AU2011202620B2 - High-quality lipids and methods for producing by enzymatic liberation from biomass - Google Patents
High-quality lipids and methods for producing by enzymatic liberation from biomass Download PDFInfo
- Publication number
- AU2011202620B2 AU2011202620B2 AU2011202620A AU2011202620A AU2011202620B2 AU 2011202620 B2 AU2011202620 B2 AU 2011202620B2 AU 2011202620 A AU2011202620 A AU 2011202620A AU 2011202620 A AU2011202620 A AU 2011202620A AU 2011202620 B2 AU2011202620 B2 AU 2011202620B2
- Authority
- AU
- Australia
- Prior art keywords
- lipid
- polyunsaturated fatty
- acid
- fatty acid
- weight percent
- 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.)
- Expired
Links
- 150000002632 lipids Chemical class 0.000 title claims abstract description 148
- 239000002028 Biomass Substances 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title abstract description 43
- 230000002255 enzymatic effect Effects 0.000 title description 10
- BHAAPTBBJKJZER-UHFFFAOYSA-N p-anisidine Chemical compound COC1=CC=C(N)C=C1 BHAAPTBBJKJZER-UHFFFAOYSA-N 0.000 claims abstract description 23
- 244000005700 microbiome Species 0.000 claims abstract description 22
- 239000000047 product Substances 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 13
- 235000013305 food Nutrition 0.000 claims abstract description 9
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 8
- 235000020244 animal milk Nutrition 0.000 claims abstract description 7
- 235000015872 dietary supplement Nutrition 0.000 claims abstract description 7
- 235000013350 formula milk Nutrition 0.000 claims abstract description 7
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 claims description 36
- 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 claims description 24
- 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 claims description 20
- YUFFSWGQGVEMMI-JLNKQSITSA-N (7Z,10Z,13Z,16Z,19Z)-docosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCCCC(O)=O YUFFSWGQGVEMMI-JLNKQSITSA-N 0.000 claims description 12
- 235000021294 Docosapentaenoic acid Nutrition 0.000 claims description 12
- 235000021342 arachidonic acid Nutrition 0.000 claims description 12
- 229940114079 arachidonic acid Drugs 0.000 claims description 12
- 235000020669 docosahexaenoic acid Nutrition 0.000 claims description 10
- 229940090949 docosahexaenoic acid Drugs 0.000 claims description 10
- 241000195493 Cryptophyta Species 0.000 claims description 7
- 241000233866 Fungi Species 0.000 claims description 7
- 150000003626 triacylglycerols Chemical class 0.000 claims description 6
- 241000235575 Mortierella Species 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 241000233675 Thraustochytrium Species 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 4
- 241000199913 Crypthecodinium Species 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 241001467333 Thraustochytriaceae Species 0.000 claims description 3
- 241000195628 Chlorophyta Species 0.000 claims description 2
- 241000206751 Chrysophyceae Species 0.000 claims description 2
- 241000199914 Dinophyceae Species 0.000 claims description 2
- 241001466451 Stramenopiles Species 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 28
- 230000001590 oxidative effect Effects 0.000 abstract description 5
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 150000001720 carbohydrates Chemical class 0.000 abstract description 2
- 230000006866 deterioration Effects 0.000 abstract 1
- 230000007515 enzymatic degradation Effects 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 40
- 108090000790 Enzymes Proteins 0.000 description 40
- 229940088598 enzyme Drugs 0.000 description 40
- 239000004094 surface-active agent Substances 0.000 description 22
- 230000008569 process Effects 0.000 description 16
- 108091005804 Peptidases Proteins 0.000 description 14
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 14
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- 230000009089 cytolysis Effects 0.000 description 10
- 238000000855 fermentation Methods 0.000 description 10
- 230000004151 fermentation Effects 0.000 description 10
- 230000003647 oxidation Effects 0.000 description 10
- 238000007254 oxidation reaction Methods 0.000 description 10
- 238000000265 homogenisation Methods 0.000 description 9
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 9
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 9
- 229940068968 polysorbate 80 Drugs 0.000 description 9
- 229920000053 polysorbate 80 Polymers 0.000 description 9
- 239000004365 Protease Substances 0.000 description 8
- 235000014113 dietary fatty acids Nutrition 0.000 description 8
- 229930195729 fatty acid Natural products 0.000 description 8
- 239000000194 fatty acid Substances 0.000 description 8
- 150000004665 fatty acids Chemical class 0.000 description 8
- 239000000796 flavoring agent Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 239000000839 emulsion Substances 0.000 description 7
- 239000003921 oil Substances 0.000 description 7
- 235000019198 oils Nutrition 0.000 description 7
- 239000004367 Lipase Substances 0.000 description 6
- 102000004882 Lipase Human genes 0.000 description 6
- 108090001060 Lipase Proteins 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 235000019634 flavors Nutrition 0.000 description 6
- 235000019421 lipase Nutrition 0.000 description 6
- 150000002978 peroxides Chemical class 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 102000005158 Subtilisins Human genes 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 210000002421 cell wall Anatomy 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 230000007717 exclusion Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 241000598397 Schizochytrium sp. Species 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- -1 and in particular Chemical class 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 125000005456 glyceride group Chemical group 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000233671 Schizochytrium Species 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Chemical class 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 235000020978 long-chain polyunsaturated fatty acids Nutrition 0.000 description 3
- 150000003904 phospholipids Chemical class 0.000 description 3
- 239000003495 polar organic solvent Substances 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 239000011786 L-ascorbyl-6-palmitate Substances 0.000 description 2
- 208000001132 Osteoporosis Diseases 0.000 description 2
- 229930182558 Sterol Natural products 0.000 description 2
- 241001491678 Ulkenia Species 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 108010089934 carbohydrase Proteins 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000003412 degenerative effect Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 150000004668 long chain fatty acids Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 229920001296 polysiloxane Polymers 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 229920005573 silicon-containing polymer Polymers 0.000 description 2
- 239000000344 soap Substances 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 235000003702 sterols Nutrition 0.000 description 2
- 229930003799 tocopherol Natural products 0.000 description 2
- 239000011732 tocopherol Substances 0.000 description 2
- 125000002640 tocopherol group Chemical class 0.000 description 2
- 235000019149 tocopherols Nutrition 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- GJJVAFUKOBZPCB-ZGRPYONQSA-N (r)-3,4-dihydro-2-methyl-2-(4,8,12-trimethyl-3,7,11-tridecatrienyl)-2h-1-benzopyran-6-ol Chemical class OC1=CC=C2OC(CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)(C)CCC2=C1 GJJVAFUKOBZPCB-ZGRPYONQSA-N 0.000 description 1
- ZORQXIQZAOLNGE-UHFFFAOYSA-N 1,1-difluorocyclohexane Chemical compound FC1(F)CCCCC1 ZORQXIQZAOLNGE-UHFFFAOYSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 description 1
- QAQJMLQRFWZOBN-UHFFFAOYSA-N 2-(3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxyethyl hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)C1OC(=O)C(O)=C1O QAQJMLQRFWZOBN-UHFFFAOYSA-N 0.000 description 1
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 239000004261 Ascorbyl stearate Substances 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical class OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 240000004355 Borago officinalis Species 0.000 description 1
- 235000007689 Borago officinalis Nutrition 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000011293 Brassica napus Nutrition 0.000 description 1
- 239000004255 Butylated hydroxyanisole Substances 0.000 description 1
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical class CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 241000283153 Cetacea Species 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 241000199912 Crypthecodinium cohnii Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 150000000996 L-ascorbic acids Chemical class 0.000 description 1
- 235000000072 L-ascorbyl-6-palmitate Nutrition 0.000 description 1
- QAQJMLQRFWZOBN-LAUBAEHRSA-N L-ascorbyl-6-palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](O)[C@H]1OC(=O)C(O)=C1O QAQJMLQRFWZOBN-LAUBAEHRSA-N 0.000 description 1
- 229940127470 Lipase Inhibitors Drugs 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 241000907999 Mortierella alpina Species 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000219925 Oenothera Species 0.000 description 1
- 235000004496 Oenothera biennis Nutrition 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 208000020547 Peroxisomal disease Diseases 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 208000005107 Premature Birth Diseases 0.000 description 1
- 244000178231 Rosmarinus officinalis Species 0.000 description 1
- 241000700141 Rotifera Species 0.000 description 1
- 108010056079 Subtilisins Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- JPBAVLUULZJFFO-JENHRLMUSA-N [(2s)-2-[(2r)-3,4-dihydroxy-5-oxo-2h-furan-2-yl]-2-hydroxyethyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](O)[C@H]1OC(=O)C(O)=C1O JPBAVLUULZJFFO-JENHRLMUSA-N 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 206010064930 age-related macular degeneration Diseases 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001773 anti-convulsant effect Effects 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229960003965 antiepileptics Drugs 0.000 description 1
- 239000002518 antifoaming agent Chemical class 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010385 ascorbyl palmitate Nutrition 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- 235000012216 bentonite Nutrition 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 235000019282 butylated hydroxyanisole Nutrition 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000004426 flaxseed Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- CQDGTJPVBWZJAZ-UHFFFAOYSA-N monoethyl carbonate Chemical compound CCOC(O)=O CQDGTJPVBWZJAZ-UHFFFAOYSA-N 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229940068965 polysorbates Drugs 0.000 description 1
- 201000011461 pre-eclampsia Diseases 0.000 description 1
- 208000037803 restenosis Diseases 0.000 description 1
- 238000007127 saponification reaction Methods 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000001593 sorbitan monooleate Substances 0.000 description 1
- 235000011069 sorbitan monooleate Nutrition 0.000 description 1
- 229940035049 sorbitan monooleate Drugs 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 229930003802 tocotrienol Natural products 0.000 description 1
- 239000011731 tocotrienol Substances 0.000 description 1
- 235000019148 tocotrienols Nutrition 0.000 description 1
- 229940068778 tocotrienols Drugs 0.000 description 1
- STCOOQWBFONSKY-UHFFFAOYSA-N tributyl phosphate Chemical compound CCCCOP(=O)(OCCCC)OCCCC STCOOQWBFONSKY-UHFFFAOYSA-N 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000012178 vegetable wax Substances 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
- 150000003735 xanthophylls Chemical class 0.000 description 1
- 235000008210 xanthophylls Nutrition 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Detergent Compositions (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A high-quality lipid composition is disclosed having low oxidative deterioration such as measured by low anisidine values. Also disclosed are methods of preparing the same from a lipid-containing material that include enzymatic degradation of protein and/or carbohydrate 5 components of the material. Lipid-containing materials include biomass, such as microorganisms. The invention further includes products containing the lipid compositions, such as dietary supplements, food products, pharmaceutical formulations, humanized animal milk, and infant formula.
Description
AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention title: High-quality lipids and methods for producing by enzymatic liberation from biomass The following statement is a full description of this invention, including the best method of performing it known to us: 2 HIGH-QUALITY LIPIDS AND METHODS FOR PRODUCING BY ENZYMATIC LIBERATION FROM BIOMASS 5 FIELD OF THE INVENTION The present invention is directed to high-quality lipids, and in particular, lipids with low anisidine values. Methods are provided for producing high-quality lipids that include the step of liberating lipids from biomass, such as algal biomass, using enzymatic treatment. 10 BACKGROUND OF THE INVENTION Various methods have been employed for extracting lipids from biomass. Techniques include direct extraction of the biomass with solvents, heating, pressure waves generated via electric arcs, direct saponification via KOH and ethanol, sonication, freezing and grinding and bead mills. For example, the biomass can be dried and the lipid extracted with a solvent such 15 as hexane. Alternatively, a microbial fermentation broth can be subjected to extreme conditions of pH and/or temperature or additional equipment such as a homogenizer can be used to disrupt the cells. Problems with prior methods include poor product quality due to chemically aggressive conditions of high temperature and high pH, high costs due to the need to dry the 20 biomass or for additional equipment such as homogenizers and pressure vessels. The "fishy" and "painty" flavors associated with many polyunsaturated fatty acids (PUFAs) found in lipids are primarily due to oxidation of the double bonds in the fatty acids. These flavor and odor notes are normally considered defects that can preclude their use in foods or other applications. The oxidative state and stability of a lipid or lipid-containing 25 material can be measured in a number of ways. Standard measurement techniques include "anisidine value," "peroxide value," "oxidative stability index," "Rancimat," and gas chromatograph headspace analysis for oxidation products. Information on these different techniques is available from the AOCS (American Oil Chemists' Society) as well as from other sources. 30 The oxidative state of the lipid or lipid-containing material is strongly impacted by the processing conditions used to make the material. For food materials, the conditions during processing as well as the actual ingredients and quality of the ingredients will affect the 3 oxidation state. For fermentation-derived lipids (e.g., lipids obtained from 5 microbes grown in fermentors, ponds, etc.), the ingredients (fermentation and postfermentation) used as well as the conditions during the lipid extraction and fermentation will affect the quality. Other sources of PUF As, such as agricultural crops and animal sources, will also be affected by the 5 processing conditions used to obtain the lipids and lipid-containing materials. SUMMARY OF THE INVENTION In a first aspect, the present invention provides a refined, bleached, or deodorized microbial or plant lipid comprising polyunsaturated fatty acid, wherein the lipid has an 10 anisidine value of 1.5 or less, wherein the lipid has been liberated from biomass enzymatically, and wherein the polyunsaturated fatty acid present in the lipid comprises at least 20 weight percent docosahexaenoic acid, at least 5 weight percent docosapentaenoic acid, or at least 20 weight percent arachidonic acid. Preferably, the lipid anisidine value is 1 or less. More preferably, the lipid anisidine 15 value is 0.5 or less. Preferably, the polyunsaturated fatty acid is docosahexaenoic acid or docosapentaenoic acid or arachidonic acid. Preferably, the polyunsaturated fatty acid comprises at least 30 weight percent docosahexaenoic acid or at least 35 weight percent docosahexaenoic acid. 20 Preferably, the polyunsaturated fatty acid comprises at least 10 weight percent docosapentaenoic acid or at least 15 weight percent docosapentaenoic acid or at least 20 weight percent docosapentaenoic acid. Preferably, the polyunsaturated fatty acid comprises at least 30 weight percent arachidonic acid or at least 40 weight percent arachidonic acid or at least 50 weight percent 25 arachidonic acid. In a second aspect, the present invention provides a product selected from the group consisting of dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, wherein the product comprises the lipid of the first aspect of the present invention. 30 In a third aspect, the present invention provides a method of making a product selected from the group consisting of a dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, comprising adding the lipid of the 4 first aspect of the present invention to the dietary supplement, food product, pharmaceutical formulation, humanized animal milk, or infant formula. In accordance with another embodiment of the present invention, provides a lipid comprising polyunsaturated fatty acid wherein the lipid has an anisidine value of 2 or less, and 5 in various embodiments the anisidine value can be as low as 0.3 or less. The polyunsaturated fatty acid in the lipid 15 can be a long chain polyunsaturated fatty acid, having a chain length of at least 20 or at least 22, and can have at least three or at least four double bonds. More particularly, the polyunsaturated fatty acid can be docosahexaenoic acid, docosapentaenoic acid, or arachidonic acid. 10 The lipid can be obtained from biomass, for example, from a plant or 20 microorganism. For example, the lipid can be obtained from algae, bacteria, fungi or protists. In preferred embodiments, the lipid can be obtained from microorganisms of the genus Mortierella, genus Crypthecodinium, or order Thraustochytriales. Further, the lipid can comprise a monoacylglyceride, a diacylglyceride, or a triacylglyceride. 15 Further embodiments of the present invention include products selected from 25 dietary supplements, food products, pharmaceutical formulations, humanized animal milk or infant formula wherein the products include a lipid comprising polyunsaturated fatty acid and having an anisidine value of 2 or less. A further embodiment of the present invention is a method of obtaining a 20 polyunsaturated fatty acid-containing lipid which includes providing a biomass 30 containing a polyunsaturated-containing fatty acid, contacting the biomass with an enzyme, and recovering the lipid. A further method of the present invention is a method for liberating a lipid from a biomass comprising liberating the lipid at a temperature of about lOC to about 80C at a 25 pH level of from about pH 5 to about pH 9 This method is conducted in the substantial absence of an extraction solvent. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not 30 the exclusion of any other element, integer or step, or group of elements, integers or steps. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for 4a the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this specification. 5 DETAILED DESCRIPTION OF THE INVENTION In accordance with one embodiment of the present invention, a high-quality lipid is provided. In particular, the lipid has a low anisidine value. Preferably, the anisidine value is 2 or less, more preferably 1.5 or less, more preferably I or less, more preferably 0.5 or less and more preferably 0.3 or less. Anisidine value can be thought of as a measure of the 10 oxidative history of a lipid. Higher values indicate a lipid that has experienced more oxidative stress. As a lipid is oxidized, it is typically converted to a peroxide. This peroxide typically gets converted to an aldehyde or ketone. The anisidine value is a measure of these secondary oxidation products. Polyunsaturated fatty acid-containing lipids are very sensitive to oxidation and this oxidation can lead to off-flavors. Methods have been employed to remove these off 15 flavors, but these methods do not remove all of the oxidation products that can then act as off flavor, and oxidation, precursors. As a result, these flavor improvement methods only lead to temporary improvement of the flavor. The anisidine value is a measurement of these oxidation and off-flavor precursors. The analytical method for measuring anisidine value is available from the AOCS (American Oil Chemists' Society). 20 In accordance with another embodiment of the present invention, a process for liberating lipids is provided. The process includes a step of liberating lipids using an enzymatic treatment for, e.g., degradation of cell walls of the lipid-containing material. Preferably, the lipids are liberated from a lipid-containing biomass using a protease enzyme whereby protein components of the lipid-containing material are proteolyzed, or other 25 enzyme that is appropriate for breaking down or reacting with the lipid-containing material. A further embodiment of the present invention is a process that utilizes surfactants in addition to the enzymes to liberate the lipids from the lipid-containing material. The inventors have surprisingly found that the use of surfactants together with enzymatic treatment can allow for milder reaction conditions than with enzymes alone for liberation of the lipids. 30 Surfactants, such as Polysorbate 80, mono- and diglycerides, or other surfactants, are preferably added at approximately the same time as the enzyme. Alternatively, surfactant can be added before or after the enzyme. In this embodiment, 5 the lipid is preferably liberated from the biomass without using extreme conditions of temperature or pH and without using additional equipment such as a homogenizer or drying the biomass prior to lipid removal. For example, the enzymatic treatment can be conducted at temperatures below about 80C, more preferably below about 70C, and even 5 more preferably, below about 65C, and at pH conditions of approximately 5-9. The use of protease enzymes, or protease enzymes in combination with surfactants, provides an economical and simple way of releasing the lipid from the biomass under mild conditions conducive to making high-quality lipid. The lipid can then be isolated from the rest of the fermentation broth by centrifugation of the mixture. 10 In some cases, the lipid will be incorporated into an emulsion. For some applications, the emulsion itself might be the final product. For other applications, the emulsion would be treated to release the lipid for recovery separately. Techniques are taught in U.S. Patent Application No. 09/766,500 and include, but are not limited to, dilution, addition of a solvent, temperature shifts, and freezing. 15 The use of a protease enzyme can help break down emulsion-stabilizing proteins present, thereby aiding in the breaking of an emulsion. In addition, the successful use of a protease for lipid liberation from microalgae is surprising because, microalgae tend to have a low protein content (-15-22% compared to -55% for E. coli), and have very robust cellular structure due to the presence of silica and polysaccharides such as 20 cellulose. This processing step also allows the production of lipids containing long chain polyunsaturated fatty acids (PUFAs) of exceedingly high quality as measured by anisidine value. The reason is that the process can easily be done under an inert atmosphere, with low temperatures, and non-reactive conditions. 25 In a particular embodiment, using proteolytic treatment of the lipid-containing material without a surfactant, the proteolytic treatment can be conducted at higher temperatures, sufficient to achieve desirable levels of lipid liberation. For example, in this embodiment, the enzymatic treatment is conducted at temperatures of at least about 30C, more preferably at least about 40C, and most preferably at least about 50C. It 30 should be recognized however, that at higher temperatures, degradation of lipids can occur. Therefore, a temperature must be selected such that adequate lipid liberation is achieved without unacceptable levels of lipid degradation.
6 Preferred polyunsaturated fatty acid sources can be any sources that are capable of liberation by enzymes as in the present invention. Preferred polyunsaturated fatty acids sources include biomass sources, such as animal, plant and/or microbial sources. As used herein, the term "lipid" includes phospholipids; free fatty acids; esters of fatty acids; 5 triacylglycerols; diacylglycerides; monoacylglycerides; lysophospholipids; soaps; phosphatides; sterols and sterol esters; carotenoids; xanthophylls (e.g., oxycarotenoids); hydrocarbons; and other lipids known to one of ordinary skill in the art. Examples of animal sources include aquatic animals (e.g., fish, marine mammals, crustaceans, rotifers, etc.) and lipids extracted from animal tissues (e.g., brain, liver, eyes, etc.). Examples of 10 plant sources include macroalgae, flaxseeds, rapeseeds, corn, evening primrose, soy and borage. Examples of microorganisms include algae, protists, bacteria and fungi (including yeast). The use of a microorganism source, such as algae, can provide organoleptic advantages, i.e., fatty acids from a microorganism source may not have the fishy taste and smell that fatty acids from a fish source tend to have. More preferably, the 15 long-chain fatty acid source comprises algae. Preferably, when microorganisms are the source of long-chain fatty acids, the microorganisms are cultured in a fermentation medium in a fermentor. Alternatively, the microorganisms can be cultured photosynthetically in a photobioreactor or pond. Preferably, the microorganisms are lipid-rich microorganisms, more preferably, the 20 microorganisms are selected from the group consisting of algae, bacteria, fungi and protists, more preferably, the microorganisms are selected from the group consisting of golden algae, green algae, dinoflagellates, yeast, fungi of the genus Mortierellaand Stramenopiles. Preferably, the microorganisms comprise microorganisms of the genus Crypthecodinium and order Thraustochytriales and filamentous fungi of the genus 25 Mortierella, and more preferably, microorganisms are selected from the genus Thraustochytrium, Schizochytrium or mixtures thereof, more preferably, the microorganisms are selected from the group consisting of microorganisms having the identifying characteristics of ATCC number 20888, ATCC number 20889, ATCC number 20890, ATCC number 20891 and ATCC number 20892, strains of Mortierella 30 schmuckeri and Mortierella alpina, strains of Crypthecodinium cohnii, mutant strains derived from any of the foregoing, and mixtures thereof. It should be noted that many experts agree that Ulkenia is not a separate genus from the genera Thraustochytrium and Schizochytrium. Accordingly, as used herein, the genera Thraustochytrium and 7 Schizochytrium will include Ulkenia. Information regarding such algae can be found in U.S. Patent Nos. 5,407,957, 5,130,242 and 5,340,594, which are incorporated herein by reference in their entirety. Lipids recovered by the present invention include lipids comprising a 5 polyunsaturated fatty acid, more particularly, a long chain polyunsaturated fatty acid, and even more particularly, a polyunsaturated fatty acid present in said lipid having a carbon chain length of at least 20 or 22. Such polyunsaturated fatty acid present can have at least 3 or at least 4 double bonds. More particularly, the polyunsaturated fatty acid can include docosahexaenoic acid (at least 10, 20, 30 or 35 weight percent), docosapentaenoic acid (at 10 least 5, 10, 15, or 20 weight percent), and/or arachidonic acid (at least 20, 30, 40 or 50 weight percent). Polyunsaturated fatty acids include free fatty acids and compounds comprising PUFA residues, including phospholipids; esters of fatty acids; triacylglycerols; diacylglycerides; monoacylglycerides; lysophospholipids; phosphatides; etc. 15 For different oil-containing materials, different enzymes and reaction conditions can be employed. For these different materials, an important enzyme selection criterion is to select an enzyme that will attack and degrade a portion of the material (such as the proteins, polysaccharides, cell wall, cell outer membrane, peptidoglycan layer, cellulose, chitin, hemicellulose, lignin, lignin-related compounds, etc.) that is otherwise impeding 20 recovery of the oil. Preferably, nonspecific protease enzymes such as trypsin, chymotrypsin, or the like are used to degrade protein components of the oil-containing materials and carbohydrase enzymes such as amylase can be used to degrade carbohydrate components of the oil-containing materials. The selection of reaction conditions, including enzyme type, enzyme concentration, temperature, pH, water 25 activity, other reagent concentration, reaction time, etc. will depend in part on the specific enzyme and material that the lipid is being liberated from. These conditions can be readily determined from information about the enzyme (and typically available from the supplier or in the literature), or determined by somebody skilled in the art. Typical temperatures may range between approximately 20-80*C, although some special enzymes 30 may be sufficiently active and stable for use outside of this range. Typical enzyme concentrations can be as low as 0.01% to several percent. The reaction rate is related to the enzyme concentration with higher concentrations allowing for shorter reaction times. In some situations, it may be possible to use an even lower concentration, such as when a 8 particular enzyme is extremely active or stable or when very long reaction times may be practical. Preferably the lipids are effectively liberated from Schizochytrium sp organisms by treating the cells with a protease enzyme. It is surprising that this particular class of 5 enzymes is effective for this organism due to the relatively small amount of protein normally found in the cell wall of this organism. Lipids can be liberated from biomass, and preferably microorganisms, by treating the cells with enzymes or other agents or by other methods that attack other components of the cell wall, such as polysaccharides, or the lipid bilayer. This treatment with enzymes provides one method under mild 10 conditions that allows for recovery of high quality lipids. Other methods for liberation of lipids that can be used, alone or in combination with enzymatic treatment, include treatment with detergents, osmotic shock, freezing/thaw cycling, autolysis,. homogenization, sonication, and mild heat treatment.) One preferred embodiment of the process of the present invention includes: 15 . Obtaining lipid-bearing single cell organisms . Treating with protease or a combination of surfactant and protease . Separating the lipid from the broth (may be an emulsion) o May require additional treatment with a polar organic solvent, salt, precipitating agent, another enzyme (protease or other kind), heating, 20 cooling. . If the lipid from the above step is in the form of an emulsion, this product can be used "as is" or dried and used or treated to release the lipid from the emulsion o Treatment can include treatment with a polar organic solvent, salt, precipitating agent, another enzyme (protease or other kind), heating, 25 cooling, etc. . The lipid can then be dried, refined, bleached, deodorized and/or reacted as needed. The lipid can also be treated with antioxidants and/or metal ion capturing agents (such as chelating agents, ion exchange resin, precipitating agents) at any point before, during 30 or after the process. As noted above, the present invention encompasses the use of a protease in the presence of a surfactant to recover lipid from a biomass.Suitable surfactants include, but are not limited to: phospholipid, lysophospholipid, monoglyceride, diglycerides, mixed 9 glycerides, partial glycerides, soaps, fatty acids, salts of fatty acids, amines, antifoam, acids or salts of sulfonic acid, detergents, polysorbates (e.g., polyethylene sorbitan monooleate), aliphatic acids and esters, polar organic molecules, alcohols, sulfates and sulfonates, nitrogen-containing compounds (e.g. amines, amides, polyamides), 5 phosphates (e.g. alkyl-alkylene diphosphate, tributyl phosphate), silicones (e.g. tri- and tetra-alkyl silanes, silicone polymer containing silica, dimethyl silicone polymers, methyl silicones), sulfides and thio derivatives, halogenated compounds, triacylglycerols, long chain fatty waxes (e.g. vegetable oils and waxes), mineral oils, sulfated derivative of triacylglycerols and mineral oils, bentonite, and monosodium phosphate mixed with boric 10 acid and ethyl carbonate. In a further embodiment, the process can be conducted with a combination of enzymes. More specifically, a protease and a lipase can be used. A lipase is an enzyme that hydrolyzes glycerides. Therefore, care needs to be taken to avoid unacceptable levels of degradation of glycerides in the lipid product. For example, a lipase will hydrolyze a 15 triglyceride producing a free fatty acid and a diglyceride. This mechanism is believed, without intending to be bound by theory, to be beneficial to an extent because products of the enzymatic. degradation function as surfactants having the benefits described above in the embodiment of the invention involving direct use of surfactants. However, there is the potential that the lipid product could be unacceptably degraded by the lipase. 20 Therefore, additional embodiments involve the use of small amounts of lipase or conditions under which the lipase is only active a small amount of the time. Such control of lipase activity could be controlled for example, by the use of temperature sensitive enzymes or the introduction of lipase inhibitors. In another embodiment of the present invention, the processes of the present 25 invention are combined with further oxidation-reducing techniques, including one or more of: exclusion of air (and oxygen) and other oxidizing agents, processing with mild conditions (moderate temperature, moderate pH, short processing times, etc.), exclusion of metal ions such as copper and iron, exclusion of previously oxidized lipids (even if subsequently purified), exclusion of oxidation precursors, and the presence of antioxidant 30 compounds (such as tocopherols, tocotrienols, BHA, carnisol, camosic acid, ascorbic acid, L-ascorbic acid esters (including L-ascorbyl palmitate, L-ascorbyl stearate, L ascorbyl oleate), rosemary, etc. as well as esters or derivatives of these compounds), to obtain minimally oxidized lipids.
10 In some cases, after the lipids are liberated from the biomass, the lipids can be separated directly from the undesired materials (e.g., cellular debris), such as by centrifugation, or other appropriate methods. In other cases, an agent such as an alcohol or other polar organic solvent can be added to facilitate the separation of the liberated 5 lipid from the other material. In still other cases, a solvent can be added that will dissolve the lipid and facilitate the separation of the liberated lipid from the other material, e.g., by solvent extraction. Techniques for separating the lipids from undesired materials can be found in U.S. Patent Number 5,928,696, U.S. Patent Application Number 09/766,500, and PCT Application Numbers US01/12047 and US01/12049, all of which are 10 incorporated herein by reference in their entirety.Another embodiment of the invention involves the use of a combination of the enzyme treatment, or the enzyme plus surfactant treatment, along with homogenization. This combination in some cases can achieve higher quality and/or higher yield than with homogenization or enzyme treatment alone. It is believed that homogenization can facilitate the enzyme reaction by allowing more 15 intimate contact between the enzyme and its substrate. It is also believed that enzyme treatment can facilitate homogenization by weakening the cell walls and allowing the use of less extreme (pressure or shear) homogenization conditions. The use of homogenization with the enzyme or enzyme-surfactant process can allow the use of conditions that are more chemically mild than would be possible without the 20 homogenization. In other cases, this combined process can allow use of a lower pressure (and also lower cost) homogenization. In accordance with a further embodiment of the present invention, the processes previously described can be employed on lipid-bearing material that has been dried prior to lipid removal. While the highest quality and lowest cost process would normally be 25 expected from material that has not been dried, there are cases where it would be advantageous to dry the material either prior to or at some intermediate point during the process, prior to lipid separation. Use of the previously described processes with drying can provide a partial improvement in quality and/or cost over processes that include drying and do not include the invented processes. Some examples of when this drying 30 step would be appropriate are when the facility for lipid separation is located remote from the fermentation or other upstream facility, or when there are scheduling difficulties between the lipid separation facility and the upstream facility, or when the lipid containing material must be stored prior to separating the lipid.
11 In one aspect of the present invention, the lipid is used in an endproduct selected from the group consisting of a dietary supplement, a food product, a pharmaceutical formulation, a humanized animal milk, and an infant formula. A pharmaceutical formulation can include, but is not limited to: an anti-inflammatory formulation, a 5 chemotherapeutic agent, an active excipient, an osteoporosis drug, an anti-depressant, an anti-convulsant, an anti-Helicobactor pylori drug, a drug for treatment of neurodegenerative disease, a drug for treatment of degenerative liver disease, an antibiotic, and a cholesterol lowering formulation. In one aspect, the endproduct is used to treat a condition selected from the group consisting of: chronic inflammation, acute 10 inflammation, gastrointestinal disorder, cancer, cachexia, cardiac restenosis, neurodegenerative disorder, degenerative disorder of the liver, blood lipid disorder, osteoporosis, osteoarthritis, autoimmune disease, preeclampsia, preterm birth, age related maculopathy, pulmonary disorder, schizophrenia, depression, weight maintenance and peroxisomal disorder. The following examples are provided for the purpose of 15 illustration and are not intended to limit the scope of the present invention. EXAMPLES Example 1. Schizochytrium sp. fermentation broth was diluted and buffered as follows: 25 ml 20 of broth was combined with 65 ml DI water, then .10 ml of pH 6.0 buffer (1.0 M 2-[N morpholino] ethanesulfonic acid) was added. To different aliquots of this broth mixture different combinations of enzyme and surfactant were added. After the enzyme and surfactant additions, the samples were incubated at 45 C for 1.5 hr, and then examined by microscope for degree of lysis. The 25 results are shown below: 12 Enzyme* Surfactant Degree of lysis None Polysorbate 80 No lysis Viscozyme@ L Polysorbate 80 No lysis Alcalase@ 2.4L FG, Polysorbate 80 Mostly lysed Viscozyme@ L Alcalase@ 2.4L FG Polysorbate 80 Virtually all lysed Viscozyme® L None No lysis Alcalase@ 2.4L FG None Mostly unlysed *The enzymes are both from Novozymes North America, Inc. of Franklinton, NC. This example demonstrates both the successful lysis of the organism with 5 enzymes and the improvement of lysis with the inclusion of a surfactant, Polysorbate 80. Example 2. Schizochytrium sp. fermentation broth was diluted and buffered as in Example 1. A commercial protease (Alcalase@ 2.4 L FG, available from Novozymes North 10 America, Inc. of Franklinton, NC) and a commercial carbohydrase (Viscozyme@ L, available from Novozymes North America, Inc. of Franklinton, NC) were added to the diluted and buffered broth. This broth mixture was divided and different. surfactants were added as follows: 1. Polysorbate 80 15 2. Sodium lauryl sulfate 3. Mono and diglycerides (Dimodan CO-K from Danisco of New Century, KA) After the surfactant addition, each sample was heated in a hot water bath (75 C) for approx. 5 min. Each sample was then held overnight at room temperature with mixing on a Fisher Hematology/Chemistry Mixer. The samples were examined under a microscope 20 for degree of lysis. The results are shown below: Surfactant Degree of lysis Polysorbate 80 -100% Sodium lauryl sulfate ~40-60% Dimodan CO-K ~100% 13 This example demonstrates that different surfactants can be used. In this case both Polysorbate 80 and mono and diglycerides (Dimodan) were successful. Sodium lauryl sulfate was not as successful due to this particular chemical attacking the enzymes. 5 Example 3. Schizochytrium sp. fermentation broth was treated with antioxidants (ascorbyl palmitate and tocopherols) and drum dried. This dried biomass was then treated as follows: 10 . Added to distilled water (51 g of biomass to 300 g water) . The pH was adjusted to the range of 6.9-7.3 with 2N H2SO4 - The mixture was heated to 60 C in a water bath . 1.5 ml of Alcalase 2.4L FG was added . The broth mixture was then purged with nitrogen to exclude oxygen and incubated 15 at 60C for 15 hours . 120 ml of isopropanol (99.9%) was added with gentle mixing . The broth-alcohol mixture was then centrifuged at 4000 RPM for 5 minutes . The lipid phase (supernatant) was collected The collected lipid was tested for anisidine and peroxide value per AOCS 20 (American Oil Chemists Society) methods Cd 8-53 and Cd 18-90. A sample of the dried biomass was also hexane extracted by combining with hexane and ball milled in a Swedish tube extraction system. The lipid collected was tested for anisidine value and peroxide value as the other lipid sample. The test results of the lipid collected by the two methods are shown below: 25 Sample Peroxide Value Anisidine Value Enzyme, isopropanol <0.1 0.8 method Hexane extracted <0.1 3.0 This example demonstrates the successful lysis of the cells with enzymes, the isolation of the lipid that was present in the cells and the very high quality of the lipid (very low anisidine value).
14 The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other 5 variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or 10 equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.
Claims (20)
1. A refined, bleached, or deodorized microbial or plant lipid comprising polyunsaturated fatty acid, wherein the lipid has an anisidine value of 1.5 or less, wherein the lipid has been liberated from biomass enzymatically, and wherein the polyunsaturated fatty acid 5 present in the lipid comprises at least 20 weight percent docosahexaenoic acid, at least 5 weight percent docosapentaenoic acid, or at least 20 weight percent arachidonic acid.
2. The lipid of claim 1, wherein the lipid anisidine value is 1 or less.
3. The lipid of claim 1, wherein the lipid anisidine value is 0.5 or less.
4. The lipid of claim 1, wherein the polyunsaturated fatty acid is docosahexaenoic acid. 10
5. The lipid of claim 1, wherein the polyunsaturated fatty acid is docosapentaenoic acid.
6. The lipid of claim 1, wherein the polyunsaturated fatty acid is arachidonic acid.
7. The lipid of claim 1, wherein the polyunsaturated fatty acid comprises at least 30 weight percent docosahexaenoic acid.
8. The lipid of claim 1, wherein the polyunsaturated fatty acid comprises at least 35 15 weight percent docosahexaenoic acid.
9. The lipid of claim 1, wherein the polyunsaturated fatty acid comprises at least 10 weight percent docosapentaenoic acid.
10. The lipid of claim 1, wherein the polyunsaturated fatty acid comprises at least 15 weight percent docosapentaenoic acid. 20
11. The lipid of claim 1, wherein the polyunsaturated fatty acid comprises at least 20 weight percent docosapentaenoic acid.
12. The lipid of claim 1, wherein the polyunsaturated fatty acid comprises at least 30 weight percent arachidonic acid.
13. The lipid of claim 1, wherein the polyunsaturated fatty acid comprises at least 40 25 weight percent arachidonic acid.
14. The lipid of claim 1, wherein the polyunsaturated fatty acid comprises at least 50 weight percent arachidonic acid.
15. The lipid of anyone of claims 1-14, wherein the lipid is obtained from at least one of the group consisting of algae, bacteria, fungi and protists. 30 16. The lipid of any one of claims 1-15, wherein the lipid is obtained from microorganisms selected from the group consisting of golden algae, green algae, dinoflagellates, yeast, fungi of the genus Mortierella, and Stramenopiles.
16
17. The lipid of any one of claims 1-15, wherein the lipid is obtained from microorganisms selected from the group consisting of the genus Mortierella, genus Crypthecodinium, and order Thraustochytriales.
18. The lipid of any one of claims 1-15, wherein the lipid is obtained from microorganisms 5 selected from the group consisting of the genus Thraustochytrium, genus Schkochytrium or mixtures thereof.
19. The lipid of any one of claims 1-18, wherein the lipid comprises a triacylglyceride.
20. A product selected from the group consisting of dietary supplement, food product, pharmaceutical formulation, humanized animal milk, and infant formula, wherein the 10 product comprises the lipid of any one of claims 1-19.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2011202620A AU2011202620B2 (en) | 2002-05-03 | 2011-06-02 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
AU2013273736A AU2013273736B2 (en) | 2002-05-03 | 2013-12-19 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
AU2015243079A AU2015243079B2 (en) | 2002-05-03 | 2015-10-16 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US60/377,550 | 2002-05-03 | ||
AU2008252072A AU2008252072B2 (en) | 2002-05-03 | 2008-12-04 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
AU2011202620A AU2011202620B2 (en) | 2002-05-03 | 2011-06-02 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2008252072A Division AU2008252072B2 (en) | 2002-05-03 | 2008-12-04 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2013273736A Division AU2013273736B2 (en) | 2002-05-03 | 2013-12-19 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
Publications (2)
Publication Number | Publication Date |
---|---|
AU2011202620A1 AU2011202620A1 (en) | 2011-06-23 |
AU2011202620B2 true AU2011202620B2 (en) | 2013-09-26 |
Family
ID=45398519
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2011202620A Expired AU2011202620B2 (en) | 2002-05-03 | 2011-06-02 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
AU2013273736A Ceased AU2013273736B2 (en) | 2002-05-03 | 2013-12-19 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
AU2015243079A Expired AU2015243079B2 (en) | 2002-05-03 | 2015-10-16 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2013273736A Ceased AU2013273736B2 (en) | 2002-05-03 | 2013-12-19 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
AU2015243079A Expired AU2015243079B2 (en) | 2002-05-03 | 2015-10-16 | High-quality lipids and methods for producing by enzymatic liberation from biomass |
Country Status (1)
Country | Link |
---|---|
AU (3) | AU2011202620B2 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5969169A (en) * | 1993-04-27 | 1999-10-19 | Cargill, Incorporated | Non-hydrogenated canola oil for food applications |
US6270828B1 (en) * | 1993-11-12 | 2001-08-07 | Cargrill Incorporated | Canola variety producing a seed with reduced glucosinolates and linolenic acid yielding an oil with low sulfur, improved sensory characteristics and increased oxidative stability |
-
2011
- 2011-06-02 AU AU2011202620A patent/AU2011202620B2/en not_active Expired
-
2013
- 2013-12-19 AU AU2013273736A patent/AU2013273736B2/en not_active Ceased
-
2015
- 2015-10-16 AU AU2015243079A patent/AU2015243079B2/en not_active Expired
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5969169A (en) * | 1993-04-27 | 1999-10-19 | Cargill, Incorporated | Non-hydrogenated canola oil for food applications |
US6270828B1 (en) * | 1993-11-12 | 2001-08-07 | Cargrill Incorporated | Canola variety producing a seed with reduced glucosinolates and linolenic acid yielding an oil with low sulfur, improved sensory characteristics and increased oxidative stability |
Also Published As
Publication number | Publication date |
---|---|
AU2013273736A1 (en) | 2014-03-06 |
AU2011202620A1 (en) | 2011-06-23 |
AU2015243079B2 (en) | 2017-07-13 |
AU2013273736B2 (en) | 2015-07-16 |
AU2015243079A1 (en) | 2015-11-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2008252072B2 (en) | High-quality lipids and methods for producing by enzymatic liberation from biomass | |
WO2006046943A2 (en) | Methods for producing lipids by liberation from biomass | |
AU2001293711B2 (en) | Isolation of microbial oils | |
AU2014203384B2 (en) | Solventless extraction process | |
EP1513922B2 (en) | Pasteurisation process for microbial cells and microbial oil | |
AU2001293711A1 (en) | Isolation of microbial oils | |
AU2011202620B2 (en) | High-quality lipids and methods for producing by enzymatic liberation from biomass |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PC1 | Assignment before grant (sect. 113) |
Owner name: DSM IP ASSETS B.V. Free format text: FORMER APPLICANT(S): MARTEK BIOSCIENCES CORPORATION |
|
FGA | Letters patent sealed or granted (standard patent) | ||
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |