11-30 | Jfs Envirohealth Ltd. | Concentration and purification of polyunsaturated fatty acid esters by distillation-enzymatic transesterification coupling |
JP4170542B2 (en) * | 1999-11-18 | 2008-10-22 | 日油株式会社 | Process for producing highly unsaturated fatty acid derivative and high-purity eicosapentaenoic acid derivative |
PL356587A1 (en) * | 2000-01-19 | 2004-06-28 | Omegatech, Inc. | Solventless extraction process |
US20050129739A1 (en) * | 2001-05-14 | 2005-06-16 | Gerhard Kohn | Production and use of a polar lipid-rich fraction containing omega-3 and/or omega-6 highly unsaturated fatty acids from microbes, genetically modified plant seeds and marine organisms |
TWI352121B (en) | 2002-10-11 | 2011-11-11 | Nippon Suisan Kaisha Ltd | Process for producing a crude oil |
GB0301701D0 (en) | 2003-01-24 | 2003-02-26 | Ensay Ltd | Psoriasis and Eicosapentaenoic acid |
WO2006031699A2 (en) * | 2004-09-10 | 2006-03-23 | Diversa Corporation | Compositions and methods for making and modifying oils |
EP1833313A2 (en) * | 2004-10-15 | 2007-09-19 | Corporation Limited Photonz | Compositions containing high omega-3 and low saturated fatty acid levels |
US20070225370A1 (en) * | 2006-03-21 | 2007-09-27 | Joar Opheim | Nutritional Compositions and Methods |
-
2007
- 2007-07-05 JP JP2009518026A patent/JP5658876B2/en active Active
- 2007-07-05 EP EP07808670A patent/EP2044208A4/en not_active Withdrawn
- 2007-07-05 CA CA2656311A patent/CA2656311C/en active Active
- 2007-07-05 AU AU2007270135A patent/AU2007270135B9/en active Active
- 2007-07-05 WO PCT/NZ2007/000172 patent/WO2008004900A1/en active Application Filing
- 2007-07-05 US US12/307,532 patent/US8877465B2/en active Active
- 2007-07-05 NZ NZ573719A patent/NZ573719A/en unknown
-
2009
- 2009-02-03 NO NO20090531A patent/NO20090531L/en not_active Application Discontinuation
-
2014
- 2014-11-03 US US14/531,783 patent/US20150057353A1/en not_active Abandoned
-
2017
- 2017-02-27 US US15/444,027 patent/US20170326090A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
US20150057353A1 (en) | 2015-02-26 |
US20100069492A1 (en) | 2010-03-18 |
NO20090531L (en) | 2009-02-03 |
CA2656311C (en) | 2016-06-21 |
NZ573719A (en) | 2011-08-26 |
AU2007270135B2 (en) | 2013-06-20 |
AU2007270135A1 (en) | 2008-01-10 |
EP2044208A1 (en) | 2009-04-08 |
US8877465B2 (en) | 2014-11-04 |
JP2009542205A (en) | 2009-12-03 |
AU2007270135B9 (en) | 2013-06-27 |
CA2656311A1 (en) | 2008-01-10 |
WO2008004900A1 (en) | 2008-01-10 |
EP2044208A4 (en) | 2012-02-22 |
JP5658876B2 (en) | 2015-01-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8877465B2 (en) | Production of ultrapure EPA and polar lipids from largely heterotrophic culture | |
Ward et al. | Omega-3/6 fatty acids: alternative sources of production | |
JP3538418B2 (en) | Method for producing docosahexaenoic acid and docosapentaenoic acid | |
JP4165827B2 (en) | Docosahexaenoic acid and compounds containing docosahexaenoic acid | |
US10844346B2 (en) | Compositions comprising eicosapentaenoic acid suitable for high purification | |
JP4522257B2 (en) | Process for producing transesterified oil or fat or triglyceride | |
KR20050055761A (en) | Process for producing microbial fat or oil having lowered unsaponifiable matter content and said fat or oil | |
CN101037641A (en) | Process for producing fat comprising triglyceride containing highly unsaturated fatty acid | |
Zhang et al. | Advances in enzyme biocatalysis for the preparation of functional lipids | |
JP4283351B2 (en) | Production method and use of novel oil and fat composition | |
JP2007089522A (en) | Method for producing fatty acid composition containing specific highly unsaturated fatty acid in concentrated state | |
KR100850646B1 (en) | Manufacturing method of phospholipid composition | |
JPH0913075A (en) | Oil and fat for diminishing lipid in blood | |
JP2006061021A (en) | Method for producing triglyceride formed out of three residues of highly unsaturated fatty acid of one kind and utilization of the same | |
Khosravi‐Darani et al. | Production of Single‐Cell Oil Containing Omega‐3 and Omega‐6 Fatty Acids | |
JPH0913077A (en) | Oil and fat highly tending to be accumulated in internal organ |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: FERMENTALG, FRANCE Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PHOTONZ CORPORATION LIMITED;REEL/FRAME:044747/0546 Effective date: 20170917 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |