JP2014508507A - Method for producing lauric acid-containing fat and oil and lauric acid or ester thereof - Google Patents
Method for producing lauric acid-containing fat and oil and lauric acid or ester thereof Download PDFInfo
- Publication number
- JP2014508507A JP2014508507A JP2013538376A JP2013538376A JP2014508507A JP 2014508507 A JP2014508507 A JP 2014508507A JP 2013538376 A JP2013538376 A JP 2013538376A JP 2013538376 A JP2013538376 A JP 2013538376A JP 2014508507 A JP2014508507 A JP 2014508507A
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- Prior art keywords
- strain
- chroomonas
- algae
- lauric acid
- algal
- 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.)
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Links
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 title claims abstract description 191
- 239000005639 Lauric acid Substances 0.000 title claims abstract description 98
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- 150000002148 esters Chemical class 0.000 title claims abstract description 17
- 241000195493 Cryptophyta Species 0.000 claims abstract description 106
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 58
- 229930195729 fatty acid Natural products 0.000 claims abstract description 58
- 239000000194 fatty acid Substances 0.000 claims abstract description 58
- 239000003925 fat Substances 0.000 claims abstract description 55
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 55
- 239000003921 oil Substances 0.000 claims abstract description 49
- 238000000034 method Methods 0.000 claims abstract description 36
- 241000195492 Chroomonas Species 0.000 claims abstract description 35
- 241001468587 Chroomonas nordstedtii Species 0.000 claims abstract description 30
- 239000000203 mixture Substances 0.000 claims abstract description 25
- 241001474408 Chroomonas mesostigmatica Species 0.000 claims abstract description 22
- 241000718366 Chroomonas placoidea Species 0.000 claims abstract description 19
- 241001501882 Rhodomonas Species 0.000 claims abstract description 13
- 239000000470 constituent Substances 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 241000512224 Rhodomonas salina Species 0.000 claims description 18
- 241000949074 Gymnochlora stellata Species 0.000 claims description 14
- 241000588538 Amorphochlora amoebiformis Species 0.000 claims description 12
- 241001279861 Lotharella Species 0.000 claims description 12
- -1 lauric acid ester Chemical class 0.000 claims description 12
- 241000305336 Bigelowiella natans Species 0.000 claims description 11
- 241000305038 Lotharella globosa Species 0.000 claims description 11
- 241001279849 Lotharella vacuolata Species 0.000 claims description 11
- 241000305337 Bigelowiella Species 0.000 claims description 10
- 241000949079 Gymnochlora Species 0.000 claims description 10
- UQDUPQYQJKYHQI-UHFFFAOYSA-N methyl laurate Chemical group CCCCCCCCCCCC(=O)OC UQDUPQYQJKYHQI-UHFFFAOYSA-N 0.000 claims description 8
- 241001147674 Chlorarachniophyceae Species 0.000 claims description 5
- 150000001450 anions Chemical class 0.000 claims description 5
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 claims description 5
- 229960004630 chlorambucil Drugs 0.000 claims description 5
- 235000019197 fats Nutrition 0.000 description 45
- 239000002609 medium Substances 0.000 description 42
- 235000019198 oils Nutrition 0.000 description 41
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 38
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 21
- 239000000243 solution Substances 0.000 description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 210000003763 chloroplast Anatomy 0.000 description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 7
- 150000002632 lipids Chemical class 0.000 description 7
- 239000013535 sea water Substances 0.000 description 7
- FLIACVVOZYBSBS-UHFFFAOYSA-N Methyl palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC FLIACVVOZYBSBS-UHFFFAOYSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 238000009825 accumulation Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000001149 (9Z,12Z)-octadeca-9,12-dienoate Substances 0.000 description 4
- HPEUJPJOZXNMSJ-UHFFFAOYSA-N Methyl stearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC HPEUJPJOZXNMSJ-UHFFFAOYSA-N 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 description 4
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 4
- 239000001307 helium Substances 0.000 description 4
- 229910052734 helium Inorganic materials 0.000 description 4
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 4
- ZAZKJZBWRNNLDS-UHFFFAOYSA-N methyl tetradecanoate Chemical compound CCCCCCCCCCCCCC(=O)OC ZAZKJZBWRNNLDS-UHFFFAOYSA-N 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000029553 photosynthesis Effects 0.000 description 4
- 238000010672 photosynthesis Methods 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 210000003495 flagella Anatomy 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000008107 starch Substances 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
- WTTJVINHCBCLGX-UHFFFAOYSA-N (9trans,12cis)-methyl linoleate Natural products CCCCCC=CCC=CCCCCCCCC(=O)OC WTTJVINHCBCLGX-UHFFFAOYSA-N 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 2
- DVWSXZIHSUZZKJ-UHFFFAOYSA-N 18:3n-3 Natural products CCC=CCC=CCC=CCCCCCCCC(=O)OC DVWSXZIHSUZZKJ-UHFFFAOYSA-N 0.000 description 2
- XUQBFMJGHHYFCP-UHFFFAOYSA-N 2-(2-chloroethyl)-3,4,5,6-tetrahydro-1h-2-benzazocine;hydrochloride Chemical compound Cl.C1N(CCCl)CCCCC2=CC=CC=C21 XUQBFMJGHHYFCP-UHFFFAOYSA-N 0.000 description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 2
- LNJCGNRKWOHFFV-UHFFFAOYSA-N 3-(2-hydroxyethylsulfanyl)propanenitrile Chemical compound OCCSCCC#N LNJCGNRKWOHFFV-UHFFFAOYSA-N 0.000 description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 2
- 229910015900 BF3 Inorganic materials 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241001147675 Chlorarachnion Species 0.000 description 2
- PKIXXJPMNDDDOS-UHFFFAOYSA-N Methyl linoleate Natural products CCCCC=CCCC=CCCCCCCCC(=O)OC PKIXXJPMNDDDOS-UHFFFAOYSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 239000005642 Oleic acid Substances 0.000 description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- QWDCYFDDFPWISL-UHFFFAOYSA-N UNPD207407 Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(=O)OC QWDCYFDDFPWISL-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- QWDCYFDDFPWISL-JEBPEJKESA-N cis-5,8,11,14,17-eicosapentaenoic acid methyl ester Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)OC QWDCYFDDFPWISL-JEBPEJKESA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 229910001873 dinitrogen Inorganic materials 0.000 description 2
- VCDLWFYODNTQOT-UHFFFAOYSA-N docosahexaenoic acid methyl ester Natural products CCC=CCC=CCC=CCC=CCC=CCC=CCCC(=O)OC VCDLWFYODNTQOT-UHFFFAOYSA-N 0.000 description 2
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- CAMHHLOGFDZBBG-UHFFFAOYSA-N epoxidized methyl oleate Natural products CCCCCCCCC1OC1CCCCCCCC(=O)OC CAMHHLOGFDZBBG-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 239000013505 freshwater Substances 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 150000004702 methyl esters Chemical class 0.000 description 2
- DVWSXZIHSUZZKJ-YSTUJMKBSA-N methyl linolenate Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(=O)OC DVWSXZIHSUZZKJ-YSTUJMKBSA-N 0.000 description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
- VUXXXYMMZBAUSE-UHFFFAOYSA-N pentadecan-7-one Chemical compound CCCCCCCCC(=O)CCCCCC VUXXXYMMZBAUSE-UHFFFAOYSA-N 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 150000004671 saturated fatty acids Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- NNMALANKTSRILL-LXENMSTPSA-N 3-[(2z,5e)-2-[[3-(2-carboxyethyl)-5-[(z)-[(3e,4r)-3-ethylidene-4-methyl-5-oxopyrrolidin-2-ylidene]methyl]-4-methyl-1h-pyrrol-2-yl]methylidene]-5-[(4-ethyl-3-methyl-5-oxopyrrol-2-yl)methylidene]-4-methylpyrrol-3-yl]propanoic acid Chemical compound O=C1C(CC)=C(C)C(\C=C\2C(=C(CCC(O)=O)C(=C/C3=C(C(C)=C(\C=C/4\C(\[C@@H](C)C(=O)N\4)=C\C)N3)CCC(O)=O)/N/2)C)=N1 NNMALANKTSRILL-LXENMSTPSA-N 0.000 description 1
- 241000224489 Amoeba Species 0.000 description 1
- 241000233788 Arecaceae Species 0.000 description 1
- 101710184216 Cardioactive peptide Proteins 0.000 description 1
- 241000199913 Crypthecodinium Species 0.000 description 1
- 241000199914 Dinophyceae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000362749 Ettlia oleoabundans Species 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 239000003225 biodiesel Substances 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
- 239000004202 carbamide Substances 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 229930002868 chlorophyll a Natural products 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
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- DGNIJJSSARBJSH-NLJAFYFLSA-L magnesium (E)-3-[(3R)-16-ethenyl-11-ethyl-3-methoxycarbonyl-12,17,21,26-tetramethyl-4-oxo-7,24-diaza-23,25-diazanidahexacyclo[18.2.1.15,8.110,13.115,18.02,6]hexacosa-1(22),2(6),5(26),7,9,11,13,15(24),16,18,20-undecaen-22-yl]prop-2-enoic acid Chemical compound [Mg++].CCc1c(C)c2cc3nc(cc4[n-]c(c(\C=C\C(O)=O)c4C)c4[C@@H](C(=O)OC)C(=O)c5c(C)c(cc1[n-]2)nc45)c(C)c3C=C DGNIJJSSARBJSH-NLJAFYFLSA-L 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 210000001577 neostriatum Anatomy 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
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- 239000011664 nicotinic acid Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000003346 palm kernel oil Substances 0.000 description 1
- 235000019865 palm kernel oil Nutrition 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- 230000029264 phototaxis Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 229960002901 sodium glycerophosphate Drugs 0.000 description 1
- PVGBHEUCHKGFQP-UHFFFAOYSA-N sodium;n-[5-amino-2-(4-aminophenyl)sulfonylphenyl]sulfonylacetamide Chemical compound [Na+].CC(=O)NS(=O)(=O)C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=C1 PVGBHEUCHKGFQP-UHFFFAOYSA-N 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004808 supercritical fluid chromatography Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000001228 trophic effect Effects 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 230000009105 vegetative growth Effects 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
-
- 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
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
藻類を用いた、ラウリン酸を構成脂肪酸として含有する油脂の供給方法、及び当該油脂を用いたラウリン酸又はそのエステルの供給方法の提供。
クロララクニオン藻綱の藻類、並びにChroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類、及びRhodomonas属藻類からなるクリプト藻綱の藻類よりなる群から選ばれる一種以上を培地中で培養し、培養物から脂肪酸組成におけるラウリン酸の含有割合が３質量％以上である油脂を採取し、所望により採取された油脂中のラウリン酸をエステル化した後、ラウリン酸又はそのエステルを分離、取得することを特徴とする、ラウリン酸又はそのエステルの製造方法。The provision of the supply method of the fats and oils which contain lauric acid as a constituent fatty acid using algae, and the supply method of lauric acid or its ester using the said fats and oils.
Chlorolacunion algae and one or more selected from the group consisting of Chroomonas diplococca , Chroomonas mesostigmatica , Chroomonas nordstedtii and Chroomonas placoidea selected from the group consisting of Chlomononas algae and Rhodomonas algae Culturing, collecting oil and fat having a content of lauric acid in the fatty acid composition of 3% by mass or more from the culture, esterifying lauric acid in the collected oil and fat as desired, and then separating lauric acid or its ester, A method for producing lauric acid or an ester thereof, characterized by comprising:
Description
本発明は、藻類を用いた、ラウリン酸を構成脂肪酸として含有する油脂（以下、単に「ラウリン酸含有油脂」とも称する）、及びそれを用いたラウリン酸又はそのエステルの製造方法に関する。 The present invention relates to an oil and fat containing lauric acid as a constituent fatty acid (hereinafter also simply referred to as “lauric acid-containing fat and oil”) using algae, and a method for producing lauric acid or an ester thereof using the same.
ラウリン酸は、ヤシ油やパーム核油に多く含まれる主な脂肪酸で、各種界面活性剤の原料や食品などに使用される。
ラウリン酸は、その供給源がヤシやパーム核に限定されており、そのため栽培地域が限定される。さらに、ラウリン酸の原料として耕地を利用することは、今後バイオディーゼルや、食料用途と競合することも懸念される。また、熱帯雨林の破壊という問題も付随する。
従って、ヤシやパーム核に頼らない、ラウリン酸供給技術の開発が望まれていた。
Lauric acid is a major fatty acid that is abundant in coconut oil and palm kernel oil, and is used as a raw material for various surfactants and foods.
The source of lauric acid is limited to palms and palm kernels, which limits the cultivation area. Furthermore, there is a concern that the use of arable land as a raw material for lauric acid will compete with biodiesel and food applications in the future. There is also the problem of rainforest destruction.
Therefore, development of lauric acid supply technology that does not rely on palm or palm kernels has been desired.
一方、藻類は面積あたりの油脂生産性が、植物などに比べ１０倍ほど高く、高効率であることが知られており（非特許文献１）、ラウリン酸供給生物として、光合成を行わず従属栄養で増殖する渦鞭毛藻であるCrypthecodinium choniiがラウリン酸を高含有（１５．７％／総脂質中）することが報告されている（非特許文献２）。 On the other hand, algae are known to have high oil and fat productivity per area about 10 times higher than plants and the like (Non-Patent Document 1), and as a lauric acid supplying organism, photosynthesis is not performed and heterotrophic nutrition is performed. It has been reported that Crypthecodinium chonii , a dinoflagellate that proliferates in potato, contains a high content of lauric acid (15.7% / in total lipid) (Non-patent Document 2).
炭素源コストなどの観点より、光合成（独立栄養）により増殖でき、且つラウリン酸をより多く含有する藻類が望まれるが、斯かる藻類では、ラウリン酸を１〜２％程度含有するNeochloris oleoabundansが知られているだけで（非特許文献３）、ラウリン酸をそれ以上含有する藻類はこれまでに知られていない。 From the viewpoint of carbon source costs, algae that can grow by photosynthesis (autotrophic) and contain more lauric acid are desired. For such algae, Neochloris oleoabundans containing about 1 to 2% lauric acid is known. However, no algae containing more lauric acid has been known so far (Non-patent Document 3).
本発明は、クロララクニオン藻綱の藻類、並びにChroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類、及びRhodomonas属藻類からなるクリプト藻綱の藻類よりなる群から選ばれる一種以上を培地中で培養し、培養物から脂肪酸組成におけるラウリン酸の含有割合が３質量％以上である油脂を採取し、所望により採取された油脂中のラウリン酸をエステル化した後、ラウリン酸又はそのエステルを分離、取得することを特徴とする、ラウリン酸又はそのエステルの製造方法を提供するものである。
また、本発明は、Chroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類の一種以上を培地中で培養し、培養物から脂肪酸組成におけるラウリン酸の含有割合が３質量％以上である油脂を採取することを含む、ラウリン酸を構成脂肪酸として含有する油脂の製造方法を提供するものである。
The present invention, algae chlorambucil Lac anion Motsuna and Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas nordstedtii and Chroomonas Chroomonas algae selected from Placoidea, and one or more selected from the group consisting of algae crypto Motsuna consisting Rhodomonas algae Cultivated in a medium, and from the culture, oil and fat having a content of lauric acid in the fatty acid composition of 3% by mass or more is collected, and if desired, lauric acid in the collected oil and fat is esterified, and then lauric acid or its The present invention provides a process for producing lauric acid or an ester thereof, characterized by separating and obtaining an ester.
In addition, the present invention includes culturing at least one kind of Chroomonas algae selected from Chroomonas diplococca , Chroomonas mesostigmatica , Chroomonas nordstedtii and Chroomonas placoidea in a medium, and the content of lauric acid in the fatty acid composition is 3% by mass or more from the culture. The present invention provides a method for producing fats and oils containing lauric acid as a constituent fatty acid, which comprises collecting certain fats and oils.
本発明は、藻類を用いた、ラウリン酸を構成脂肪酸として含有する油脂の供給方法、及び当該油脂を用いたラウリン酸又はそのエステルの供給方法を提供することに関する。 The present invention relates to a method for supplying fats and oils containing lauric acid as a constituent fatty acid using algae, and a method for supplying lauric acid or esters thereof using the fats and oils.
本発明者らは、ラウリン酸供給生物について検討したところ、光合成をするタイプの藻類のうち、単細胞藻類であるクロララクニオン藻綱（Chlorarachniophyceae）の藻類、並びにクリプト藻綱（Cryptophyceae）の藻類のうち、Chroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類、又はRhodomonas属藻類がラウリン酸を高濃度で含有し、これを用いることにより、構成脂肪酸としてラウリン酸を高含有する油脂、更にはラウリン酸又はそのエステルを効率良く製造できることを見出した。 The present inventors have studied lauric acid-supplying organisms. Among the types of algae that perform photosynthesis, the algae of Chlorarachniophyceae, which is a unicellular algae, and the algae of Cryptophyceae are among the algae of Cryptophyceae. Chroomonas diplococca , Chroomonas mesostigmatica , Chroomonas nordstedtii and Chroomonas placoidea Chroomonas genus algae, or Rhodomonas genus algae contain lauric acid at a high concentration, by using this, fats and oils containing lauric acid as a constituent fatty acid, Furthermore, it discovered that lauric acid or its ester could be manufactured efficiently.
本発明の方法によれば、容易に増殖可能な藻類を用いることから、ヤシやパーム核の如く栽培地域が限定されたり、食料用途等の競合が発生することなく、効率良くラウリン酸を構成脂肪酸として高含有する油脂を製造することができる。また、本発明の方法によれば、熱帯雨林の破壊も回避可能である。 According to the method of the present invention, since algae that can be easily propagated is used, the cultivated fatty acid can be efficiently formed without limiting the cultivation area such as palm and palm kernels or causing competition for food use. As a result, it is possible to produce fats and oils that are highly contained. Moreover, according to the method of the present invention, it is possible to avoid the destruction of the rainforest.
本発明のラウリン酸含有油脂の製造方法は、Chroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類並びにRhodomonas属藻類よりなる群から選ばれるクリプト藻綱藻類の一種以上を培地中で培養し、培養物から脂肪酸組成におけるラウリン酸の含有割合が３質量％以上である油脂を採取するものである。
また、本発明のラウリン酸又はそのエステルの製造方法は、クロララクニオン藻綱（Chlorarachniophyceae）の藻類、並びにChroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類、及びRhodomonas属藻類からなるクリプト藻綱の藻類よりなる群から選ばれる一種以上を培地中で培養し、培養物から脂肪酸組成におけるラウリン酸の含有割合が３質量％以上である油脂を採取し、所望により採取された油脂中のラウリン酸をエステル化した後、ラウリン酸又はそのエステルを分離、取得するものである。
ここで、脂肪酸組成におけるラウリン酸の含有割合が３質量％以上である油脂としては、油脂中の全構成脂肪酸中のラウリン酸の含有割合が３質量％以上、好ましくは３〜６０質量％、より好ましくは５〜６０質量％、更に好ましくは６〜６０質量％、更に好ましくは７〜６０質量％、更に好ましくは８〜６０質量％、更に好ましくは９〜６０質量％、更に好ましくは１０〜６０質量％、更に好ましくは１１〜５０質量％、更に好ましくは１２〜４０質量％の油脂が挙げられる。
The method of manufacturing lauric acid-containing fats of the present invention, Chroomonas diplococca, Chroomonas mesostigmatica, in Chroomonas nordstedtii and Chroomonas Chroomonas genus selected from placoidea algae and Rhodomonas spp medium in one or more kinds of crypto Motsuna algae selected from the group consisting of algae Culturing is performed, and fats and oils in which the content of lauric acid in the fatty acid composition is 3% by mass or more are collected from the culture.
The manufacturing method of lauric acid or an ester of the invention, algae chlorambucil Lac anions Motsuna (Chlorarachniophyceae), and Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas algae selected from Chroomonas nordstedtii and Chroomonas placoidea, and from Rhodomonas algae One or more selected from the group consisting of the algae of Cryptophyceae is cultured in a medium, and oil and fat having a lauric acid content in the fatty acid composition of 3% by mass or more is collected from the culture, and the oil and fat collected as desired After esterifying lauric acid therein, lauric acid or its ester is separated and obtained.
Here, as fats and oils in which the content ratio of lauric acid in the fatty acid composition is 3% by mass or more, the content ratio of lauric acid in all the constituent fatty acids in the fats and oils is 3% by mass or more, preferably 3 to 60% by mass. Preferably it is 5-60 mass%, More preferably, it is 6-60 mass%, More preferably, it is 7-60 mass%, More preferably, it is 8-60 mass%, More preferably, it is 9-60 mass%, More preferably, it is 10-60 The oil and fat of mass%, More preferably, 11-50 mass%, More preferably, 12-40 mass% is mentioned.
本発明において用いられるクロララクニオン藻綱（Chlorarachniophyceae）に属する藻類は、脂肪酸組成におけるラウリン酸の含有割合が３質量％以上の油脂生産能を有するものであれば何れの株でもよい。
本発明において用いられるクリプト藻綱藻類は、Chroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類又はRhodomonas属藻類であり、より好ましくはChroomonas diplococca、Chroomonas mesostigmatica、及びChroomonas nordstedtiiから選ばれるChroomonas属藻類であり、脂肪酸組成におけるラウリン酸の含有割合が３質量％以上の油脂生産能を有するものであれば何れの株でもよい。
The algae belonging to Chlorarachniophyceae used in the present invention may be any strain as long as it has an oil-and-fat producing ability with a lauric acid content in the fatty acid composition of 3% by mass or more.
Crypto Motsuna algae used in the present invention, Chroomonas diplococca, Chroomonas mesostigmatica, a Chroomonas algae or Rhodomonas algae selected from Chroomonas nordstedtii and Chroomonas placoidea, more preferably selected from Chroomonas diplococca, Chroomonas mesostigmatica, and Chroomonas nordstedtii Any strain may be used as long as it is an algae of the genus Chroomonas and has an oil-and-fat producing ability in which the content of lauric acid in the fatty acid composition is 3% by mass or more.
本発明の藻類は、例えば、次のようなスクリーニング法に従って選択することができる。
ｉ）培養容器に滅菌した培地（淡水用としてＷＡ培地（表２参照）、海水用としてダイゴＩＭＫ培地（表３参照））を分注する。
ii）藻類株を培地に接種し、室温（２２℃〜２４℃）、照度約３０００ルクス、１２時間明暗条件下で静置培養する。
iii）藻体を回収し、油脂を抽出し、脂肪酸をメチルエステル化し、その組成を分析して、ラウリン酸含有油脂を産生している藻類株を選択する。
iv）油脂中に全脂肪酸あたり、ラウリン酸を３質量％以上含有する藻類株を選択する。
The algae of the present invention can be selected, for example, according to the following screening method.
i) Dispense sterilized medium (WA medium (see Table 2) for fresh water and Daigo IMK medium (see Table 3) for seawater) into a culture vessel.
ii) The algae strain is inoculated into the medium, followed by static culture at room temperature (22 ° C. to 24 ° C.), illumination intensity of about 3000 lux, and light / dark conditions for 12 hours.
iii) Algal bodies are collected, fats and oils are extracted, fatty acids are methyl esterified, the composition is analyzed, and an algal strain producing lauric acid-containing fats and oils is selected.
iv) Select an algal strain containing 3% by mass or more of lauric acid per total fatty acid in the fats and oils.
クロララクニオン藻綱に属する藻類としては、例えば、Chlorarachnion属、Lotharella属、Gymnochlora属、Cryptochlora属、Bigelowiella属の藻類が挙げられ、好ましくはLotharella属、Gymnochlora属、Bigelowiella属である。
更に好適なクロララクニオン藻綱藻類としては、例えば、Lotharella属では、Lotharella globosa、Lotharella amoeboformis、Lotharella vacuolata等が挙げられ、Gymnochlora属では、Gymnochlora stellata等が挙げられる。Bigelowiella属では、Bigelowiella natans等が挙げられる。このうち、Lotharella globosaとしては、より好ましくはLotharella globosa CCMP1729株、Lotharella amoebiformisとしては、より好ましくはLotharella amoebiformis CCMP2058株、Lotharella vacuolataとしては、より好ましくはLotharella vacuolata CCMP240株、Gymnochlora stellataとしては、より好ましくはGymnochlora stellata CCMP2057株、Bigelowiella natansとしては、より好ましくはBigelowiella natans CCMP621株、CCMP2757株（これらは、The Provasoli-Guillard National Center for Culture of Marine Phytoplankton(CCMP)等より入手可能）、又は当該藻類株と実質的に同一の藻類学的性質を有する株を挙げることができる。例えば、Lotharella amoebiformisは、近年Amorphochlora amoebiformisという新属に属することが提唱されているが、これらは同一の藻類学的性質を有する株とみなすことができる。これらのうち、Lotharella globosa がより好ましく、Lotharella globosa CCMP1729株又は当該藻類株と実質的に同一の菌学的性質を有する藻類株が更に好ましい。
Examples of the algae belonging to the chloracranion algae include algae of the genus Chlorarachnion , Lotharella , Gymnochlora , Cryptochlora , and Bigelowiella , preferably Lotharella , Gymnochlora , and Bigelowiella .
Examples of more suitable Chloralachniophyceae algae include Lotharella globosa , Lotharella amoeboformis , Lotharella vacuolata and the like in the genus Lotharella , and Gymnochlora stellata and the like in the genus Gymnochlora . Bigelowiella genus includes Bigelowiella natans . Among these, as Lotharella globosa , more preferably Lotharella globosa CCMP1729 strain, Lotharella amoebiformis , more preferably Lotharella amoebiformis CCMP2058 strain, Lotharella vacuolata , more preferably Lotharella vacuolata CCMP240 strain, Gymnochlora stellata , more preferably As Gymnochlora stellata CCMP2057 strain, Bigelowiella natans , more preferably Bigelowiella natans CCMP621 strain, CCMP2757 strain (these are available from The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) etc.), or substantially the algal strain And strains having identical algaeological properties. For example, Lotharella amoebiformis has recently been proposed to belong to the new genus Amorphochlora amoebiformis , which can be regarded as strains having the same algaeological properties. Of these, Lotharella globosa is more preferable, and Lotharella globosa CCMP1729 strain or an algal strain having substantially the same mycological properties as the algal strain is more preferable.
Lotharella amoebiformis CCMP2058株と実質的に同一の藻類学的性質を有する株としては、例えば、Lotharella amoebiformis Ryukyu株等が挙げられ、Lotharella vacuolata CCMP240株と実質的に同一の藻類学的性質を有する株としては、例えば、Lotharella vacuolata FK18G株等が挙げられ、Gymnochlora stellata CCMP2057株と実質的に同一の藻類学的性質を有する株としては、例えば、Gymnochlora stellata Guam-1株等が挙げられ、Bigelowiella natans CCMP621株、CCMP2757株と実質的に同一の藻類学的性質を有する株としては、例えばBigelowiella natans A11株、490株、VA3株などが挙げられる。 Lotharella The amoebiformis CCMP2058 strain substantially strains with identical PHYCOLOGY properties, for example, Lotharella amoebiformis Ryukyu strain and the like, as Lotharella vacuolata CCMP240 strain substantially strains with identical PHYCOLOGY properties are For example, the Lotharella vacuolata FK18G strain and the like, and the strain having substantially the same algal characteristics as the Gymnochlora stellata CCMP2057 strain include, for example, the Gymnochlora stellata Guam-1 strain, the Bigelowiella natans CCMP621 strain, Examples of the strain having substantially the same algal characteristics as the CCMP2757 strain include Bigelowiella natans A11 strain, 490 strain, and VA3 strain.
斯かる藻類株の藻類学的性質は、以下のとおりである。これらの各藻類株と同一の種に属する菌株、各藻類株と実質的に同一の菌学的性質を有する菌株は、斯かる性質に基づいて同定することができる。
＜クロララクニオン藻綱に属する藻類の藻類学的性質＞
ｉ）クロロフィルa、bを有する
ii）葉緑体包膜は4枚である
iii）ヌクレオモルフを有する
iv）デンプンを蓄積しない
v）アメーバ相と細胞壁を持つ相が存在する
vi) 眼点を持たない
＜Chlorarachnion属に属する藻類の藻類学的性質＞
ｉ）栄養細胞はアメーバ様細胞である
ii）ヌクレオモルフはピレノイド内部に位置する
＜Cryptochlora属に属する藻類の藻類学的性質＞
ｉ）栄養細胞は球状細胞である
ii) ピレノイド構造は不明
＜Lotharella属に属する藻類の藻類学的性質＞
ｉ）深裂型のピレノイドを有する
ii）ヌクレオモルフはピレノイド基部近くにある葉緑体周縁区画内に位置する
＜Gymnochlora属に属する藻類の藻類学的性質＞
ｉ）葉緑体包膜の最内膜が管状にピレノイドマトリックス内に陥入している
ii）ヌクレオモルフはピレノイド基部近くにある葉緑体周縁区画内に位置する
＜Bigelowiella属に属する藻類の藻類学的性質＞
ｉ）遊泳細胞が無性的に増殖できる
ii) ピレノイドは浅裂型である
iii) ヌクレオモルフはピレノイドの基部にある
＜Lotharella globosa CCMP1729株の藻類学的性質＞
i）栄養細胞は球状であり、増殖時にアメーバ様細胞は出現しない
＜Lotharella amoebiformis CCMP2058株の藻類学的性質＞
i）栄養細胞はアメーバ様細胞である
＜Lotharella vacuolata CCMP240株の藻類学的性質＞
i) 生活環の主要ステージは球状であるが、培養令初期から中期にかけて糸状仮足を有するアメーバ状細胞が認められる
ii）他のLotharella属の藻類より、大きな液胞を有する
iii）栄養増殖はアメーバ様細胞の二分裂によって行われる
＜Gymnochlora stellata CCMP2057株の藻類学的性質＞
i）星型アメーバ状生物であり、多数の糸状仮足を有するが網状ネットワークを形成しない
ii) 生活環を通じて細胞壁を持つ細胞や遊泳細胞を生じない
＜Bigelowiella natans CCMP621株、CCMP2757株の藻類学的性質＞
i) 栄養ステージが遊泳細胞であり、アメーバ様細胞ではない
ii）長いものと短いものの2本の鞭毛を有する
iii）線条体を持たない
The algaeological properties of such algae strains are as follows. Strains belonging to the same species as these algae strains and strains having substantially the same mycological properties as each algae strain can be identified based on such properties.
<Algaeological properties of algae belonging to Chloraracunionae>
i) having chlorophyll a, b
ii) Four chloroplast envelopes
iii) Has a nucleomorph
iv) Does not accumulate starch
v) There is an amoeba phase and a phase with cell walls
vi) No eye point < Algalological properties of algae belonging to the genus Chlorarachnion >
i) Vegetative cells are amoeba-like cells
ii) Nucleomorphs are located inside the pyrenoid < Algalological properties of algae belonging to the genus Cryptochlora >
i) Vegetative cells are spherical cells
ii) The pyrenoid structure is unknown <algal properties of algae belonging to the genus Lotharella >
i) Has a deep-cracked pyrenoid
ii) Nucleomorphs are located in the peripheral chloroplast compartment near the base of the pyrenoid < Algalological properties of algae belonging to the genus Gymnochlora >
i) The innermost membrane of the chloroplast envelope is tubularly invaded into the pyrenoid matrix
ii) Nucleomorphs are located in the peripheral chloroplast compartment near the base of the pyrenoid < Algalological properties of algae belonging to the genus Bigelowiella >
i) Swim cells can grow asexually
ii) Pyrenoids are superficial
iii) Nucleomorph is located at the base of pyrenoid < Algalological properties of Lotharella globosa CCMP1729 strain>
i) Vegetative cells are spherical and no amoeba-like cells appear during growth < algal characteristics of Lotharella amoebiformis CCMP2058 >
i) Vegetative cells are amoeba-like cells < Algalological properties of Lotharella vacuolata CCMP240 strain>
i) Although the main stage of the life cycle is spherical, amoeba-like cells with filopodia are observed from early to middle culture period
ii) larger vacuoles than other Lotharella algae
iii) Vegetative growth is performed by bisection of amoeba-like cells < Algaeological properties of Gymnochlora stellata CCMP2057>
i) A star-shaped amoeba-like creature with a large number of filamentous limbs but no reticulated network
ii) Does not generate cell walls or swimming cells throughout the life cycle <Algalological properties of Bigelowiella natans CCMP621 and CCMP2757>
i) The trophic stage is swimming cells, not amoeba-like cells
ii) Long and short flagella
iii) No striatum
本発明のChroomonas属藻類のうち、Chroomonas diplococcaとしては、好ましくは例えばChroomonas diplococca UTEXLB2422株、Chroomonas mesostigmaticaとしては、好ましくは例えばChroomonas mesostigmatica NIES1370株、Chroomonas nordstedtiiとしては、好ましくは例えばChroomonas nordstedtii NIES707株、NIES710株、Chroomonas placoideaとしては、好ましくは例えばChroomonas placoidea NIES705株（これらは、The culture collection of algae at University of Texas at Austin (UTEX)、国立環境研究所(NIES)等より入手可能）、又は当該藻類株と実質的に同一の藻類学的性質を有する株を挙げることができる。
Rhodomonas属藻類としては、例えばRhodomonas salinaが好ましく、Rhodomonas salina UTEX1375、Rhodomonas salina CCMP272又は当該藻類株と実質的に同一の藻類学的性質を有する株がより好ましく、Rhodomonas salina UTEX1375株又は当該藻類株と実質的に同一の菌学的性質を有する藻類株が更に好ましい。これらはUTEX、The Provasoli-Guillard National Center for Culture of Marine Phytoplankton（CCMP）より入手可能である。
Among the Chroomonas genus algae of the present invention, as Chroomonas diplococca, for example, Chroomonas diplococca UTEXLB2422 strain, Chroomonas mesostigmatica , preferably Chroomonas mesostigmatica NIES1370 strain, Chroomonas nordstedtii strain, preferably Chroomonas nordstedtii, for example, Chroomonas nordstedtii As Chroomonas placoidea , preferably, for example, Chroomonas placoidea NIES705 strain (these are available from The Culture Collection of Algae at University of Texas at Austin (UTEX), National Institute for Environmental Studies (NIES), etc.), or the algal strain Mention may be made of strains having substantially the same algaeological properties.
As the Rhodomonas genus algae, for example, Rhodomonas salina is preferable, Rhodomonas salina UTEX1375, Rhodomonas salina CCMP272 or a strain having substantially the same algal characteristics as the algae strain is more preferable, Rhodomonas salina UTEX1375 strain or the algae strain substantially Algal strains having the same mycological properties are more preferred. These are available from UTEX, The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP).
Chroomonas mesostigmatica NIES1370株と実質的に同一の藻類学的性質を有する株としては、例えば、Chroomonas mesostigmatica TKB-112株等が挙げられ、Chroomonas nordstedtii NIES707株と実質的に同一の藻類学的性質を有する株としては、例えば、Chroomonas nordstedtii #00173株等が挙げられ、Chroomonas nordstedtii NIES710株と実質的に同一の藻類学的性質を有する株としては、例えば、Chroomonas nordstedtii #00331株等が挙げられ、Chroomonas placoidea NIES705株と実質的に同一の藻類学的性質を有する株としては、例えば、Chroomonas placoidea CCAP 978/8株等が挙げられる。
Rhodomonas salina CCMP272と実質的に同一の藻類学的性質を有する株としては、例えば、Rhodomonas salina Mel-023株等が挙げられる。
The Chroomonas mesostigmatica NIES1370 strain substantially strains with identical PHYCOLOGY properties, for example, Chroomonas mesostigmatica TKB-112 strain and the like, Chroomonas nordstedtii NIES707 strain substantially strains with identical PHYCOLOGY properties As, for example, Chroomonas nordstedtii # 00173 strain and the like, and Chroomonas nordstedtii NIES710 strain has substantially the same algal characteristics, for example, Chroomonas nordstedtii # 00331 strain, and the like, Chroomonas placoidea NIES705 Examples of the strain having substantially the same algal characteristics as the strain include Chroomonas placoidea CCAP 978/8 strain.
The Rhodomonas salina CCMP272 substantially strains with identical PHYCOLOGY properties, for example, Rhodomonas salina Mel-023 strain and the like.
斯かる藻類株の藻類学的性質は、以下のとおりである。これらの各藻類株と同一の種に属する菌株、各藻類株と実質的に同一の菌学的性質を有する菌株は、斯かる性質に基づいて同定することができる。
＜クリプト藻網の藻類学的性質＞
ｉ）フィコビリン、クロロフィルｃを有する
ii）葉緑体包膜は4枚である
iii）ヌクレオモルフを有する
iv）管状羽型と管状片羽型の鞭毛を有する
v）α-1,4デンプンを蓄積する
＜Chroomonas属の藻類学的性質＞
i) 細胞は樽状で、側溝が認められない
ii) 2つの連なった射出装置を有する
iii) 葉緑体の色は青から緑である
iv) 通常、細胞中心に眼点が認められる
＜Rhodomonas属の藻類学的性質＞
i) 細胞は卵形で、短い側溝を有する
ii) 葉緑体の色は赤から赤褐色であり、目立つピレノイドを有する
iv) 通常、細胞中心に眼点が認められる
＜Chroomonas mesostigmatica NIES1370株の藻類学的性質＞
ｉ）クロロプラスト内に多量のラメラ構造を有する
ii) 葉緑体に付随して大きなピレノイドを一つ有する
＜Chroomonas nordstedtii NIES707、NIES710株の藻類学的性質＞
ｉ）眼点を持たない
ii) ４５０nmから６５０nmの波長の光に対し、走光性を有する
＜Chroomonas placoidea NIES705株の藻類学的性質＞
ｉ）鞭毛軸受け部位に葉舌を有する
＜Rhodomonas salina UTEX1375、Rhodomonas salina CCMP272の藻類学的性質＞
ｉ）細胞亜頂端から細胞長より短い鞭毛が２本生じている。
ii) 縦溝は短く、咽頭部は2列ほどの射出装置が付随して細胞中程まで達する。
iii)赤褐色〜黄橙色の葉緑体を１個もち、背側に明瞭なデンプン鞘で囲まれたピレノイドが１個存在する。
The algaeological properties of such algae strains are as follows. Strains belonging to the same species as these algae strains and strains having substantially the same mycological properties as each algae strain can be identified based on such properties.
<Algaeological properties of cryptoalgae>
i) having phycobilin, chlorophyll c
ii) Four chloroplast envelopes
iii) Has a nucleomorph
iv) Tubular and single-wing flagellum
v) Accumulation of α-1,4 starch < Algalological properties of the genus Chroomonas >
i) Cells are barrel-shaped with no lateral grooves
ii) Having two consecutive injection devices
iii) Chloroplast color is blue to green
iv) Usually, an eye spot is observed at the cell center. <Algaeological properties of the genus Rhodomonas>
i) Cells are oval and have short gutters
ii) Chloroplast color is red to reddish brown and has a prominent pyrenoid
iv) Usually, an eye spot is observed at the cell center < Algalological properties of Chroomonas mesostigmatica NIES1370 strain>
i) Has a large amount of lamellar structure in chloroplast
ii) One large pyrenoid associated with chloroplast < Algalological properties of Chroomonas nordstedtii NIES707 and NIES710 strains>
i) No eye point
ii) It has phototaxis for light with a wavelength of 450 nm to 650 nm < Algalological properties of Chroomonas placoidea NIES705 strain>
i) Having a leaf tongue at the flagellar bearing site < Algalological properties of Rhodomonas salina UTEX1375 and Rhodomonas salina CCMP272>
i) Two flagella shorter than the cell length are generated from the cell apex.
ii) The flutes are short and the pharynx reaches the middle of the cell with two rows of injection devices.
iii) There is one pyrenoid with one reddish brown to yellow orange chloroplast and surrounded by a clear starch sheath on the back side.
また、上述した藻類株又は当該藻類株と実質的に同一の菌学的性質を有する菌株の変異株も本発明の藻類に包含される。
例えば、ラウリン酸を野性株より高含量で含む油脂を産生するように設計された変異株が包含される。
さらに本発明のクロララクニオン藻綱藻類由来の遺伝子及びクリプト藻綱藻類由来の遺伝子は、ラウリン酸高含有油脂を産生するために利用することができる。
Moreover, the algal strain of the present invention also includes the above-mentioned algal strains or mutant strains of strains having substantially the same mycological properties as the algal strains.
For example, mutant strains designed to produce fats and oils containing a higher content of lauric acid than wild strains are included.
Furthermore, the gene derived from Chloralachunion algae and the gene derived from Cryptophyceae algae of the present invention can be used for producing oils and fats with a high content of lauric acid.
本発明のクロララクニオン藻綱藻類及びクリプト藻綱藻類の培養は、天然海水又は人工海水で調製した適当な培地中で、光照射下、微細藻類に用いられる一般的培養手段を用いることにより行うことができる。 The chloralachion algae and cryptoalgae algae according to the present invention are cultured in a suitable medium prepared with natural seawater or artificial seawater by using general culture means used for microalgae under light irradiation. be able to.
培地としては、天然海水又は人工海水をベースに、窒素源、リン源、金属塩、ビタミン類等を添加した公知のものを使用できる。
ここで、例えば、窒素源としてＮａＮＯ3、ＫＮＯ3、Ｃａ（ＮＯ3）2、ＮＨ4ＮＯ3、（ＮＨ4）2ＳＯ4等、リン源としてＫ2ＨＰＯ4、ＫＨ2ＰＯ4、Ｎａ2ＨＰＯ4、ＮａＨ2ＰＯ4、グリセロリン酸ナトリウム等、金属塩としてＮａＣｌ、ＫＣｌ、ＣａＣｌ2、ＭｇＣｌ2、Ｎａ2ＳＯ4、Ｋ2ＳＯ4、ＭｇＳＯ4、Ｎａ2ＣＯ3、ＮａＨＣＯ3、Ｎａ2ＳｉＯ3、Ｈ3ＢＯ3、ＭｎＣｌ2、ＭｎＳＯ4、ＦｅＣｌ3、ＦｅＳＯ4、ＣｏＣｌ2、ＺｎＳＯ4、ＣｕＳＯ4、Ｎａ2ＭｏＯ4等、ビタミン類としてＢｉｏｔｉｎ、Ｖｉｔａｍｉｎ Ｂ１２、Ｔｈｉａｍｉｎｅ−ＨＣｌ、ニコチン酸、イノシトール、葉酸、チミン等を挙げることができる。
また、上記培地にはラウリン酸含有油脂の産生を促進するため、炭素源、微量金属等を適宜添加することができる。
As a culture medium, a known medium to which a nitrogen source, a phosphorus source, a metal salt, vitamins and the like are added based on natural seawater or artificial seawater can be used.
Here, for example, NaNO 3 , KNO 3 , Ca (NO 3 ) 2 , NH 4 NO 3 , (NH 4 ) 2 SO 4, etc. are used as the nitrogen source, and K 2 HPO 4 , KH 2 PO 4 , Na 2 are used as the phosphorus source. Metal salts such as HPO 4 , NaH 2 PO 4 , sodium glycerophosphate, NaCl, KCl, CaCl 2 , MgCl 2 , Na 2 SO 4 , K 2 SO 4 , MgSO 4 , Na 2 CO 3 , NaHCO 3 , Na 2 SiO 3 , H 3 BO 3 , MnCl 2 , MnSO 4 , FeCl 3 , FeSO 4 , CoCl 2 , ZnSO 4 , CuSO 4 , Na 2 MoO 4, etc., Biotin, Vitamin B12, Thiamine-HCl, nicotinic acid, inositol as vitamins , Folic acid, thymine and the like.
Moreover, in order to promote the production of lauric acid-containing fats and oils, a carbon source, trace metals and the like can be appropriately added to the medium.
好ましい培地としては、例えば、ダイゴＩＭＫ培地、ｆ／２培地、ＥＳＭ培地、Ｌ１培地、ＭＮＫ培地等が挙げられる。 Examples of preferable medium include Daigo IMK medium, f / 2 medium, ESM medium, L1 medium, and MNK medium.
上記の培地は、調製後、適当な酸又は塩基を加えることによりｐＨを７．０〜８．０の範囲内に調整した後、オートクレーブにより殺菌して使用することが好ましい。 The above medium is preferably used after preparation, after adjusting the pH within the range of 7.0 to 8.0 by adding an appropriate acid or base, and then sterilizing with an autoclave.
培養は、培地に接種する藻類の量は特に限定されないが、好ましくは培養培地当り１．０〜１０．０％（vol/vol）が好ましく、１．０〜５．０％（vol/vol）がより好ましい。 The amount of algae inoculated into the medium is not particularly limited for the culture, but preferably 1.0 to 10.0% (vol / vol), preferably 1.0 to 5.0% (vol / vol) per culture medium. Is more preferable.
培養温度は、本発明藻類の増殖に悪影響を与えない範囲であれば特に制限されないが、一般的には１０〜３０℃で行うのが好ましく、１５〜２５℃がより好ましい。 The culture temperature is not particularly limited as long as it does not adversely affect the growth of the alga of the present invention, but is generally preferably 10 to 30 ° C, more preferably 15 to 25 ° C.
光照射は、光合成が可能な条件であれば良く、人工光又は太陽光の何れでもよい。
照度は、１００〜５００００ルクスの範囲が好ましく、３００〜１００００ルクスがより好ましい。
The light irradiation may be any conditions that allow photosynthesis, and may be artificial light or sunlight.
The illuminance is preferably in the range of 100 to 50000 lux, more preferably 300 to 10000 lux.
培養時のｐＨは、一般的には６．５〜８．５であり、好ましくはｐＨ７．０〜８．０ である。 The pH during the culture is generally 6.5 to 8.5, preferably pH 7.0 to 8.0.
培養期間は、ラウリン酸含有油脂を高濃度に蓄積する藻体が高い濃度で増殖するよう行えばよく、例えば７〜１２０日間、好ましくは７〜３０日間、通気攪拌培養、振とう培養又は静置培養すればよい。 The culture period may be such that algal bodies that accumulate lauric acid-containing fats and oils in a high concentration grow at a high concentration, for example, 7 to 120 days, preferably 7 to 30 days, aeration and agitation culture, shaking culture, or stationary. What is necessary is just to culture.
培養終了後、遠心分離法や濾過などの常法により、藻体を分離し、分離藻体をそのままもしくは超音波やダイノミル等によって破砕した後、例えば、クロロホルム、ヘキサン、ブタノール、メタノール、酢酸エチル等の有機溶剤による溶剤抽出を行うことにより、ラウリン酸含有油脂を採取することができる。 After completion of the culture, the algal bodies are separated by a conventional method such as centrifugation or filtration, and the separated algal bodies are crushed as they are or by ultrasonic waves, dynomill, etc., for example, chloroform, hexane, butanol, methanol, ethyl acetate, etc. By performing solvent extraction with an organic solvent, lauric acid-containing fats and oils can be collected.
Gymnochlora stellata CCMP2057株、乾燥藻体１００ｇ当たりのラウリン酸含有油脂の含有量は、５〜１０ｇ程度であり、培地１リットル当たりのラウリン酸含有油脂の生産量は、０．０２〜０．０５ｇ程度に達する。
またこの場合、油脂の脂肪酸組成におけるラウリン酸の含有割合は、４．０〜８．５質量％と高濃度である。従って、培地１リットル当たりのラウリン酸の生産量としては、０．０００８〜０．００４３ｇ程度と高い。
Gymnochlora stellata CCMP2057, the content of lauric acid-containing fats and oils per 100 g of dry algae is about 5 to 10 g, and the production amount of lauric acid-containing fats and fats per liter of medium is about 0.02 to 0.05 g. Reach.
In this case, the content of lauric acid in the fatty acid composition of the fat is as high as 4.0 to 8.5% by mass. Therefore, the production amount of lauric acid per liter of the medium is as high as about 0.0008 to 0.0043 g.
Chroomonas diplococca UTEXLB2422株を用いた場合、乾燥藻体１００ｇ当たりのラウリン酸含有油脂の含有量は、3〜4ｇ程度であり、培地１リットル当たりのラウリン酸含有油脂の生産量は、０．００７〜０．０１６ｇ程度に達する。
またこの場合、油脂の脂肪酸組成におけるラウリン酸の含有割合は、５．０〜１７．０質量％と高濃度である。従って、培地１リットル当たりのラウリン酸の生産量としては、０．０００４〜０．００２７ｇ程度と高い。
When Chroomonas diplococca UTEXLB2422 strain is used, the content of fat and oil containing lauric acid per 100 g of dried alga is about 3 to 4 g, and the amount of oil and fat containing lauric acid per liter of medium is 0.007 to 0. It reaches about .016g.
In this case, the content of lauric acid in the fatty acid composition of the oil and fat is as high as 5.0 to 17.0% by mass. Therefore, the production amount of lauric acid per liter of the medium is as high as about 0.0004 to 0.0027 g.
ラウリン酸含有油脂からラウリン酸の分離及び取得は、常法により混合脂肪酸あるいは脂肪酸エステルの状態とした後、尿素付加法、冷却分離法、高速液体クロマトグラフィー法あるいは超臨界クロマトグラフィー法などにより濃縮採取することにより行うことができる。
また、ラウリン酸含有油脂について、当該油脂中のラウリン酸をエステル化することにより、ラウリン酸エステルを分離、取得することができる。
例えば、ラウリン酸含有油脂とメタノール等のアルコールとをアルカリ触媒の存在下で反応させ、反応生成物よりラウリン酸エステルを分離、取得することができる。
ここで、ラウリン酸エステルとしては、メチルエステル、エチルエステル等の低級アルキルエステルが挙げられ、メチルエステルが好ましい。
また、ラウリン酸含有油脂について、当該油脂中のラウリン酸を還元することにより、ラウリルアルコールを分離、取得することもできる。
例えば、ラウリン酸含有油脂を水素化触媒の存在下で水素化し、反応生成物よりラウリルアルコールを分離、取得することができる。
Separation and acquisition of lauric acid from lauric acid-containing fats and oils is made into a mixed fatty acid or fatty acid ester state by a conventional method, and then concentrated and collected by urea addition method, cooling separation method, high performance liquid chromatography method or supercritical chromatography method, etc. This can be done.
Moreover, about lauric acid containing fats and oils, lauric acid ester can be isolate | separated and acquired by esterifying lauric acid in the said fats and oils.
For example, lauric acid-containing oil and fat can be reacted with alcohol such as methanol in the presence of an alkali catalyst, and lauric acid ester can be separated and obtained from the reaction product.
Here, examples of the lauric acid ester include lower alkyl esters such as methyl ester and ethyl ester, and methyl ester is preferable.
Moreover, about lauric acid containing fats and oils, lauryl alcohol can also be isolate | separated and acquired by reduce | restoring the lauric acid in the said fats and oils.
For example, lauric acid-containing fats and oils can be hydrogenated in the presence of a hydrogenation catalyst, and lauryl alcohol can be separated and obtained from the reaction product.
上述した実施形態に応じ、本発明は、下記［１］のラウリン酸又はそのエステルの製造方法、及び下記[１２]のラウリン酸を構成脂肪酸として含有する油脂の製造方法を開示するものであり、更に好適には、［２］〜［１１］のラウリン酸又はそのエステルの製造方法、及び[１２]〜［１４］のラウリン酸を構成脂肪酸として含有する油脂の製造方法を開示するものである。
[１]クロララクニオン藻綱の藻類、並びにChroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類、及びRhodomonas属藻類からなるクリプト藻綱の藻類よりなる群から選ばれる一種以上を培地中で培養し、培養物から脂肪酸組成におけるラウリン酸の含有割合が３質量％以上である油脂を採取し、所望により採取された油脂中のラウリン酸をエステル化した後、ラウリン酸又はそのエステルを分離、取得することを特徴とする、ラウリン酸又はそのエステルの製造方法。
[２]クロララクニオン藻綱（Chlorarachniophyceae）の藻類が、Lotharella属、Gymnochlora属又はBigelowiella属の藻類である、[１]の方法。
[３]Lotharella属藻類が、Lotharella globosa、Lotharella amoebiformis又はLotharella vacuolataであり、Gymnochlora属藻類が、Gymnochlora stellataであり、Bigelowiella属藻類が、Bigelowiella natansである、[１]又は[２]の方法。
[４]Lotharella属藻類が、Lotharella globosa CCMP1729株、Lotharella amoebiformis CCMP2058株、Lotharella vacuolata CCMP240株又は当該藻類株と実質的に同一の菌学的性質を有する藻類株である、[３]の方法。
[５]Gymnochlora属藻類が、Gymnochlora stellata CCMP2057株又は当該藻類株と実質的に同一の菌学的性質を有する藻類株である、[３]の方法。
[６]Bigelowiella 属藻類が、Bigelowiella natans CCMP621株、CCMP2757株又は当該藻類株と実質的に同一の菌学的性質を有する藻類株である、[３]の方法。
[７]Chroomonas属藻類が、Chroomonas diplococca UTEXLB2422株、Chroomonas mesostigmatica NIES1370株、Chroomonas nordstedtii NIES707株、Chroomonas nordstedtii NIES710株、Chroomonas placoidea NIES705株、又はこれらの藻類株と実質的に同一の菌学的性質を有する藻類株である、[１]の方法。
[８]Rhodomonas属藻類がRhodomonas salinaである、[１]の方法。
[９]Rhodomonas salina がRhodomonas salina UTEX1375、Rhodomonas salina CCMP272又はこれらの藻類株と実質的に同一の菌学的性質を有する藻類株である、[８]の方法。
[１０]照度３００〜１００００ルクスの光照射下で、７〜１２０日間培養する[１]〜[９]の方法。
[１１]ラウリン酸エステルがラウリン酸メチルである、[１]〜[１０]の方法。
[１２]Chroomonas diplococca、Chroomonas mesostigmatica、Chroomonas nordstedtii及びChroomonas placoideaから選ばれるChroomonas属藻類の一種以上を培地中で培養し、培養物から脂肪酸組成におけるラウリン酸の含有割合が３質量％以上である油脂を採取することを含む、ラウリン酸を構成脂肪酸として含有する油脂の製造方法。
[１３]Chroomonas属藻類が、Chroomonas diplococca UTEXLB2422株、Chroomonas mesostigmatica NIES1370株、Chroomonas nordstedtii NIES707株、Chroomonas nordstedtii NIES710株、Chroomonas placoidea NIES705株、又はこれらの藻類株と実質的に同一の菌学的性質を有する藻類株である、[１２]の方法。
[１４]照度３００〜１００００ルクスの光照射下で、７〜１２０日間培養する[１２]又は[１３]の方法。
According to the embodiment described above, the present invention discloses a method for producing lauric acid or ester thereof according to [1] below, and a method for producing fats and oils containing lauric acid according to [12] below as a constituent fatty acid, More preferably, a method for producing [2] to [11] lauric acid or an ester thereof and a method for producing an oil or fat containing [12] to [14] lauric acid as a constituent fatty acid are disclosed.
[1] chlorambucil easy anions Motsuna algae, as well as Chroomonas diplococca, Chroomonas mesostigmatica, Chroomonas algae selected from Chroomonas nordstedtii and Chroomonas placoidea, and one or more selected from the group consisting of algae crypto Motsuna consisting Rhodomonas algae After culturing in a medium, collecting oil and fat having a content of lauric acid in the fatty acid composition of 3% by mass or more from the culture, esterifying lauric acid in the collected oil and fat, and then lauric acid or its ester A process for producing lauric acid or an ester thereof, characterized in that
[2] algae chlorambucil Lac anions Motsuna (Chlorarachniophyceae) is, Lotharella genus algae of Gymnochlora genus or Bigelowiella genus, method [1].
[3] The method according to [1] or [2], wherein the Lotharella algae is Lotharella globosa , Lotharella amoebiformis or Lotharella vacuolata , the Gymnochlora algae is Gymnochlora stellata , and the Bigelowiella algae is Bigelowiella natans .
[4] The method according to [3], wherein the algae of the genus Lotharella is a Lotharella globosa CCMP1729 strain, a Lotharella amoebiformis CCMP2058 strain, a Lotharella vacuolata CCMP240 strain or an algal strain having substantially the same mycological properties as the algal strain.
[5] The method according to [3], wherein the Gymnochlora algae is Gymnochlora stellata CCMP2057 strain or an algal strain having substantially the same mycological properties as the algal strain.
[6] The method according to [3], wherein the Bigelowiella genus algae is a Bigelowiella natans CCMP621 strain, CCMP2757 strain, or an algal strain having substantially the same mycological properties as the algal strain.
[7] Chroomonas genus algae having Chrononas diplococca UTEXLB2422 strain, Chroomonas mesostigmatica NIES1370 strain, Chroomonas nordstedtii NIES707 strain, Chroomonas nordstedtii NIES710 strain, Chroomonas placoidea NIES705 strain, or an algal strain substantially identical to these The method of [1], which is an algae strain.
[8] Rhodomonas algae is Rhodomonas salina, method [1].
[9] The method of [8], wherein Rhodomonas salina is Rhodomonas salina UTEX1375, Rhodomonas salina CCMP272, or an algal strain having substantially the same mycological properties as these algal strains.
[10] The method of [1] to [9], wherein the cells are cultured for 7 to 120 days under irradiation with light having an illuminance of 300 to 10,000 lux.
[11] The method of [1] to [10], wherein the lauric acid ester is methyl laurate.
[12] One or more Chroomonas algae selected from Chroomonas diplococca , Chroomonas mesostigmatica , Chroomonas nordstedtii, and Chroomonas placoidea are cultured in the medium, and the fat and oil having a lauric acid content in the fatty acid composition of 3% by mass or more is cultured from the culture. The manufacturing method of the fats and oils which contain lauric acid as a constituent fatty acid including collecting.
[13] Chroomonas genus algae having Chrononas diplococca UTEXLB2422 strain, Chroomonas mesostigmatica NIES1370 strain, Chroomonas nordstedtii NIES707 strain, Chroomonas nordstedtii NIES710 strain, Chroomonas placoidea NIES705 strain, or an algal strain substantially identical to these algal strains The method of [12], which is an algae strain.
[14] The method according to [12] or [13], wherein the cells are cultured for 7 to 120 days under irradiation with light having an illuminance of 300 to 10,000 lux.
実施例１ クロララクニオン藻綱藻類の培養と脂肪酸組成分析
実験に用いた藻類株として、The Provasoli-Guillard National Center for Culture of Marine Phytoplankton(CCMP)より以下の6株を入手した。
Example 1 The following 6 strains were obtained from The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) as algal strains used for culturing chloracranion algae and fatty acid composition analysis experiments.
藻類の培養は、以下のように行った。海水用として、市販培地であるダイゴＩＭＫ培地（日本製薬製、組成は表２参照）を用いた。 The culture of algae was performed as follows. A commercially available medium, Daigo IMK medium (manufactured by Nippon Pharmaceutical Co., Ltd., see Table 2 for the composition) was used for seawater.
培養容器として滅菌した１６ｍｍ×１５０ｍｍのカルチャーチューブ（ＶＷＲ製）とスポンジ栓（６０８８２−１６７：ＶＷＲ製）を使用し、滅菌した培地を１０ｍＬ分注した。藻類株は液体培地からは１００μＬ、固体培地からは１白金耳相当新しい培地に接種し、室温（２２℃〜２４℃）、蛍光灯下、照度約３０００ ｌｕｘ、１２時間明暗条件下で静置培養した。
藻類の培養液から３０００ｒｐｍ、３０分にて遠心分離した沈殿画分を得た。沈殿画分を８０℃にて約３時間から１６時間乾燥させ、乾燥藻体とした。乾燥藻体の重量を測定後、０．５ｍＬの１％食塩水にて懸濁し、内部標準として５ｍｇ／ｍＬの７−ペンタデカノンを１０μＬ添加後、０．５ｍＬのクロロホルムおよび１ｍＬのメタノールを培養液に添加して激しく攪拌後３０分間放置し、その後さらに０．５ｍＬのクロロホルムおよび０．５ｍＬの１．５％ＫＣｌを添加して攪拌後、３０００ｒｐｍ、１５分遠心分離を行い、パスツールピペットにてクロロホルム層（下層）を回収した。
A sterile 16 mm × 150 mm culture tube (manufactured by VWR) and a sponge stopper (60882-167: manufactured by VWR) were used as culture containers, and 10 mL of the sterilized medium was dispensed. Algae strains are inoculated into 100 μL of liquid medium and one medium equivalent to 1 platinum ear from solid medium, and statically cultured at room temperature (22 ° C. to 24 ° C.), under fluorescent light, at an illumination intensity of about 3000 lux, for 12 hours under light and dark conditions did.
A precipitate fraction was obtained by centrifugation at 3000 rpm for 30 minutes from the algae culture solution. The precipitate fraction was dried at 80 ° C. for about 3 to 16 hours to obtain dried alga bodies. After measuring the weight of the dried alga, suspended in 0.5 mL of 1% saline, added 10 μL of 5 mg / mL 7-pentadecanone as an internal standard, then added 0.5 mL of chloroform and 1 mL of methanol to the culture solution. Add and leave vigorously stirred for 30 minutes, then add 0.5 mL of chloroform and 0.5 mL of 1.5% KCl, and after stirring, centrifuge at 3000 rpm for 15 minutes. Chloroform with Pasteur pipette The layer (lower layer) was collected.
調製した脂質画分約５００μＬを窒素にて乾固後、０．５Ｎ 水酸化カリウム／メタノール溶液７００μＬを添加し、８０℃で３０分恒温した。続いて１ｍＬの１４％三フッ化ホウ素溶液（ＳＩＧＭＡ製）を添加し８０℃にて２０分恒温し、その後ヘキサン、飽和食塩水を各１ｍＬ添加し室温にて３０分放置後、上層であるヘキサン層を回収した後、ＧＣにて分析した。 About 500 μL of the prepared lipid fraction was dried with nitrogen, 700 μL of 0.5N potassium hydroxide / methanol solution was added, and the temperature was constant at 80 ° C. for 30 minutes. Subsequently, 1 mL of 14% boron trifluoride solution (manufactured by SIGMA) was added, and the temperature was kept constant at 80 ° C. for 20 minutes. After that, 1 mL each of hexane and saturated saline was added and allowed to stand at room temperature for 30 minutes. The layer was collected and analyzed by GC.
装置はＨＰ ７８９０Ａ ＧＣ−ＦＩＤ（Ａｇｉｌｅｎｔ製）、カラムはＤＢ−１ ｍｓ ３０ｍ×２００μｍ×０．２５μｍ（Ｊ＆Ｗ ｓｃｉｅｎｔｉｆｉｃ製）、移動相に高純度ヘリウムを用い、流量１ｍＬ／分、昇温プログラムは、１００℃（１分）、５℃／分、２８０℃（２０分）で行った。飽和脂肪酸のコントロールとして、ラウリン酸メチル（Ｃ１２）、ミリスチン酸メチル（Ｃ１４）、パルミチン酸メチル（Ｃ１６）、ステアリン酸メチル（Ｃ１８）を、不飽和脂肪酸はパルミトレイン酸メチル（Ｃ１６：１）、オレイン酸メチル（Ｃ１８：１）、リノール酸メチル（Ｃ１８：２）、リノレン酸メチル（Ｃ１８：３）、エイコサペンタエン酸メチル（Ｃ２０：５）、ドコサヘキサエン酸メチル（Ｃ２２：６）を購入し（全てＳＩＧＭＡ製）、分析した。脂肪酸の同定は、これら標準物質とのリテンションタイムと同一かどうかにより判断した。また、ラウリン酸に関してはＧＣ−ＭＳによる同定も行った。鎖長数１６の多価不飽和脂肪酸に関しては、ＧＣ−ＭＳによる解析結果から推定した。表記はC16:x(x=2または3)とし、xは不飽和結合数を示した。装置はＨＰ ７８９０Ａ ＧＣ及び５９７５Ｃ ＭＳ（Ａｇｉｌｅｎｔ製）、カラムはＤＢ−１ ｍｓ ３０ｍ×２００μｍ×０．２５μｍ（Ｊ＆Ｗ ｓｃｉｅｎｔｉｆｉｃ製）、移動相に高純度ヘリウムを用い、流量１ｍＬ／分、昇温プログラムは、１００℃（１分）、５℃／分、２８０℃（２０分）で行った。ＧＣ分析にて検出した脂肪酸エステル量を、内部標準を基準に算出し、その総量を総脂肪酸量とした。また総脂肪酸量を乾燥藻体量で除し１００を乗じた値を脂肪酸含量（％）とした。
各種藻類の脂肪酸組成データを表３に示した。
The apparatus is HP 7890A GC-FID (manufactured by Agilent), the column is DB-1 ms 30 m × 200 μm × 0.25 μm (manufactured by J & W scientific), high purity helium is used for the mobile phase, the flow rate is 1 mL / min, and the temperature raising program is It was carried out at 100 ° C. (1 minute), 5 ° C./minute, and 280 ° C. (20 minutes). As saturated fatty acid controls, methyl laurate (C12), methyl myristate (C14), methyl palmitate (C16), methyl stearate (C18), unsaturated fatty acids methyl palmitate (C16: 1), oleic acid Purchase methyl (C18: 1), methyl linoleate (C18: 2), methyl linolenate (C18: 3), methyl eicosapentaenoate (C20: 5), methyl docosahexaenoate (C22: 6) (all made by SIGMA) ),analyzed. The identification of the fatty acid was judged by whether it was the same as the retention time with these standard substances. In addition, lauric acid was also identified by GC-MS. The polyunsaturated fatty acid having a chain length of 16 was estimated from the analysis result by GC-MS. The notation was C16: x (x = 2 or 3), and x represents the number of unsaturated bonds. The equipment is HP 7890A GC and 5975C MS (manufactured by Agilent), the column is DB-1 ms 30 m × 200 μm × 0.25 μm (manufactured by J & W scientific), high purity helium is used for the mobile phase, the flow rate is 1 mL / min, and the temperature raising program is , 100 ° C. (1 minute), 5 ° C./minute, and 280 ° C. (20 minutes). The amount of fatty acid ester detected by GC analysis was calculated based on the internal standard, and the total amount was taken as the total amount of fatty acid. The total fatty acid amount was divided by the dry algal body amount and multiplied by 100 to obtain the fatty acid content (%).
Table 3 shows fatty acid composition data of various algae.
Lotharella globosa CCMP1729株、Lotharella amoebiformis CCMP2058株、Lotharella vacuolata CCMP240株、Gymnochlora stellata CCMP2057株、Bigelowiella natans CCMP621株、CCMP2757株において、総脂肪酸中３％以上のラウリン酸の蓄積が認められた。特にGymnochlora stellata CCMP2057において、高い脂肪酸生産性、及び総脂肪酸中約８．５％と高いラウリン酸の蓄積が認められた。 In the Lotharella globosa CCMP1729 strain, Lotharella amoebiformis CCMP2058 strain, Lotharella vacuolata CCMP240 strain, Gymnochlora stellata CCMP2057 strain, Bigelowiella natans CCMP621 strain and CCMP2757 strain, accumulation of lauric acid of 3% or more in total fatty acids was observed. In particular, Gymnochlora stellata CCMP2057 showed high fatty acid productivity and high accumulation of lauric acid, about 8.5% of the total fatty acids.
実施例２ クロララクニオン藻綱藻類を用いたラウリン酸の生産
Gymnochlora stellata CCMP2057を、１６ｍｍ×１５０ｍｍのカルチャーチューブ（ＩＭＫ培地１０ｍＬ仕込み）にて室温（２２℃〜２４℃）、照度約３０００ ｌｕｘ、１２時間明暗条件下で４週間静置培養した種培養液を、２００ｍＬ容三角フラスコ（ＩＭＫ培地１００ｍＬ仕込み）に２％（ｖ／ｖ）植菌し、室温（２２〜２４℃）、照度約３０００ ｌｕｘ、１２時間明暗条件下で３１日間静置培養した。培養液を３０００ｒｐｍで３０分間遠心して集菌し、１％（ｗ／ｖ）塩化ナトリウム水溶液で１回洗菌を行った。
Example 2 Production of Lauric Acid Using Chloraracnion Algae
A seed culture solution in which Gymnochlora stellata CCMP2057 was cultivated in a 16 mm × 150 mm culture tube (10 mL of IMK medium) at room temperature (22 ° C. to 24 ° C.), illuminance of about 3000 lux, and 12 hours light / dark conditions for 4 weeks, A 200 mL Erlenmeyer flask (100 mL of IMK medium) was inoculated with 2% (v / v) and statically cultured at room temperature (22-24 ° C.), illuminance of about 3000 lux, and 12 hours light / dark conditions for 31 days. The culture broth was collected by centrifugation at 3000 rpm for 30 minutes and washed once with a 1% (w / v) aqueous sodium chloride solution.
１００ｍＬの培養液から回収した藻体を、８０℃にて約１６時間乾燥させた。乾燥藻体に２ｍＬのクロロホルムおよび４ｍＬのメタノールを培養液に添加して激しく攪拌後３０分間放置し、その後さらに２ｍＬのクロロホルムおよび２ｍＬの１．５％ＫＣｌを添加して攪拌後、３０００ｒｐｍ、１５分遠心分離を行い、パスツールピペットにてクロロホルム層（下層）を回収した。回収したクロロホルム層より１００μＬを窒素ガス噴霧により乾固させ１０μＬのクロロホルムに溶解させた後、うち１μＬをイアトロスキャン（三菱化学ヤトロン社製）に供し中性脂質量を測定したところ、培養液１００ｍＬから１．２ｍｇの中性脂質を得た。 Algae bodies recovered from 100 mL of the culture solution were dried at 80 ° C. for about 16 hours. Add 2 mL of chloroform and 4 mL of methanol to the dried algae and stir vigorously for 30 minutes, then add 2 mL of chloroform and 2 mL of 1.5% KCl, stir and then 3000 rpm, 15 minutes Centrifugation was performed, and the chloroform layer (lower layer) was collected with a Pasteur pipette. After 100 μL of the recovered chloroform layer was dried by nitrogen gas spraying and dissolved in 10 μL of chloroform, 1 μL of the solution was subjected to Iatroscan (Mitsubishi Chemical Yatron Co., Ltd.) and the amount of neutral lipid was measured. 1.2 mg of neutral lipid was obtained.
先に回収したクロロホルム層のうち５００μＬを実施例１記載と同様にメチルエステル化し分析したところ、培養液１００ｍＬから得た総脂肪酸は６．２ｍｇ、総脂肪酸中のラウリン酸の割合は４．９％であった。これにより１００ｍＬの培養液より、ラウリン酸０．３ｍｇを得た。 When 500 μL of the previously recovered chloroform layer was methylesterified and analyzed in the same manner as described in Example 1, the total fatty acid obtained from 100 mL of the culture solution was 6.2 mg, and the proportion of lauric acid in the total fatty acid was 4.9%. Met. Thereby, 0.3 mg of lauric acid was obtained from 100 mL of the culture solution.
実施例３ Chromonas属藻類の培養と脂肪酸組成分析
実験に用いたChromonas属の藻類株として、The culture collection of algae at University of Texas at Austin (UTEX)、及び国立環境研究所(NIES)より以下の９株を入手した。
Example 3 Culture of Chromonas Algae and Analysis of Fatty Acid Composition As an algae strain of the genus Chromonas used in the experiment, the following 9 from the Culture Collection of algae at University of Texas at Austin (UTEX) and National Institute for Environmental Studies (NIES) The stock was obtained.
藻類の培養は、以下のように行った。淡水用としてＣ（組成は表５参照）、またはＷＡ培地（組成は表６参照）を、海水用として、f/2（組成は表７参照）または市販培地であるダイゴＩＭＫ培地（日本製薬製、組成は表８参照）を用いた。 The culture of algae was performed as follows. For fresh water use C (see Table 5 for composition) or WA medium (see Table 6 for composition), for seawater use f / 2 (see Table 7 for composition) or Daigo IMK medium (Nippon Pharmaceutical) The composition was as shown in Table 8.
培養容器として滅菌した１６ｍｍ×１５０ｍｍのカルチャーチューブ（ＶＷＲ製）とスポンジ栓（６０８８２−１６７：ＶＷＲ製）を使用し、滅菌した培地を１０ｍＬ分注した。藻類株は液体培地からは１００μＬ、固体培地からは１白金耳相当新しい培地に接種し、室温（２２℃〜２４℃）、蛍光灯下、照度約３０００ ｌｕｘ、１２時間明暗条件下で静置培養した。
藻類の培養液から３０００ｒｐｍ、３０分にて遠心分離した沈殿画分を得た。沈殿画分を８０℃にて約３時間から１６時間乾燥させ、乾燥藻体とした。乾燥藻体の重量を測定後、０．５ｍＬの１％食塩水にて懸濁し、内部標準として５ｍｇ／ｍＬの７−ペンタデカノンを１０μＬ添加後、０．５ｍＬのクロロホルムおよび１ｍＬのメタノールを培養液に添加して激しく攪拌後３０分間放置し、その後さらに０．５ｍＬのクロロホルムおよび０．５ｍＬの１．５％ＫＣｌを添加して攪拌後、３０００ｒｐｍ、１５分遠心分離を行い、パスツールピペットにてクロロホルム層（下層）を回収した。
A sterile 16 mm × 150 mm culture tube (manufactured by VWR) and a sponge stopper (60882-167: manufactured by VWR) were used as culture containers, and 10 mL of the sterilized medium was dispensed. Algae strains are inoculated into 100 μL of liquid medium and one medium equivalent to 1 platinum ear from solid medium, and statically cultured at room temperature (22 ° C. to 24 ° C.), under fluorescent light, at an illumination intensity of about 3000 lux, for 12 hours under light and dark conditions did.
A precipitate fraction was obtained by centrifugation at 3000 rpm for 30 minutes from the algae culture solution. The precipitate fraction was dried at 80 ° C. for about 3 to 16 hours to obtain dried alga bodies. After measuring the weight of the dried alga, suspended in 0.5 mL of 1% saline, added 10 μL of 5 mg / mL 7-pentadecanone as an internal standard, then added 0.5 mL of chloroform and 1 mL of methanol to the culture solution. Add and leave vigorously stirred for 30 minutes, then add 0.5 mL of chloroform and 0.5 mL of 1.5% KCl, and after stirring, centrifuge at 3000 rpm for 15 minutes. Chloroform with Pasteur pipette The layer (lower layer) was collected.
調製した脂質画分約５００μＬを窒素にて乾固後、０．５Ｎ 水酸化カリウム／メタノール溶液７００μＬを添加し、８０℃で３０分恒温した。続いて１ｍＬの１４％三フッ化ホウ素溶液（ＳＩＧＭＡ製）を添加し８０℃にて２０分恒温し、その後ヘキサン、飽和食塩水を各１ｍＬ添加し室温にて３０分放置後、上層であるヘキサン層を回収した後、ＧＣにて分析した。 About 500 μL of the prepared lipid fraction was dried with nitrogen, 700 μL of 0.5N potassium hydroxide / methanol solution was added, and the temperature was constant at 80 ° C. for 30 minutes. Subsequently, 1 mL of 14% boron trifluoride solution (manufactured by SIGMA) was added, and the temperature was kept constant at 80 ° C. for 20 minutes. After that, 1 mL each of hexane and saturated saline was added and allowed to stand at room temperature for 30 minutes. The layer was collected and analyzed by GC.
装置はＨＰ ７８９０Ａ ＧＣ−ＦＩＤ（Ａｇｉｌｅｎｔ製）、カラムはＤＢ−１ ｍｓ ３０ｍ×２００μｍ×０．２５μｍ（Ｊ＆Ｗ ｓｃｉｅｎｔｉｆｉｃ製）、移動相に高純度ヘリウムを用い、流量１ｍＬ／分、昇温プログラムは、１００℃（１分）、５℃／分、２８０℃（２０分）で行った。飽和脂肪酸のコントロールとして、ラウリン酸メチル（Ｃ１２）、ミリスチン酸メチル（Ｃ１４）、パルミチン酸メチル（Ｃ１６）、ステアリン酸メチル（Ｃ１８）を、不飽和脂肪酸はパルミトレイン酸メチル（Ｃ１６：１）、オレイン酸メチル（Ｃ１８：１）、リノール酸メチル（Ｃ１８：２）、リノレン酸メチル（Ｃ１８：３）、エイコサペンタエン酸メチル（Ｃ２０：５）、ドコサヘキサエン酸メチル（Ｃ２２：６）を購入し（全てＳＩＧＭＡ製）、分析した。脂肪酸の同定は、これら標準物質とのリテンションタイムと同一かどうかにより判断した。また、ラウリン酸に関してはＧＣ−ＭＳによる同定も行った。鎖長数１６の多価不飽和脂肪酸に関しては、ＧＣ−ＭＳによる解析結果から推定した。表記はC16:x(x=2または3)とし、xは不飽和結合数を示した。装置はＨＰ ７８９０Ａ ＧＣ及び５９７５Ｃ ＭＳ（Ａｇｉｌｅｎｔ製）、カラムはＤＢ−１ ｍｓ ３０ｍ×２００μｍ×０．２５μｍ（Ｊ＆Ｗ ｓｃｉｅｎｔｉｆｉｃ製）、移動相に高純度ヘリウムを用い、流量１ｍＬ／分、昇温プログラムは、１００℃（１分）、５℃／分、２８０℃（２０分）で行った。ＧＣ分析にて検出した脂肪酸エステル量を、内部標準を基準に算出し、その総量を総脂肪酸量（ｇ）とした。総脂肪酸量を培養液量（Ｌ）で除した値を生産性(ｇ/Ｌ，又はｍｇ/Ｌ)とした。また各脂肪酸量を総脂肪酸量で除し１００を乗じた値を脂肪酸含量（％）とした。
各種藻類の脂肪酸組成データを表９に示した。
The apparatus is HP 7890A GC-FID (manufactured by Agilent), the column is DB-1 ms 30 m × 200 μm × 0.25 μm (manufactured by J & W scientific), high purity helium is used for the mobile phase, the flow rate is 1 mL / min, and the temperature raising program is It was carried out at 100 ° C. (1 minute), 5 ° C./minute, and 280 ° C. (20 minutes). As saturated fatty acid controls, methyl laurate (C12), methyl myristate (C14), methyl palmitate (C16), methyl stearate (C18), unsaturated fatty acids methyl palmitate (C16: 1), oleic acid Purchase methyl (C18: 1), methyl linoleate (C18: 2), methyl linolenate (C18: 3), methyl eicosapentaenoate (C20: 5), methyl docosahexaenoate (C22: 6) (all made by SIGMA) ),analyzed. The identification of the fatty acid was judged by whether it was the same as the retention time with these standard substances. In addition, lauric acid was also identified by GC-MS. The polyunsaturated fatty acid having a chain length of 16 was estimated from the analysis result by GC-MS. The notation was C16: x (x = 2 or 3), and x represents the number of unsaturated bonds. The equipment is HP 7890A GC and 5975C MS (manufactured by Agilent), the column is DB-1 ms 30 m × 200 μm × 0.25 μm (manufactured by J & W scientific), high purity helium is used for the mobile phase, the flow rate is 1 mL / min, and the temperature raising program is , 100 ° C. (1 minute), 5 ° C./minute, and 280 ° C. (20 minutes). The amount of fatty acid ester detected by GC analysis was calculated based on the internal standard, and the total amount was defined as the total amount of fatty acid (g). The value obtained by dividing the total amount of fatty acid by the amount of culture solution (L) was defined as productivity (g / L or mg / L). Further, the value obtained by dividing the amount of each fatty acid by the total amount of fatty acids and multiplying by 100 was defined as the fatty acid content (%).
Table 9 shows the fatty acid composition data of various algae.
Chroomonas diplococca UTEXLB2422株、Chroomonas mesostigmatica NIES1370株、Chroomonas nordstedtii NIES707株、Chroomonas nordstedtii NIES 710株、Chroomonas placoidea NIES705株において、総脂肪酸中５％以上のラウリン酸の蓄積が認められた。特にChroomonas diplococca LB2422株において、総脂肪酸中約１７％と非常に高いラウリン酸の蓄積が認められた。 In Chroomonas diplococca UTEXLB2422 strain, Chroomonas mesostigmatica NIES1370 strain, Chroomonas nordstedtii NIES707 strain, Chroomonas nordstedtii NIES 710 strain and Chroomonas placoidea NIES705 strain, accumulation of lauric acid of 5% or more in total fatty acids was observed. In particular, in Chroomonas diplococca LB2422 strain, a very high accumulation of lauric acid, about 17% of the total fatty acids, was observed.
実施例４ Chromonas属藻類を用いたラウリン酸の生産
Chroomonas diplococca(LB2422株)を、１６ｍｍ×１５０ｍｍのカルチャーチューブ（ＩＭＫ培地１０ｍＬ仕込み）にて、室温（２２℃〜２４℃）、照度約３０００ ｌｕｘ、１２時間明暗条件下で４週間静置培養した種培養液を、２００ｍＬ容三角フラスコ（ＩＭＫ培地１００ｍＬ仕込み）に２％（ｖ／ｖ）植菌し、室温（２２〜２４℃）、照度約３０００ ｌｕｘ、１２時間明暗条件下で３１日間静置培養した。培養液を３０００ｒｐｍで３０分間遠心して集菌し、１％（ｗ／ｖ）塩化ナトリウム水溶液で１回洗菌を行った。
Example 4 Production of Lauric Acid Using Chromonas Algae
Species obtained by stationary culture of Chroomonas diplococca (LB2422 strain) in a 16 mm x 150 mm culture tube (10 mL of IMK medium) at room temperature (22 ° C to 24 ° C), illuminance of about 3000 lux, and light / dark conditions for 12 hours. Inoculate 2% (v / v) of the culture solution into a 200 mL Erlenmeyer flask (100 mL of IMK medium), and statically culture for 31 days at room temperature (22-24 ° C.), illuminance of about 3000 lux, 12 hours light / dark conditions did. The culture broth was collected by centrifugation at 3000 rpm for 30 minutes and washed once with a 1% (w / v) aqueous sodium chloride solution.
１００ｍＬの培養液から回収した藻体を、８０℃にて約１６時間乾燥させた。乾燥藻体に２ｍＬのクロロホルムおよび４ｍＬのメタノールを培養液に添加して激しく攪拌後３０分間放置し、その後さらに２ｍＬのクロロホルムおよび２ｍＬの１．５％ＫＣｌを添加して攪拌後、３０００ｒｐｍ、１５分遠心分離を行い、パスツールピペットにてクロロホルム層（下層）を回収した。回収したクロロホルム層より１００μＬを窒素ガス噴霧により乾固させ１０μＬのクロロホルムに溶解させた後、うち１μＬをイアトロスキャン（三菱化学ヤトロン社製）に供し中性脂質量を測定したところ、培養液１００ｍＬから０．６４ｍｇの中性脂質を得た。 Algae bodies recovered from 100 mL of the culture solution were dried at 80 ° C. for about 16 hours. Add 2 mL of chloroform and 4 mL of methanol to the dried algae and stir vigorously for 30 minutes, then add 2 mL of chloroform and 2 mL of 1.5% KCl, stir and then 3000 rpm, 15 minutes Centrifugation was performed, and the chloroform layer (lower layer) was collected with a Pasteur pipette. After 100 μL of the recovered chloroform layer was dried by nitrogen gas spraying and dissolved in 10 μL of chloroform, 1 μL of the solution was subjected to Iatroscan (Mitsubishi Chemical Yatron Co., Ltd.) and the amount of neutral lipid was measured. 0.64 mg of neutral lipid was obtained.
先に回収したクロロホルム層のうち５００μＬを実施例３記載と同様にメチルエステル化し分析したところ、培養液１００ｍＬから得た総脂肪酸は３．５ｍｇ、総脂肪酸中のラウリン酸の割合は５．７％であった。これにより１００ｍＬの培養液より、ラウリン酸０．２ｍｇを得た。 When 500 μL of the previously collected chloroform layer was methylesterified and analyzed in the same manner as described in Example 3, the total fatty acid obtained from 100 mL of the culture solution was 3.5 mg, and the proportion of lauric acid in the total fatty acid was 5.7%. Met. Thereby, 0.2 mg of lauric acid was obtained from 100 mL of the culture solution.
実施例５ Rhodomonas属藻類の培養と脂肪酸組成分析
Rhodomonas属の藻類として、The culture collection of algae at University of Texas at Austin (UTEX)、及びThe Provasoli-Guillard National Center for Culture of Marine Phytoplankton（CCMP）より、Rhodomonas salina UTEX1375、Rhodomonas salina CCMP272を購入し、IMK培地にて実施例３と同様の試験を行った。表９記載の培養時間にて、培養時間以外は実施例３と同じ条件で試験を行い、総脂肪酸の生産性及び各脂肪酸の割合を測定した。その結果、両藻類とも３％以上のラウリン酸の割合を示し、それぞれ９．４％及び８．８％であった。
Example 5 Cultivation and fatty acid composition analysis of Rhodomonas algae
Purchased Rhodomonas salina UTEX1375 and Rhodomonas salina CCMP272 from The culture collection of algae at University of Texas at Austin (UTEX) and The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP) The same test as in Example 3 was performed on the medium. Tests were carried out under the same culture conditions as in Example 3 except for the culture time, and the total fatty acid productivity and the ratio of each fatty acid were measured. As a result, both algae showed a ratio of lauric acid of 3% or more, which were 9.4% and 8.8%, respectively.
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