WO2011018896A1 - アスタキサンチン含有組成物の製造方法 - Google Patents
アスタキサンチン含有組成物の製造方法 Download PDFInfo
- Publication number
- WO2011018896A1 WO2011018896A1 PCT/JP2010/005036 JP2010005036W WO2011018896A1 WO 2011018896 A1 WO2011018896 A1 WO 2011018896A1 JP 2010005036 W JP2010005036 W JP 2010005036W WO 2011018896 A1 WO2011018896 A1 WO 2011018896A1
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- WIPO (PCT)
- Prior art keywords
- astaxanthin
- hdco
- genus
- composition
- relative amount
- Prior art date
Links
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 title claims abstract description 139
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 title claims abstract description 139
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 139
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 139
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 139
- 239000000203 mixture Substances 0.000 title claims abstract description 75
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 38
- 239000000284 extract Substances 0.000 claims description 38
- 241001000247 Xanthophyllomyces Species 0.000 claims description 30
- 230000002378 acidificating effect Effects 0.000 claims description 26
- 244000005700 microbiome Species 0.000 claims description 11
- 239000012264 purified product Substances 0.000 claims description 8
- 235000013305 food Nutrition 0.000 claims description 7
- 235000013373 food additive Nutrition 0.000 claims description 7
- 239000002778 food additive Substances 0.000 claims description 7
- 241000589158 Agrobacterium Species 0.000 claims description 3
- 241000131407 Brevundimonas Species 0.000 claims description 3
- 241000195585 Chlamydomonas Species 0.000 claims description 3
- 241000190844 Erythrobacter Species 0.000 claims description 3
- 241000168525 Haematococcus Species 0.000 claims description 3
- 241001478792 Monoraphidium Species 0.000 claims description 3
- 241001057811 Paracoccus <mealybug> Species 0.000 claims description 3
- 230000008827 biological function Effects 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract 1
- JQSLJBXSPJMQQS-ZGZGHAEGSA-N 3-Hydroxy-3',4'-didehydro-beta,psi-caroten-4-one Chemical compound CC(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)C(=O)C(O)CC1(C)C JQSLJBXSPJMQQS-ZGZGHAEGSA-N 0.000 description 92
- 210000004027 cell Anatomy 0.000 description 67
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 40
- 239000002609 medium Substances 0.000 description 39
- 239000000243 solution Substances 0.000 description 30
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 27
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 18
- 239000002904 solvent Substances 0.000 description 18
- 239000011521 glass Substances 0.000 description 13
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 12
- 239000011324 bead Substances 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 150000001875 compounds Chemical class 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 238000010306 acid treatment Methods 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 6
- 241000222057 Xanthophyllomyces dendrorhous Species 0.000 description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 4
- 241001542817 Phaffia Species 0.000 description 4
- 235000021466 carotenoid Nutrition 0.000 description 4
- 150000001747 carotenoids Chemical class 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000004254 Ammonium phosphate Substances 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 3
- 235000019289 ammonium phosphates Nutrition 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000012046 mixed solvent Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 description 3
- 235000011009 potassium phosphates Nutrition 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000007218 ym medium Substances 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001491670 Labyrinthula Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 210000005056 cell body Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- -1 staxanthin Species 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000233675 Thraustochytrium Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910000272 alkali metal oxide Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 229910001860 alkaline earth metal hydroxide Inorganic materials 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/01—Hydrocarbons
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/174—Vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/179—Colouring agents, e.g. pigmenting or dyeing agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- 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
- C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
Definitions
- the present invention relates to a method for producing an astaxanthin-containing composition.
- it relates to a method for reducing the relative amount of HDCO to astaxanthin in an astaxanthin-containing composition comprising 3-hydroxy-3 ′, 4′-didehydro- ⁇ , ⁇ -caroten-4-one (HDCO).
- HDCO 3-hydroxy-3 ′, 4′-didehydro- ⁇ , ⁇ -caroten-4-one
- Astaxanthin is a naturally occurring carotenoid, widely used as an additive for feed to improve the color of meat and skin of cultured fish, and has recently attracted attention as a functional food material.
- Astaxanthin is derived from the genus Xanthophyllomyces (formerly Phaffia), Brevundimonas, Haematococcus, Chlamydomonas, and Monoraphidium. ), Erythrobacter genus, Agrobacterium genus, Paracoccus genus, Labyrinthulea, etc. It is also possible to manufacture it.
- HDCO 3-Hydroxy-3 ', 4'-didehydro- ⁇ , ⁇ -carotene-4-one
- Patent Document 1 describes both a high astaxanthin content and a low HDCO content among yeast strains of the genus Xanthophyllomyces. To obtain an astaxanthin-containing composition from the mutant strain.
- Patent Document 1 as a method for obtaining a target mutant strain, from a solid group of red colonies (having a high astaxanthin content and a relatively high HDCO content relative to astaxanthin), a high astaxanthin content and a relatively high relative to astaxanthin. It is described to visually select intensely colored orange colonies with low HDCO content. However, since mutants with a lower astaxanthin content have a weaker colony redness, it is very difficult to select a target strain based only on the color of the colony, and the achievement of the purpose is affected by chance, and it takes a lot of time and effort. .
- an object of the present invention is to provide an extremely simple method that can reduce the relative amount of HDCO to astaxanthin in an astaxanthin-containing composition containing HDCO.
- the inventors of the present application have brought the composition containing astaxanthin and HDCO into contact with an acidic medium having a pH of 3 or less and / or a basic medium having a pH of 9 or more. It has been found that the relative amount of HDCO to astaxanthin in the product can be reduced, and the present invention has been completed.
- the present invention is a method in which a composition containing astaxanthin and HDCO is brought into contact with an acidic medium having a pH of 3 or less and / or a basic medium having a pH of 9 or more to reduce the relative amount of HDCO to astaxanthin in the composition. is there.
- the composition containing astaxanthin and HDCO includes a step of bringing the composition containing astaxanthin and HDCO into contact with an acidic medium having a pH of 3 or less and / or a basic medium having a pH of 9 or more to reduce the relative amount of HDCO to astaxanthin in the composition. It is a manufacturing method of a composition. Furthermore, it is a feed, a food, a food additive, and a pharmaceutical containing the astaxanthin-containing composition obtained by the above method.
- the relative amount of HDCO to astaxanthin in the composition containing astaxanthin and HDCO can be efficiently reduced.
- Astaxanthin-containing compositions having a reduced relative amount of HDCO with respect to astaxanthin are useful, for example, as feeds, foods, food additives, and pharmaceuticals.
- composition containing astaxanthin and HDCO used in the present invention is not particularly limited as long as it contains both compounds, but for example, a culture solution or culture of cells that simultaneously produce both compounds Supernatant, cell bodies, and crushed cells, dried products, extracts and the like, and partially purified products thereof are further exemplified.
- a composition containing astaxanthin produced by chemical synthesis corresponds to the composition containing astaxanthin and HDCO used in the present invention as long as it contains HDCO.
- Examples of cells that produce both compounds simultaneously include, for example, the genus Xanthophyllomyces (formerly Phaffia), the genus Brevundimonas, the genus Haematococcus, and the Chlamydomonas.
- Microorganisms belonging to the genus, Monoraphidium genus, Erythrobacter genus, Agrobacterium genus, Paracoccus genus, Labyrinthula (Labyrinthulea), etc., these mutants, these Examples include, but are not limited to, genetically modified strains and cells such as these self-cloning strains.
- microorganism belonging to Labyrinthulea examples include microorganisms belonging to the genus Thraustochytrium, the genus Schizothytrium, and the genus Labyrinthula.
- the acidic medium used in the present invention is a pH of 3 or less, preferably 2 or less, more preferably 1 or less, still more preferably 0.5 or less, and even more preferably 0.1 or less when pH is measured by the glass electrode method.
- it is obtained by dissolving an acidic substance in a suitable solvent.
- the acidic substance is not particularly limited as long as the effects of the present invention can be obtained. Examples thereof include inorganic acids such as hydrogen chloride, sulfuric acid, phosphoric acid and nitric acid, and organic acids such as formic acid and acetic acid. Hydrogen and sulfuric acid are particularly preferred. These acidic substances may be used alone or in any combination.
- the basic medium used in the present invention is a pH of 9 or more, preferably 10 or more, more preferably 11 or more, more preferably 12 or more, still more preferably 13 or more, and more preferably when pH is measured by the glass electrode method.
- the liquid substance showing 13.5 or more, more preferably 13.9 or more is indicated.
- the basic substance is not particularly limited, but examples include alkali metal or alkaline earth metal hydroxides such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, Ammonia, amines and the like can be mentioned, and sodium hydroxide is particularly preferable. These basic substances may be used alone or in combination.
- the solvent for dissolving the acidic substance and / or the basic substance is not particularly limited as long as the effect of the present invention can be obtained. Examples thereof include water, ethanol, acetone, methanol, and a mixed solvent thereof. Water is preferred. It is also possible to use the solvent contained in the composition containing astaxanthin and HDCO as a solvent constituting the acidic medium and / or the basic medium.
- the acidic medium has a pH of 3 or less, preferably 2 or less, more preferably 1 or less, still more preferably 0.5 or less, and even more preferably 0.1 or less when measured by the above method, and the effects of the present invention can be obtained.
- compounds other than the acidic substance and the solvent may be included.
- the basic medium has a pH of 9 or more, preferably 10 or more, more preferably 11 or more, still more preferably 12 or more, still more preferably 13 or more, still more preferably 13.5 or more, more preferably when measured by the above method. Indicates 13.9 or more, and may contain compounds other than the basic substance and the solvent as long as the effects of the present invention can be obtained.
- the step of bringing the composition containing astaxanthin and HDCO into contact with the acidic medium and / or the basic medium is not particularly limited as long as the effect of the present invention is obtained, but includes, for example, astaxanthin and HDCO. It can be carried out by putting the composition, acidic medium and / or basic medium in a suitable container, reaction can, etc., and mixing as necessary. Mixing can also be carried out in piping.
- the upper limit temperature is usually 140 ° C., preferably 100 ° C., more preferably 90 ° C., and further preferably 70 ° C.
- the lower limit temperature is usually ⁇ 20 ° C., preferably 0 ° C., more preferably 10 ° C., and further preferably 30 ° C. If the upper limit temperature is exceeded, astaxanthin tends to be unstable, and if the lower limit temperature is not reached, operation tends to be difficult.
