WO1998029558A1 - Milieu destine a la culture de micro-organismes et procede permettant de produire des acides gras insatures ou des lipides renfermant ceux-ci - Google Patents
Milieu destine a la culture de micro-organismes et procede permettant de produire des acides gras insatures ou des lipides renfermant ceux-ci Download PDFInfo
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- WO1998029558A1 WO1998029558A1 PCT/JP1997/004898 JP9704898W WO9829558A1 WO 1998029558 A1 WO1998029558 A1 WO 1998029558A1 JP 9704898 W JP9704898 W JP 9704898W WO 9829558 A1 WO9829558 A1 WO 9829558A1
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- Prior art keywords
- ion
- acid
- ions
- calcium
- phosphate
- Prior art date
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- 244000005700 microbiome Species 0.000 title claims abstract description 56
- 235000021122 unsaturated fatty acids Nutrition 0.000 title claims abstract description 45
- 150000004670 unsaturated fatty acids Chemical class 0.000 title claims abstract description 44
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- 238000012258 culturing Methods 0.000 title claims abstract description 23
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229910001424 calcium ion Inorganic materials 0.000 claims abstract description 29
- 241000235575 Mortierella Species 0.000 claims abstract description 25
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- 229910001415 sodium ion Inorganic materials 0.000 claims abstract description 22
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 21
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- 229910001414 potassium ion Inorganic materials 0.000 claims abstract description 11
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 claims description 46
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 44
- 239000002609 medium Substances 0.000 claims description 36
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- 150000003839 salts Chemical class 0.000 claims description 33
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 23
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical class [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 claims description 3
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
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- 239000000126 substance Substances 0.000 claims description 2
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000004641 brain development Effects 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 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
- 235000015165 citric acid Nutrition 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 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
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 210000003792 cranial nerve Anatomy 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 238000004332 deodorization Methods 0.000 description 1
- 238000004807 desolvation Methods 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- VLCINIKIVYNLPT-UHFFFAOYSA-J dicalcium;hydrogen phosphate Chemical compound [Ca+2].[Ca+2].OP(O)([O-])=O.[O-]P([O-])([O-])=O VLCINIKIVYNLPT-UHFFFAOYSA-J 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- KAQKFAOMNZTLHT-VVUHWYTRSA-N epoprostenol Chemical compound O1C(=CCCCC(O)=O)C[C@@H]2[C@@H](/C=C/[C@@H](O)CCCCC)[C@H](O)C[C@@H]21 KAQKFAOMNZTLHT-VVUHWYTRSA-N 0.000 description 1
- 229960001123 epoprostenol Drugs 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 150000002232 fructoses Chemical class 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229960002337 magnesium chloride Drugs 0.000 description 1
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 description 1
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229940038384 octadecane Drugs 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 235000003441 saturated fatty acids Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- RZWIIPASKMUIAC-VQTJNVASSA-N thromboxane Chemical compound CCCCCCCC[C@H]1OCCC[C@@H]1CCCCCCC RZWIIPASKMUIAC-VQTJNVASSA-N 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
-
- 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
- C12N1/14—Fungi; 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
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
- C12P7/6432—Eicosapentaenoic acids [EPA]
Definitions
- the present invention relates to a novel culture medium for culturing microorganisms, and a method for producing an unsaturated fatty acid-containing oil or fat obtained by culturing a microorganism belonging to the genus Mortierella having the ability to produce unsaturated fatty acids in the medium.
- Unsaturated fatty acids such as arachidonic acid, dihomo-a-linolenic acid, eicosapentaenoic acid, and mead acid are powerful and have a wide variety of physiological activities, such as prostaglandin, thromboxane, prostacyclin, and leucot. It is said to be a precursor of Lien and has been attracting attention in recent years. For example, arachidonic acid is being studied rapidly along with DHA (docosahexaenoic acid) as a component particularly necessary for the development of infants, and Lantin et al.
- DHA docosahexaenoic acid
- a method for producing mead acid using a mutant obtained by subjecting a Mortierella microorganism to a mutation treatment and having a reduced or deleted ⁇ 12 desaturation activity is also known ( Japanese Patent Application Laid-Open No. 5-9188 8). Also, a method for producing a large amount of dihomo-arinolenic acid using a mutant strain obtained by subjecting a microorganism of the genus Mortierella to a mutation and having a reduced or deleted ⁇ 5 desaturation activity.
- a filamentous fungus such as Mortierella sp.
- the viscosity of the culture solution often increases due to the growth of cells.
- the present invention relates to a method for producing an unsaturated fatty acid-containing fat or oil by fermentation of a microorganism belonging to the genus Mortierella, wherein unsaturated fatty acid productivity
- the purpose of the present invention is to provide an unsaturated fatty acid-containing fat or oil in an economical and stable manner by improving the growth of non-saturated fatty acids, the accumulation of unsaturated fatty acids, and the accumulation of total lipids.
- the present invention has an advantage that, for example, unsaturated fatty acids can be obtained in a high yield, and aims to provide an inexpensive medium for culturing microorganisms.
- the present inventors have comprehensively studied not only the unsaturated fatty acid yield but also the bacterial morphology and lipid composition change in the effect of adding salts to the culture medium. It has been found that it is extremely effective to add all ions of calcium, sodium, calcium, magnesium and phosphoric acid at a predetermined concentration and in a good balance, and have completed the present invention. Was.
- the present invention relates to phosphates, potassium ions, sodium ions, magnesium ions and calcium ions, which are 5 to 60 mM, 5 to 60 mM, 2 to 5 mM, respectively. 0 mM, 0.5 to 9 mM, 0.5 to 12 mM, and a medium for culturing microorganisms, and a filamentous fungus in the medium, particularly to the genus Mortierella.
- a method for producing an unsaturated fatty acid or a lipid containing the same which has improved productivity by culturing a microorganism belonging thereto.
- the unsaturated fatty acid refers to a fatty acid having 16 or more carbon atoms and one or more double bonds, and among them, a fatty acid having 18 or more carbon atoms and two or more double bonds.
- polyunsaturated fatty acids such as arlinoleic acid, dihomoarinolenic acid, arachidonic acid, eicosapentaenoic acid, mead acid, 6, 9-octadecadienic acid, 8, 1 1 — Eicosadic acid and the like.
- the medium for culturing microorganisms of the present invention contains phosphate ions, calcium ions, sodium ions, magnesium ions, and calcium ions in the medium at 5 to 60 mM, 5 to 60 mM, 5 to 60 mM, respectively. 2 to 50 mM, 0.5 to 9 mM, 0.5 to 12 mM, preferably 10 to 45 mM, 10 to 45 mM, 5 to 40 mM, l to 40 mM, respectively It is in the range of 6 mM and 1 to 9 mM, and can be used for culturing microorganisms, for example, filamentous fungi.
- the medium of the present invention contains phosphate ions, potassium ions, and sodium ions.
- Components other than magnesium ions and calcium ions eg, a carbon source, a nitrogen source, a micronutrient source, etc. can be appropriately combined depending on the microorganism used.
- any microorganism used for producing unsaturated fatty acid-containing fats and oils can be used as long as it is a microorganism belonging to the genus Mortierella.
- a microorganism for example, a strain described in MYCOTAXON, Vol. XL1V, No. 2, pp. 257-265 (1992) can be used.
- strains belonging to these type cultures or strains isolated from nature can be used as they are, but natural mutations that differ in properties from the original strains obtained by one or more growth and / or isolation steps. Stocks can also be used.
- the microorganism used in the present invention is a mutant or recombinant strain of a microorganism belonging to the genus Mortierella (wild strain), that is, the amount of the microorganism in the oil and fat compared to the amount produced by the original wild strain when cultured using the same substrate. And those designed to increase the specific and Z or total unsaturated fatty acid content, or to increase the total fat content, or both.
- a mutant strain designed to have a high content of a specific unsaturated fatty acid a Mortierella alpina SAM1861 lacking ⁇ 12 desaturation activity (Shin-Keken Co., Ltd. No. 3) No. 590, FERMBP — 359 0), and Mortierella 'Alpina SAM 186 0 0 lacking ⁇ 5 desaturation activity (Microe Kenjo No. 3589, FERMBP-35) 8 9).
- microorganisms that are designed to produce the same amount of unsaturated fatty acids as the corresponding wild-type strain by efficiently using cost-effective substrates.
- the microorganisms belonging to the genus Mortierella can be produced in a conventional manner except that the concentrations of phosphate, potassium, sodium, magnesium and potassium ions in the culture medium are adjusted to a predetermined range. Therefore, it can be cultured.
- a spore, a hypha or a pre-culture solution obtained by pre-culturing the above strain is inoculated into a liquid medium or a solid medium and cultured.
- Glucose, fructoses, xylose as carbon sources Saccharose, maltose, soluble starch, molasses, glycerol, mannitol, citric acid, cornstarch, etc.
- Maltose, fructose, corn starch, glycerol, citrate are preferred.
- Nitrogen sources include organic nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor and urea, and inorganic nitrogen sources such as ammonium nitrate and ammonium sulfate. Although it can be used, particularly by using a nitrogen source obtained from soybean alone or in combination, or in combination with the nitrogen source, a more preferable synergistic effect of salt addition can be obtained. As a nitrogen source obtained from soybeans, it is desirable that the nitrogen content per component excluding water is 2% or more, preferably 3% or more, and more preferably 5% or more.
- Examples of the nitrogen source obtained from soybeans include defatted soybeans or heat-treated; acid-treated; alcohol-treated; enzymatic-treated; chemical-modified; or denaturation by chemical and / or physical treatments including the treatment. And / or regeneration; removal of some components using water and / or organic solvents; filtration and removal of some components by Z or centrifugation; freezing; crushing; drying; and processing such as Z or sieving.
- Untreated or defatted soybeans can be used singly or in combination, and commonly used are soybeans, defatted soybeans, soybean flakes, edible soybean tamper Boiled soybeans, soymilk, soy milk, and kinako, etc. Soluble components removed Things are preferred.
- precursors of unsaturated fatty acids include, for example, hydrocarbons such as hexadecane or octadecane; oleic acid or linoleic acid.
- Fatty acids such as carboxylic acid or salts thereof, or fatty acid esters, for example, ethyl esters, glycerin fatty acid esters, sorbitan fatty acid esters; or olive oil, soybean oil, rapeseed oil, cottonseed oil or coconut oil. Such fats and oils can be used alone or in combination.
- the amount of the substrate to be added is 0.0001 to 10%, preferably 0.5 to 10%, based on the medium.
- these substrates may be cultured as the sole carbon source.
- the phosphate ion in the medium is 5 to 60 mM
- the calcium ion is 5 to 6 OmM
- the sodium ion is Range from 2 to 5 O mM
- magnesium ion ranges from 0.5 to 9 mM
- calcium ions range from 0.5 to 12 mM, and preferably 10 to 45 mM phosphate ions in the culture medium.
- mM potassium ions are in the range of 10 to 45 mM
- sodium ions are in the range of 5 to 40 mM
- magnesium ions are in the range of 1 to 6 mM
- calcium ions are in the range of 1 to 9 mM.
- phosphate ions are, for example, dicalcium hydrogen phosphate, monolithium dihydrogen phosphate, sodium dihydrogen phosphate and Z or diphosphate.
- Salts such as hydrogen sodium and potassium, for example, salts such as potassium dihydrogen phosphate, potassium monohydrogen phosphate and / or potassium chloride
- Sodium ions include, for example, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and salts such as sodium chloride or Z or sodium sulfate, and magnesium ion
- salts such as magnesium chloride and Z or magnesium sulfate, and potassium ion can be obtained by adding salts such as calcium chloride and / or calcium carbonate to a culture medium. There is no particular limitation as long as it is not limited and does not inhibit the growth of bacteria.
- These salts may be either hydrates or anhydrides.
- the above salts are used in an appropriate combination so as to be within the range of the ion concentration of the present invention.
- Li phosphate dihydrogen Ichika Li um KH 2 P0 4
- sulfuric acid Na Application Benefits um anhydrous Na 2 S0 4
- magnesium chloride hexahydrate MgC 1 2 - 6H 2 0
- calcium chloride dihydrate by adding a predetermined amount by blending a four hydrate (CaC ⁇ 2H 2 0)
- the ions of the present invention can be a child prepared to a predetermined concentration.
- the addition of such salts greatly increases the unsaturated fatty acid yield.
- the effect on the cell morphology in liquid culture cannot be specified unconditionally because of the effects of the medium components other than salts and the strain used.
- the percentage of bacteria in the form of pellets increases with the increase in the amount of sodium, calcium, or magnesium added. Propagation in pulp increases the viscosity of the culture, causing a decrease in fluidity and dissolved oxygen concentration, leading to a decrease in yield.
- the above-mentioned carbon source, nitrogen source and other medium components can be added to the medium before the start of the culture and / or the culture solution during the culture. These medium components can be added all at once, or they can be added continuously or in multiple portions over time. These medium components can be sterilized and added individually or in a premixed state, and the sterilizing method and the order of addition are not particularly limited.
- the carbon and nitrogen sources are preferably sterilized separately, and the salts are added by the end of log growth, more preferably before mid-log growth.
- Other medium components that do not affect the concentrations of phosphate, calcium, sodium, magnesium, and canoledium are not particularly limited as long as they do not inhibit the growth of the bacteria.
- the total amount of the carbon source is generally 0.1 to 40% by weight, preferably 1 to 25% by weight, and the total amount of the nitrogen source is 0.01 to 10% by weight, preferably.
- the concentration is 0.1 to 10% by weight, more preferably, the initial carbon source addition amount is 1 to 5% by weight, and the initial nitrogen source addition amount is 0.1 to 6% by weight.
- the medium is fed with a carbon source and a nitrogen source during the cultivation, and more preferably, only with the carbon source.
- the culture temperature is 5 to 40 ° C, preferably 20 to 30 ° C. After the cells are grown at 20 to 30 ° C, the culture is performed at 5 to 20 ° C. The production of unsaturated fatty acids can be continued.
- the culture medium is adjusted to pH 4 to 10, preferably 5 to 8, for aeration, stirring, shaking, or stationary culture. Culture is usually performed for 2 to 20 days. This By culturing as described above, lipids containing unsaturated fatty acids are produced and accumulated in the cells. In the production of unsaturated fatty acids, aeration and stirring culture in a liquid medium is preferred.
- the target lipid may be a culture medium during the production of lipids by culturing or a sterilized culture medium, a culture medium after the cultivation or a sterilized culture medium, or a culture collected from each.
- the cells can be obtained from the cells or the dried product according to a conventional method.
- the target lipid is collected from the cultured cells by, for example, the following method.
- the cultured cells are obtained from the culture solution by conventional solid-liquid separation means such as centrifugation and Z or filtration.
- Cultured cells are preferably washed, crushed and dried. Drying can be performed by freeze drying, air drying, or the like.
- the dried cells are extracted with an organic solvent, preferably under a stream of nitrogen.
- organic solvent ether, hexane, methanol, ethanol, cross-sectional form, dichloromethane, petroleum ether, etc. can be used. Good results can also be obtained by alternate extraction or extraction with a single solvent of chloroform-methanol / water, preferably with hexane.
- a high concentration of unsaturated fatty acid-containing lipid can be obtained.
- extraction can be performed using wet cells instead of the above method.
- a water-compatible solvent such as methanol or ethanol, or a solvent compatible with water and
- Mortiere 11a alpina CBS754.68 was used as the arachidonic acid producing bacterium. Glucose 2%, soybean oil 0.
- Triglyceride content 97.23 ⁇ 4 97.4% 97.03 ⁇ 4 96.03 ⁇ 4 88.33 ⁇ 4
- Yeast extract TASTONE 154 AG manufactured by Universal Foods
- Soy protein Ajinomoto S-San Meat Special
- Mortiere 11a alpina CBS754.68 was used as the arachidonic acid producing bacterium.
- 50 mL of the preculture was inoculated, and aeration-stirred culture was performed for 8 days at a temperature of 28 ° C, aeration of 0 vvm, stirring at 300 rpm, and a tank pressure of 200 kPa.
- Glucose concentration was maintained at 1-2% by fed-batch until day 4 and 0.5-1% thereafter.
- Mortiere 11a alpina CBS754.68 was used as the arachidonic acid-producing bacterium. Prepare 4 types of 25 L medium containing glucose 2%, defatted soy flour 1.5%, soybean oil 0.1%, and the salt components shown in Table 3 in a 50L culture tank, and adjust the initial pH to 6 0. 50 mL of the preculture was inoculated, and aeration-stirred culture was carried out for 8 days at a temperature of 28 ° C., an aeration rate of 1.0 vvm, stirring at 300 rpm, and a tank pressure of 200 kPa. Glucose concentration was maintained at ⁇ 2% by fed-batch until day 4 and 0.5-1% thereafter.
- the bacterial morphology grew in a mixed state of pellets and pulp, and most of the pellets were rice grain type, about 0.5 to 1.5 mm. In the medium containing only phosphate, very fine pulp was grown, and the fluidity of the medium was greatly reduced. On the other hand, in a medium supplemented with magnesium, calcium, and sodium salts, most of the bacteria are spherical and pellets with a diameter of about 1 to 2 mm, and the fluidity is high but the lipid content per bacterial cell is high. The result was low.
- Mortielle 11a elongata IF08570, Morti erel la exigua IF08571, Moronereti elera ) IF05941 was used.
- Medium of 6 types (2 types of X3 strains) containing 2% glucose, 1.5% edible soy protein (Essan Protein SS, manufactured by Ajinomoto Co.), 0.1% rapeseed oil and the components shown in Table 4 Each 5 L was prepared in a 50 L culture tank, and the initial pH was adjusted to 5.8.
- Ortiere 1 la alpina CBS754.68 was used as the arachidonic acid producing bacterium.
- 100 mL of the preculture was inoculated, and aeration and agitation cultivation was performed for 8 days at a temperature of 24 ° C, an aeration rate of 1.0 vvm, agitation of 200 rpm, and a tank pressure of 200 kPa.
- Glucose concentration was maintained at 1-2% by fed-batch until day 4 and 0.5-1% thereafter.
- conditions 3 and 4 in Table 5 must be maintained at 300 rpm with stirring.
- Conditions 5 and 6 were increased to 400 rpm.
- Mortierella alpina SAM1861 (Fishenjo No. 3590, FERM BP-3590) as a middle acid producing bacterium
- Mortierella alpina SAM1860 (Motherella monolinolenic acid producing bacterium)
- Wei-Ken Kenjo No. 3589, FERM BP-3589 4 types of 5 L medium containing 2% initial glucose, 1.5% soybean protein (Ajinomoto Co., Essan Meat Co., Ltd.), 0.1% olive oil, and salt components shown in Table 6 (2 Species X 2 strain) was prepared in a 10 L culture tank, and the initial pH was adjusted to 6.0.
- Inoculated with 1 0 0 ml preculture was done temperature 2 8 ° C, aeration rate 1. 0 vvm, aeration spinner culture for 3 0 0 8 days at r P m.
- the culture temperature was reduced to 20 ° C on the second day.
- the glucose concentration was maintained at 1-2% by the fed-batch method.
- MorUerera alpina CBS754.68 was used as the arachidonic acid-producing bacterium.
- Initial glucose 2%, large edible ... MPt Npaku 4%, soybean oil 0 1%, KH 2 P ( 0 3% N Na 2 S0 4 0 1%, MgCl 2 -. 6H 2 0 0 0 5%, CaCl 2 - 2H 2 0 0 0.5% culture medium 600 liters
- Aeration and agitation culture was started at a volume of 0.5 V vm, agitation of 30 rpm, and a tank pressure of 20 OkPa From the first day of culture, the aeration amount and the number of revolutions were adjusted to maintain the dissolved oxygen concentration. Also, 18% glucose was added in multiple portions over the first to fifth days of the culture.
- the amount of arachidonic acid produced was 13 g ZL.
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Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1019997005817A KR100653107B1 (ko) | 1996-12-27 | 1997-12-26 | 불포화 지방산 또는 이를 함유하는 지질의 제조방법 |
ES97950433.9T ES2446982T3 (es) | 1996-12-27 | 1997-12-26 | Medios para cultivar microorganismos y método para producir ácidos grasos insaturados o lípidos que los contienen |
US09/331,759 US6746857B2 (en) | 1996-12-27 | 1997-12-26 | Media for culturing microorganisms and process for producing unsaturated fatty acids or lipids containing the same |
JP52987298A JP4197744B2 (ja) | 1996-12-27 | 1997-12-26 | 微生物培養用培地、並びに不飽和脂肪酸またはこれを含有する脂質の製造方法 |
DK97950433.9T DK0960943T3 (en) | 1996-12-27 | 1997-12-26 | MEDIA FOR CULTIVATION OF MICRO-ORGANISMS AND METHOD FOR PRODUCING unsaturated fatty acids or lipids containing DEM |
EP97950433.9A EP0960943B1 (en) | 1996-12-27 | 1997-12-26 | Media for culturing microorganisms and process for producing unsaturated fatty acids or lipids containing the same |
CA002276179A CA2276179C (en) | 1996-12-27 | 1997-12-26 | Media for culturing microorganisms and process for producing unsaturated fatty acids or lipids containing the same |
AU53414/98A AU740811C (en) | 1996-12-27 | 1997-12-26 | Media for culturing microorganisms and process for producing unsaturated fatty acids or lipids containing the same |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8/349541 | 1996-12-27 | ||
JP34954196 | 1996-12-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998029558A1 true WO1998029558A1 (fr) | 1998-07-09 |
Family
ID=18404424
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1997/004898 WO1998029558A1 (fr) | 1996-12-27 | 1997-12-26 | Milieu destine a la culture de micro-organismes et procede permettant de produire des acides gras insatures ou des lipides renfermant ceux-ci |
Country Status (9)
Country | Link |
---|---|
US (1) | US6746857B2 (ja) |
EP (2) | EP2308988A1 (ja) |
JP (1) | JP4197744B2 (ja) |
KR (1) | KR100653107B1 (ja) |
CA (1) | CA2276179C (ja) |
DK (1) | DK0960943T3 (ja) |
ES (1) | ES2446982T3 (ja) |
PT (1) | PT960943E (ja) |
WO (1) | WO1998029558A1 (ja) |
Cited By (5)
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WO1999040215A1 (en) * | 1998-02-07 | 1999-08-12 | Avecia Limited | Mortierella |
US7247468B2 (en) | 2004-04-30 | 2007-07-24 | Daiso Co., Ltd. | Method for producing optically active 1,2-diols by microorganism culturing |
JP2007527233A (ja) * | 2003-10-02 | 2007-09-27 | マーテック バイオサイエンシーズ コーポレーション | 改変された量の塩化物およびカリウムを使用した微細藻類における高レベルのdhaの産生法 |
EP2239316A1 (en) | 2004-08-12 | 2010-10-13 | Nippon Suisan Kaisha, Ltd. | Method for polyunsaturated fatty acid production using novel cell preservation technique |
US8241868B2 (en) | 2005-02-08 | 2012-08-14 | Nippon Suisan Kaisha, Ltd. | Production of polyunsaturated fatty acids using cell treatment method |
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JP4088097B2 (ja) * | 2002-04-26 | 2008-05-21 | サントリー株式会社 | 高度不飽和脂肪酸含有脂質の製造方法 |
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CN101787379A (zh) | 2002-06-19 | 2010-07-28 | 帝斯曼知识产权资产管理有限公司 | 包含多不饱和脂肪酸的微生物油的制备 |
JP4849806B2 (ja) * | 2005-02-08 | 2012-01-11 | 日本水産株式会社 | 新規な菌体処理方法を用いた高度不飽和脂肪酸の製造方法 |
JP2007209272A (ja) * | 2006-02-10 | 2007-08-23 | Suntory Ltd | 微生物発酵による長鎖高度不飽和脂肪酸を構成要素として含むリン脂質の製造方法 |
US8207363B2 (en) | 2009-03-19 | 2012-06-26 | Martek Biosciences Corporation | Thraustochytrids, fatty acid compositions, and methods of making and uses thereof |
EA031136B1 (ru) | 2010-01-19 | 2018-11-30 | ДСМ АйПи АССЕТС Б.В. | Микроорганизмы, продуцирующие эйкозапентаеновую кислоту, композиции жирных кислот и способы их получения и применения |
KR20140057394A (ko) * | 2010-06-30 | 2014-05-12 | 닛폰 스이산 가부시키가이샤 | 유용 물질의 제조 방법 |
EP2655647A4 (en) | 2010-12-23 | 2016-04-27 | Exxonmobil Res & Eng Co | CULTIVATION OF A MICROORGANISM IN A MEDIUM CONTAINING A HIGH LEVEL OF A CARBOXYLATE CONTRA-ION SOURCE |
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WO2013161674A1 (ja) * | 2012-04-27 | 2013-10-31 | 花王株式会社 | 乳酸の製造方法 |
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- 1997-12-26 ES ES97950433.9T patent/ES2446982T3/es not_active Expired - Lifetime
- 1997-12-26 EP EP10181443A patent/EP2308988A1/en not_active Withdrawn
- 1997-12-26 EP EP97950433.9A patent/EP0960943B1/en not_active Expired - Lifetime
- 1997-12-26 CA CA002276179A patent/CA2276179C/en not_active Expired - Lifetime
- 1997-12-26 US US09/331,759 patent/US6746857B2/en not_active Expired - Lifetime
- 1997-12-26 WO PCT/JP1997/004898 patent/WO1998029558A1/ja active IP Right Grant
- 1997-12-26 KR KR1019997005817A patent/KR100653107B1/ko not_active IP Right Cessation
- 1997-12-26 PT PT97950433T patent/PT960943E/pt unknown
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Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1999040215A1 (en) * | 1998-02-07 | 1999-08-12 | Avecia Limited | Mortierella |
JP2013212126A (ja) * | 2003-10-02 | 2013-10-17 | Dsm Ip Assets Bv | 改変された量の塩化物およびカリウムを使用した微細藻類における高レベルのdhaの産生法 |
US7824892B2 (en) | 2003-10-02 | 2010-11-02 | Martek Biosciences Corporation | Production of DHA in microalgae in medium having modified amounts of potassium |
JP4733043B2 (ja) * | 2003-10-02 | 2011-07-27 | マーテック バイオサイエンシーズ コーポレーション | 改変された量の塩化物およびカリウムを使用した微細藻類における高レベルのdhaの産生法 |
US9249434B2 (en) | 2003-10-02 | 2016-02-02 | Dsm Ip Assets B.V. | Production of high levels of DHA in microalgae using modified amounts of chloride and potassium |
US8008050B2 (en) | 2003-10-02 | 2011-08-30 | Martek Biosciences Corporation | Methods for discouraging bacterial growth in culture medium |
US7829129B2 (en) | 2003-10-02 | 2010-11-09 | Martek Biosciences Corporation | Method to reduce corrosion during fermentation of microalgae |
US7871809B2 (en) | 2003-10-02 | 2011-01-18 | Martek Biosciences Corporation | Production of DHA in microalgae in medium having modified amounts of chloride ion and potassium ion |
JP2015130886A (ja) * | 2003-10-02 | 2015-07-23 | ディーエスエム アイピー アセッツ ビー.ブイ. | 改変された量の塩化物およびカリウムを使用した微細藻類における高レベルのdhaの産生法 |
US7745183B2 (en) | 2003-10-02 | 2010-06-29 | Martek Biosciences Corporation | Methods for the selection of low pH-tolerant, DHA producing microalgae |
JP2007527233A (ja) * | 2003-10-02 | 2007-09-27 | マーテック バイオサイエンシーズ コーポレーション | 改変された量の塩化物およびカリウムを使用した微細藻類における高レベルのdhaの産生法 |
JP2011036261A (ja) * | 2003-10-02 | 2011-02-24 | Martek Biosciences Corp | 改変された量の塩化物およびカリウムを使用した微細藻類における高レベルのdhaの産生法 |
US8669090B2 (en) | 2003-10-02 | 2014-03-11 | Dsm Ip Assets B.V. | Production of high levels of DHA in microalgae using modified amounts of chloride and potassium |
US8663953B2 (en) | 2003-10-02 | 2014-03-04 | Dsm Ip Assets B.V. | Production of high levels of DHA in microalgae using modified amounts of chloride and potassium |
US7247468B2 (en) | 2004-04-30 | 2007-07-24 | Daiso Co., Ltd. | Method for producing optically active 1,2-diols by microorganism culturing |
US8609397B2 (en) | 2004-08-12 | 2013-12-17 | Nippon Suisan Kaisha, Ltd. | Method for preserving a microorganism |
EP2239316A1 (en) | 2004-08-12 | 2010-10-13 | Nippon Suisan Kaisha, Ltd. | Method for polyunsaturated fatty acid production using novel cell preservation technique |
US8241868B2 (en) | 2005-02-08 | 2012-08-14 | Nippon Suisan Kaisha, Ltd. | Production of polyunsaturated fatty acids using cell treatment method |
Also Published As
Publication number | Publication date |
---|---|
US20010016342A1 (en) | 2001-08-23 |
EP0960943B1 (en) | 2014-02-12 |
AU740811B2 (en) | 2001-11-15 |
AU5341498A (en) | 1998-07-31 |
EP0960943A1 (en) | 1999-12-01 |
ES2446982T3 (es) | 2014-03-11 |
EP0960943A4 (en) | 2004-09-08 |
JP4197744B2 (ja) | 2008-12-17 |
PT960943E (pt) | 2014-04-29 |
DK0960943T3 (en) | 2014-02-24 |
US6746857B2 (en) | 2004-06-08 |
KR20000062343A (ko) | 2000-10-25 |
EP2308988A1 (en) | 2011-04-13 |
KR100653107B1 (ko) | 2006-12-04 |
CA2276179C (en) | 2008-01-08 |
CA2276179A1 (en) | 1998-07-09 |
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