- the time for bringing the composition containing astaxanthin and HDCO into contact with the acidic medium or the basic medium is not particularly limited as long as the effect of the present invention is obtained, but the upper limit time is usually 12000 minutes, preferably 1200 minutes. Preferably it is 300 minutes.
- the lower limit time is usually 1 minute, preferably 10 minutes, more preferably 60 minutes.
- the pH of the acidic medium and / or the basic medium for contacting the composition containing astaxanthin and HDCO, the temperature at the time of contacting, and the time for contacting can be selected from any combination, and the effects of the present invention can be obtained. As long as it is possible, the combination is not particularly limited. Depending on the form of the composition containing astaxanthin and HDCO, the type of acidic medium and / or basic medium used, the pH of the acidic medium and / or basic medium used, the mixing conditions during the contact treatment, etc. Just choose.
- the relative amount of HDCO relative to astaxanthin in the composition containing astaxanthin and HDCO is the peak area of astaxanthin and the peak of HDCO on the analysis chart obtained by analyzing the composition by HPLC and detecting the absorbance at 471 nm. From the area, it can be calculated by the following formula.
- Relative amount of HDCO to astaxanthin (%) (Peak area of HDCO / peak area of astaxanthin) ⁇ 100
- the above-mentioned analysis by HPLC uses, for example, YMC Carotenoid (4.6 ⁇ 250 mm; YMC) as the column, the column temperature is 20 ° C., and the mobile phase is liquid A: methanol / methyl-t-butyl ether / 1.
- % Phosphoric acid aqueous solution 82/15/3
- Analysis of the composition containing astaxanthin and HDCO using HPLC can be performed, for example, by dissolving the composition in an appropriate solvent as necessary and injecting the composition into an HPLC apparatus.
- the solvent for dissolving the composition for example, dimethyl sulfoxide, acetone, chloroform, methylene chloride, methanol, and a mixed solvent of any combination thereof can be used, but the composition can be dissolved. If there is no particular limitation.
- the composition contains a component insoluble in these solvents such as a culture solution, cell body, or a crushed product of cells having astaxanthin-producing ability, for example, the composition
- the analysis can be performed by injecting an extract obtained by mechanical crushing in a solvent into an HPLC apparatus. Examples of the mechanical crushing method include a method using glass beads and a pressure crushing method.
- the composition containing astaxanthin and HDCO used in the present invention is not particularly limited as long as it contains both compounds.
- the relative amount of HDCO to astaxanthin obtained by the above-mentioned method is preferably 1% or more, More preferably, the composition is 5% or more. From the viewpoint of maximizing the effects of the present invention, the relative amount of HDCO with respect to astaxanthin is more preferably 10% or more, and particularly preferably 15% or more.
- the astaxanthin-containing composition obtained from the cells of the genus Xanthophyllomyces having an astaxanthin content of 2000 ⁇ g / g dry cells or more or the cells. Things are preferred.
- xanthophyllomyces having an astaxanthin content of 3000 ⁇ g / g dry cells or more, more preferably 5000 ⁇ g / g dry cells or more, still more preferably 8000 ⁇ g / g dry cells or more, particularly preferably 10,000 ⁇ g / g dry cells or more.
- Xanthophyllomyces) microorganism cell or an astaxanthin-containing composition obtained from the cell In these Xanthophyllomyces microorganisms or astaxanthin-containing compositions obtained from the microorganisms, the relative amount of HDCO with respect to astaxanthin is preferably 10% or more, more preferably 15% or more. .
- the relative amount (%) of HDCO to astaxanthin determined by the above method is: Usually, it decreases by 0.1 percent point (pp) or more, preferably 0.5 pp or more, more preferably 1 pp or more, further preferably 2 pp or more, more preferably 3 pp or more, further preferably 4 pp or more, and further preferably 5 pp or more.
- Astaxanthin-containing compositions with a reduced relative amount of HDCO to astaxanthin prepared according to the present invention are neutralized with an acidic medium and / or a basic medium as necessary, and then, if necessary, these compositions are subjected to ordinary operations. Astaxanthin-containing compositions that can be used as feeds, foods, food additives, pharmaceuticals, and the like can be obtained. Moreover, after further refine
- a composition having a reduced relative amount of HDCO to astaxanthin according to the present invention can be dissolved and phase-separated between an acidic medium and / or a basic medium.
- a solvent present, the solution obtained by dissolving the composition in the solvent is separated from the acidic medium and / or the basic medium, washed with water, and the solvent is distilled off.
- the above-described normal operation is performed by separating the composition from an acidic medium and / or a basic medium by, for example, continuous centrifugation or filtration. It can be implemented by washing with water accordingly.
- the normal operation is not limited to these.
- the astaxanthin-containing composition thus obtained can be further processed into a product form suitable as a feed, food, food additive, or pharmaceutical product, and the product is also included in the present invention.
- feed includes, for example, aquatic animals such as fish, crustaceans and shellfish, poultry such as chickens, quails and ducks, livestock animals such as cows, pigs and sheep, pets such as dogs and cats, etc. It refers to feed and supplements to be given, but is not limited thereto.
- a foodstuff includes a coloring agent, a supplement, etc. other than a regular meal, it is not limited to these.
- the relative amount of HDCO with respect to astaxanthin in the composition containing astaxanthin and HDCO can be efficiently reduced. From the composition containing astaxanthin and HDCO, It is possible to efficiently obtain an astaxanthin-containing composition that is suitable for use as feed, food, food additives, pharmaceuticals, etc. and contains less HDCO.
- Extraction method for analyzing astaxanthin and HDCO When the composition containing astaxanthin and HDCO is, for example, microbial cells, a mechanical crushing method using glass beads can be used as a method for obtaining an extract containing astaxanthin and HDCO. The extract containing astaxanthin and HDCO obtained by this method can be subjected to analysis using an HPLC apparatus.
- Xanthophyllomyces dendrorhous NBRC 10129 strain National Institute of Technology and Evaluation, Biological Genetic Resources Division, 2-9-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818) N- After mutation treatment with methyl-N′-nitro-N-nitrosoguanidine (NTG), a strong reddish colony was selected on an agar plate to obtain a mutant strain with improved astaxanthin content. By repeating the same operation on the obtained mutant strain, a mutant strain having further improved astaxanthin content was obtained. In this way, Xanthophyllomyces dendrorhous strains KNK-01, KNK-02-1, KNK-02-2 and KNK-03, which are mutants with improved astaxane content, were obtained. did.
- Table 1 shows the astaxanthin content of the obtained mutant strain and the relative amount of HDCO with respect to astaxanthin.
- the mutant strain was cultured as follows. Place 30 ml of medium (1.3% ammonium phosphate, 0.7% potassium phosphate, 0.6% succinic acid, 0.3% yeast extract, pH 5.4) in a 500 ml Sakaguchi flask and sterilize by autoclave. did. Astaxanthin-producing strain was inoculated and cultured with shaking at 20 ° C. for 180 hours. At the start of the culture, 0.3 g of glucose was added, and thereafter 0.3 g of glucose was replenished every 12 hours.
- Preparation Example 2 10 ml of the culture solution obtained in Preparation Example 1 was dispensed into a 50 ml centrifuge tube, and after centrifugation, the supernatant was discarded to obtain a cell pellet. To this, 25 g of glass beads with a diameter of 0.5 mm and 25 ml of acetone were added and sealed, and then the cells were crushed using a multi-bead shocker (manufactured by Yasui Kikai). This was centrifuged to recover the acetone phase of the supernatant, and the solvent was distilled off under reduced pressure to obtain a cell extract containing astaxanthin and HDCO. The astaxanthin concentration in the bacterial cell extract was 6.1 mg / g.
- Preparation Example 5 50 ml of the culture solution obtained in Preparation Example 4 was dispensed into a 50 ml centrifuge tube, and after centrifugation, the supernatant was discarded to obtain a cell pellet. To this, 25 g of glass beads with a diameter of 0.5 mm and 25 ml of acetone were added and sealed, and then the cells were crushed using a multi-bead shocker (manufactured by Yasui Kikai). This was centrifuged to recover the acetone phase of the supernatant, and the solvent was distilled off under reduced pressure to obtain a cell extract containing astaxanthin and HDCO. The astaxanthin concentration in the bacterial cell extract was 5.6 mg / g.
- Preparation Example 6 Culturing was carried out in the same manner as in Preparation Example 4 except that the microorganism was Xanthophyllomyces dendrorhous KNK-02-1 strain, and a culture solution containing cells containing astaxanthin and HDCO was obtained. .
- Example 1 Treatment of cell extract of Xanthophyllomyces dendroas strain KNK-03 with sulfuric acid 10 mg of the cell extract obtained in Preparation Example 2 was put into a sealable test tube, and the concentrations shown in Table 1 below were obtained. 3 ml of an aqueous sulfuric acid solution was added and stirred. The pH of the solution after stirring was measured using an F-22 type pH meter (Horiba Seisakusho).
- Relative amount of HDCO to astaxanthin (Peak area of HDCO / peak area of astaxanthin) ⁇ 100
- YMC Carotenoid (4.6 ⁇ 250 mm; YMC) was used as the column, the column temperature was 20 ° C., and the mobile phase was liquid A: methanol / methyl-t-butyl ether / 1% phosphoric acid.
- Aqueous solution 82/15/3
- B solution: methanol / methyl-t-butyl ether / water / phosphoric acid 7/90/3 / 0.03, 0 to 30 minutes after sample injection, 100% of A solution , 30 to 90 minutes were conducted with a linear gradient from 100% of solution A to 100% of solution B, and 90 to 95 minutes of solution B being 100%. Under these conditions, astaxanthin was detected at about 13 minutes after the start of analysis, and HDCO was detected at about 80 minutes after the start of analysis.
- Table 2 shows that the relative amount of HDCO with respect to astaxanthin in the bacterial cell extract decreased by contacting the cell extract of Xanthophyllomyces dendroas strain KNK-03 containing astaxanthin and HDCO with sulfuric acid. Show.
- Example 2 Hydrochloric acid treatment of cell extract of Xanthophyllomyces dendroas strain KNK-03 The same procedure as in Example 1 was carried out except that hydrochloric acid was used instead of sulfuric acid. The results are shown in Table 3.
- Table 3 shows that the relative amount of HDCO with respect to astaxanthin in the bacterial cell extract decreased by contacting the cell extract of Xanthophyllomyces dendrous strain KNK-03 containing astaxanthin and HDCO with hydrochloric acid. Show. Moreover, from the results of Tables 2 and 3, the effects of the present invention were obtained regardless of the type of acidic medium.
- Example 3 Sulfuric acid treatment of partially purified Xanthophyllomyces dendroasu KNK-03 cell extract
- the partially purified product obtained in Preparation Example 3 was used in place of the cell extract obtained in Preparation Example 2.
- the procedure was the same as in Example 1 except that. The results are shown in Table 4.
- Table 4 shows the relative amount of HDCO with respect to astaxanthin in the cell extract by bringing a partially purified product of the cell extract of Xanthophyllomyces dendroas strain KNK-03 containing astaxanthin and HDCO into contact with sulfuric acid. Indicates a drop.
- Example 4 Sodium hydroxide treatment of cell extract of Xanthophyllomyces dendroas strain KNK-03 The same procedure as in Example 1 was conducted except that sodium hydroxide was used instead of sulfuric acid. The results are shown in Table 5.
- Table 5 shows that treatment with sodium hydroxide decreases the relative amount of HDCO to astaxanthin in the cell extract.
- Example 5 Potassium hydroxide treatment of cell extract of Xanthophyllomyces dendroas strain KNK-03 The same procedure as in Example 1 was carried out except that potassium hydroxide was used instead of sulfuric acid. The results are shown in Table 6.
- Table 6 shows that the treatment with potassium hydroxide reduces the relative amount of HDCO to astaxanthin in the cell extract. From the results of Tables 5 and 6, the effects of the present invention were obtained regardless of the type of basic medium.
- Example 6 Sodium Hydroxide Treatment of Partially Purified Xanthophyllomyces dendroas KNK-03 Cell Extract Partially Purified Product Obtained in Preparative Example 3 instead of Cell Extract Obtained in Preparative Example 2 This was carried out in the same manner as in Example 4 except that was used. The results are shown in Table 7.
- Table 7 shows the results of contacting HDCO with respect to astaxanthin in the cell extract by contacting a partially purified product of cell extract of Xanthophyllomyces dendroas strain KNK-03 containing astaxanthin and HDCO with sodium hydroxide. It indicates that the relative amount has decreased.
- Example 7 Sulfuric acid treatment of extract of Xanthophyllomyces dendroas KNK-01 strain cells Except that the cell extract obtained in Preparation Example 5 was used instead of the cell extract obtained in Preparation Example 2 The same operation as in Example 1 was performed. The results are shown in Table 8.
- Table 8 shows the relative amount of HDCO with respect to astaxanthin in the cell extract by bringing a partially purified product of the cell extract of Xanthophyllomyces dendroas strain KNK-01 containing astaxanthin and HDCO into contact with sulfuric acid. Indicates a drop.
- Example 8 Sulfuric acid treatment of Xanthophyllomyces dendrous strain KNK-03 cells Sulfuric acid was added to the culture solution obtained in Preparation Example 1 so that the final concentrations shown in Table 9 were obtained. The results of measuring the pH of the culture solution after addition of sulfuric acid in the same manner as in Example 1 are shown in Table 9. Next, dispense 10 ml of each culture solution into a sealable glass container and stir for 2 hours at 30 ° C., 50 ° C. or 70 ° C. After treatment for 2 hours, 0.05 ml of each culture solution can be sealed In a 2 ml polypropylene tube, the cells were sedimented by centrifugation.
- the cells were resuspended in 1 ml of water, the cells were sedimented by centrifugation again, and the supernatant was removed.
- 1 g of glass beads ( ⁇ 0.5 mm) and 1 ml of acetone were added, and the cells were crushed using a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.).
- the contents of the tube were filtered (Cosmonis filter S, 0.45 ⁇ m, manufactured by Millipore), and the filtrate was subjected to HPLC analysis.
- the HPLC analysis and the calculation of the relative amount of HDCO with respect to astaxanthin were performed in the same manner as in Example 1. The results are shown in Table 9.
- Example 9 Sodium hydroxide treatment of Xanthophyllomyces dendrous KNK-03 strain cells The same procedure as in Example 8 was performed except that sodium hydroxide was used instead of sulfuric acid. The results are shown in Table 10.
- Example 10 Sulfuric acid treatment of Xanthophyllomyces dendrous strain KNK-03 cells 50 ml of the culture solution obtained in Preparation Example 1 was dispensed into a sealable glass container, and sulfuric acid was added to pH 7.0 and pH3. Adjusted to 0.0. This was sealed and stirred in a 70 ° C. water bath. After 0 hour, 12 hours, 24 hours, and 48 hours, 0.05 ml was collected, and the relative amount of HDCO to astaxanthin in the cells was calculated in the same manner as in Example 8. The results are shown in Table 11.
- Example 11 Sulfuric acid treatment of cells of Xanthophyllomyces dendrous KNK-02-1 50 ml of the culture solution obtained in Preparation Example 6 was dispensed into a sealable glass container, and sulfuric acid was added to adjust the pH to 7.0. And adjusted to pH 1.0. This was sealed and stirred in a 70 ° C. water bath. 0.05 ml each was taken after 0 hour, 3 hours, and 6 hours, and the relative amount of HDCO to astaxanthin in the cells was calculated in the same manner as in Example 8.
- Tables 9 to 12 show that the effects of the present invention can be obtained even when cells of Xanthophyllomyces dendroas are used as a composition containing astaxanthin and HDCO.
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Abstract
Description
本願発明は、アスタキサンチン含有組成物の製造方法に関する。とりわけ、3-ヒドロキシ-3’,4’-ジデヒドロ-β,Ψ-カロテン-4-オン(HDCO)を含むアスタキサンチン含有組成物中の、アスタキサンチンに対するHDCOの相対量を低減する方法に関する。
アスタキサンチンに対するHDCOの相対量(%)
=(HDCOのピーク面積/アスタキサンチンのピーク面積)×100
上記のHPLCによる分析は、例えば、カラムとしてYMC Carotenoid(4.6×250mm;YMC社)を使用し、カラム温度を20℃とし、移動相として、A液:メタノール/メチル-t-ブチルエーテル/1%リン酸水溶液=82/15/3、および、B液:メタノール/メチル-t-ブチルエーテル/水/リン酸=7/90/3/0.03を用い、試料注入後0~30分はA液100%、30~90分はA液100%からB液100%へのリニアグラジエント、90~95分はB液100%の条件で、1.0ml/分の流速で通液することにより実施できる。
(処理前の組成物におけるアスタキサンチンに対するHDCOの相対量(%))-(処理後の組成物のアスタキサンチンに対するHDCOの相対量(%))
で計算される数値が1以上であることを意味する。
アスタキサンチンおよびHDCOを含む組成物が、例えば、微生物菌体等である場合には、アスタキサンチンおよびHDCOを含有する抽出物を得る方法として、ガラスビーズを用いる機械的な破砕方法を使用することができる。この方法により得られたアスタキサンチンおよびHDCOを含有する抽出物は、HPLC装置を用いた分析に供することができる。
キサントフィロマイセス・デンドロアス(Xanthophyllomyces dendrorhous)NBRC 10129株(独立行政法人製品評価技術基盤機構生物遺伝資源部門、〒292-0818千葉県木更津市かずさ鎌足2-5-8より入手)をN-メチル-N’-ニトロ-N-ニトロソグアニジン(NTG)で変異処理した後、寒天プレート上で赤みの強いコロニーを選別し、アスタキサンチン含量が向上した変異株を取得した。得られた変異株に対して同様の操作を繰り返すことで、さらにアスタキサンチン含量の向上した変異株を取得した。このようにして、アスタキサン含量が向上した変異株であるキサントフィロマイセス・デンドロアス(Xanthophyllomyces dendrorhous)KNK-01株、KNK-02-1株、KNK-02-2株およびKNK-03株を取得した。
5mlのYM培地(ポリペプトン0.5%、酵母エキス0.3%、モルトエキス0.3%、グルコース1.0%)を含む試験管4本に、キサントフィロマイセス・デンドロアス(Xanthophyllomyces dendrorhous)KNK-03株を接種し、20℃で48時間培養した。培養液を50mlのYM培地を含む500ml容の坂口フラスコ4本に移し、20℃で48時間の培養を行なった。培養液を2500mlの培地(リン酸アンモニウム1.3%、リン酸カリウム0.7%、酵母エキス0.3%、グルコース1%を含む)を含む5000ml容のジャーファーメンターに移し、20℃で培養し、アスタキサンチンおよびHDCOを含有する菌体を含む培養液を得た。培養中は、pHを4.4~5.6の間にコントロールし、溶存酸素濃度は飽和の30~80%となるようにグルコースを流加した。
調製例1で得た培養液10mlを50ml容遠心チューブに分注し、遠心分離後上清を廃棄して菌体ペレットを得た。これに、0.5mm径のガラスビーズ25gとアセトン25mlを添加して密栓後、マルチビーズショッカー(安井機械製)を用いて菌体を破砕した。これを、遠心分離して上清のアセトン相を回収し、減圧下で溶媒を留去して、アスタキサンチンおよびHDCOを含有する菌体抽出物を得た。当該菌体抽出物中のアスタキサンチン濃度は、6.1mg/gであった。
シリカゲル60(メルク社製)を充填したガラスカラム(φ40mm×600mm)をヘキサン/アセトン=3/1の混合溶媒で平衡化し、これに調製例2で得た菌体抽出物をアプライした。これを同上溶媒で溶出し、赤色を呈するフラクションを集め、減圧下で溶媒を留去して、アスタキサンチンおよびHDCOを含有する部分精製物を得た。当該菌体抽出物中のアスタキサンチン濃度は、53mg/gであった。
5mlのYM培地(ポリペプトン0.5%、酵母エキス0.3%、モルトエキス0.3%、グルコース1.0%)を含む試験管に、キサントフィロマイセス・デンドロアス(Xanthophyllomyces dendrorhous)KNK-01株を接種し、20℃で48時間、振盪培養した。この培養液0.6mlを、リン酸アンモニウム1.3%、リン酸カリウム0.7%、コハク酸0.6%、酵母エキス0.3%を含む培地30ml(pH5.4)に摂取し、500ml容の坂口フラスコにて、20℃で180時間振盪培養し、アスタキサンチンおよびHDCOを含有する菌体を含む培養液を得た。炭素源のグルコースは培養開始時に0.3g添加し、グルコースが消費された後、12時間毎に0.3gのグルコースを補給した。
調製例4で得た培養液50mlを50ml容遠心チューブに分注し、遠心分離後上清を廃棄して菌体ペレットを得た。これに、0.5mm径のガラスビーズ25gとアセトン25mlを添加して密栓後、マルチビーズショッカー(安井機械製)を用いて菌体を破砕した。これを、遠心分離して上清のアセトン相を回収し、減圧下で溶媒を留去して、アスタキサンチンおよびHDCOを含有する菌体抽出物を得た。当該菌体抽出物中のアスタキサンチン濃度は、5.6mg/gであった。
微生物をキサントフィロマイセス・デンドロアス(Xanthophyllomyces dendrorhous)KNK-02-1株とした以外は、調製例4と同様にして培養を行い、アスタキサンチンおよびHDCOを含有する菌体を含む培養液を得た。
調製例2で得た細胞抽出物10mgを密閉可能な試験管に入れ、下記の表1に示す濃度の硫酸水溶液3mlを添加、攪拌した。攪拌後の溶液のpHをF-22型pHメーター(堀場製作所)を用いて測定した。
アスタキサンチンに対するHDCOの相対量は、次の計算式により算出した。
アスタキサンチンに対するHDCOの相対量(%)
=(HDCOのピーク面積/アスタキサンチンのピーク面積)×100
また、HPLC分析は、カラムとしてYMC Carotenoid(4.6×250mm;YMC社)を使用し、カラム温度を20℃とし、移動相として、A液:メタノール/メチル-t-ブチルエーテル/1%リン酸水溶液=82/15/3、および、B液:メタノール/メチル-t-ブチルエーテル/水/リン酸=7/90/3/0.03を用い、試料注入後0~30分はA液100%、30~90分はA液100%からB液100%へのリニアグラジエント、90~95分はB液100%の条件で、1.0ml/分の流速で通液することにより実施した。本条件において、アスタキサンチンは分析開始後約13分、HDCOは分析開始後約80分経過時に検出された。
硫酸に替えて塩酸を用いた以外は実施例1と同様に実施した。結果を表3に示した。
調製例2で得た細胞抽出物に替えて、調製例3で得た部分精製物を用いた以外は実施例1と同様に実施した。結果を表4に示した。
硫酸に替えて水酸化ナトリウムを用いた以外は実施例1と同様に実施した。結果を表5に示した。
硫酸に替えて水酸化カリウムを用いた以外は実施例1と同様に実施した。結果を表6に示した。
調製例2で得た細胞抽出物に替えて、調製例3で得た部分精製物を用いた以外は実施例4と同様に実施した。結果を表7に示した。
調製例2で得た細胞抽出物に替えて、調製例5で得た細胞抽出物を用いた以外は実施例1と同様に実施した。結果を表8に示した。
調製例1で得た培養液に表9に記した終濃度となるように硫酸を添加した。硫酸添加後の培養液のpHを実施例1と同様に測定した結果を表9に示した。次に、各培養液を密閉可能なガラス容器に10mlずつ分注し、30℃、50℃、または、70℃で、2時間攪拌した
2時間の処理後、各培養液0.05mlを密閉可能な2ml容のポリプロピレン製チューブに入れ、遠心分離により細胞を沈降させた。上清を除去した後、1mlの水に細胞を再懸濁し、再度の遠心分離により細胞を沈降させ、上清を除去した。これに、1gのガラスビーズ(φ0.5mm)と1mlのアセトンを加え、マルチビーズショッカー(安井器械社製)を用いて細胞を破砕した。破砕処理後、チューブの内容物をフィルターろ過(コスモナイスフィルターS、0.45μm、ミリポア社製)し、ろ液をHPLC分析に供した。HPLC分析、およびアスタキサンチンに対するHDCOの相対量の算出は、実施例1と同様に行った。結果を表9に示した。
硫酸に替えて水酸化ナトリウムを使用した以外は、実施例8と同様に実施した。結果を表10に示した。
調製例1で得た培養液50mlを密閉可能なガラス容器に分注し、硫酸を添加してpH7.0およびpH3.0に調整した。これを密栓後、70℃の水浴中で攪拌した。0時間後、12時間後、24時間後、48時間後に各々0.05mlを分取し、実施例8と同様に、細胞中のアスタキサンチンに対するHDCOの相対量を算出した。結果を表11に示した。
調製例6で得た培養液50mlを密閉可能なガラス容器に分注し、硫酸を添加してpH7.0およびpH1.0に調整した。これを密栓後、70℃の水浴中で攪拌した。0時間後、3時間後、6時間後に各々0.05mlを分取し、実施例8と同様に、細胞中のアスタキサンチンに対するHDCOの相対量を算出した。
Claims (10)
- アスタキサンチンおよびHDCOを含有する組成物を、pH3以下の酸性媒体又は/及びpH9以上の塩基性媒体に接触せしめ、当該組成物中の、アスタキサンチンに対する3-ヒドロキシ-3’ ,4’-ジデヒドロ-β,Ψ-カロテン-4-オン(HDCO)の相対量を低下させる工程を含む、アスタキサンチン含有組成物の製造方法。
- 前記のアスタキサンチンおよびHDCOを含有する組成物が、アスタキサンチン生産能力を有する細胞、細胞の一部、細胞の抽出物、および/または、それらの部分精製物から選ばれたものである、請求項1記載の方法。
- 前記のアスタキサンチン生産能力を有する細胞が、キサントフィロマイセス(Xanthophyllomyces)属、ブレバンディモナス(Brevundimonas)属、ヘマトコッカス(Haematococcus)属、クラミドモナス(Chlamydomonas)属、モノラフィディウム(Monoraphidium)属、エリスロバクター(Erythrobacter)属、アグロバクテリウム(Agrobacterium)属、パラコッカス(Paracoccus)属、ラビリンチュラ類(Labyrinthulea)に属する微生物の細胞である請求項2に記載の方法。
- 前記のアスタキサンチン生産能力を有する細胞が、キサントフィロマイセス(Xanthophyllomyces)属に属する微生物である請求項2に記載の方法。
- 前記キサントフィロマイセス(Xanthophyllomyces)属に属する微生物のアスタキサンチン含量が2000μg/g乾燥細胞以上である請求項4記載の方法。
- 前記のアスタキサンチンおよびHDCOを含有する組成物中のアスタキサンチンに対するHDCOの相対量が1%以上である請求項1~5のいずれかに記載の方法。
- 前記酸性媒体のpHが2以下である、請求項1~6のいずれかに記載の方法。
- 前記塩基性媒体のpHが10以上である、請求項1~6のいずれかに記載の方法。
- 酸性媒体および/または塩基性媒体に接触せしめた後に、アスタキサンチンに対するHDCOの相対量が0.1パーセントポイント以上低下する、請求項1~8のいずれかに記載の方法。
- 請求項1~9のいずれかに記載の方法により得られるアスタキサンチン含有組成物を含有する飼料、食品、食品添加物、または医薬品。
Priority Applications (4)
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US13/389,924 US20120202889A1 (en) | 2009-08-11 | 2010-08-11 | Production method for astaxanthin-containing composition |
CN201080035421.8A CN102471795B (zh) | 2009-08-11 | 2010-08-11 | 含有虾青素的组合物的制备方法 |
JP2011526688A JPWO2011018896A1 (ja) | 2009-08-11 | 2010-08-11 | アスタキサンチン含有組成物の製造方法 |
EP10808081A EP2465938A1 (en) | 2009-08-11 | 2010-08-11 | Production method for astaxanthin-containing composition |
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JP2009186206 | 2009-08-11 | ||
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US (1) | US20120202889A1 (ja) |
EP (1) | EP2465938A1 (ja) |
JP (1) | JPWO2011018896A1 (ja) |
CN (1) | CN102471795B (ja) |
WO (1) | WO2011018896A1 (ja) |
Cited By (4)
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WO2013002398A1 (ja) * | 2011-06-30 | 2013-01-03 | 株式会社カネカ | カロテノイド組成物の製造方法 |
WO2014104200A1 (ja) * | 2012-12-27 | 2014-07-03 | 株式会社カネカ | カロテノイド組成物の製造方法 |
JP2017516750A (ja) * | 2014-03-28 | 2017-06-22 | ディーエスエム アイピー アセッツ ビー.ブイ. | カロテノイド産生生物有機体からのカロテノイドの単離プロセス |
CN107712354A (zh) * | 2017-11-13 | 2018-02-23 | 南昌傲农生物科技有限公司 | 一种含虾青素的仔猪饲料添加剂及其制备方法与应用 |
Families Citing this family (1)
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WO2024076289A1 (en) * | 2022-10-06 | 2024-04-11 | National Science And Technology Development Agency | Feed additive containing a non-genetically modified microorganism to create a probiotic feed for aquaculture |
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- 2010-08-11 US US13/389,924 patent/US20120202889A1/en not_active Abandoned
- 2010-08-11 WO PCT/JP2010/005036 patent/WO2011018896A1/ja active Application Filing
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WO2013002398A1 (ja) * | 2011-06-30 | 2013-01-03 | 株式会社カネカ | カロテノイド組成物の製造方法 |
CN103649328A (zh) * | 2011-06-30 | 2014-03-19 | 株式会社钟化 | 类胡萝卜素组合物的制造方法 |
JPWO2013002398A1 (ja) * | 2011-06-30 | 2015-02-23 | 株式会社カネカ | カロテノイド組成物の製造方法 |
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WO2014104200A1 (ja) * | 2012-12-27 | 2014-07-03 | 株式会社カネカ | カロテノイド組成物の製造方法 |
JP2017516750A (ja) * | 2014-03-28 | 2017-06-22 | ディーエスエム アイピー アセッツ ビー.ブイ. | カロテノイド産生生物有機体からのカロテノイドの単離プロセス |
CN107712354A (zh) * | 2017-11-13 | 2018-02-23 | 南昌傲农生物科技有限公司 | 一种含虾青素的仔猪饲料添加剂及其制备方法与应用 |
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EP2465938A1 (en) | 2012-06-20 |
JPWO2011018896A1 (ja) | 2013-01-17 |
CN102471795A (zh) | 2012-05-23 |
CN102471795B (zh) | 2014-12-10 |
US20120202889A1 (en) | 2012-08-09 |
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