JP4225783B2 - 培養培地に適切な前駆体分子を加えることによる、繊毛虫培養物からのγ−リノレン酸の製造方法 - Google Patents
培養培地に適切な前駆体分子を加えることによる、繊毛虫培養物からのγ−リノレン酸の製造方法 Download PDFInfo
- Publication number
- JP4225783B2 JP4225783B2 JP2002565119A JP2002565119A JP4225783B2 JP 4225783 B2 JP4225783 B2 JP 4225783B2 JP 2002565119 A JP2002565119 A JP 2002565119A JP 2002565119 A JP2002565119 A JP 2002565119A JP 4225783 B2 JP4225783 B2 JP 4225783B2
- Authority
- JP
- Japan
- Prior art keywords
- gla
- culture
- acid
- tetrahymena
- acetate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 235000020664 gamma-linolenic acid Nutrition 0.000 title claims abstract description 109
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 title claims description 106
- 239000002243 precursor Substances 0.000 title claims description 20
- 238000004519 manufacturing process Methods 0.000 title claims description 19
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 title claims description 15
- 229960002733 gamolenic acid Drugs 0.000 title claims description 15
- 241000223782 Ciliophora Species 0.000 title abstract description 13
- 239000001963 growth medium Substances 0.000 title 1
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 29
- 239000000194 fatty acid Substances 0.000 claims abstract description 29
- 229930195729 fatty acid Natural products 0.000 claims abstract description 29
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims description 33
- 230000004151 fermentation Effects 0.000 claims description 33
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 30
- 239000010496 thistle oil Substances 0.000 claims description 20
- 241000223892 Tetrahymena Species 0.000 claims description 13
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 12
- 239000001632 sodium acetate Substances 0.000 claims description 12
- 235000017281 sodium acetate Nutrition 0.000 claims description 12
- 241000248384 Tetrahymena thermophila Species 0.000 claims description 10
- 150000003904 phospholipids Chemical class 0.000 claims description 10
- IPCSVZSSVZVIGE-UHFFFAOYSA-N palmitic acid group Chemical group C(CCCCCCCCCCCCCCC)(=O)O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 claims description 8
- 150000003626 triacylglycerols Chemical class 0.000 claims description 7
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 6
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 6
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 239000005642 Oleic acid Substances 0.000 claims description 6
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 6
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 6
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 6
- 241001442550 Tetrahymena sp. Species 0.000 claims description 5
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 claims description 4
- 235000021314 Palmitic acid Nutrition 0.000 claims description 4
- 235000021355 Stearic acid Nutrition 0.000 claims description 4
- 150000002148 esters Chemical class 0.000 claims description 4
- 235000020778 linoleic acid Nutrition 0.000 claims description 4
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 claims description 4
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 claims description 4
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 4
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 4
- 239000008117 stearic acid Substances 0.000 claims description 4
- 241000248418 Tetrahymena pyriformis Species 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 241000248417 Tetrahymena rostrata Species 0.000 claims description 2
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 230000000977 initiatory effect Effects 0.000 claims 3
- -1 diglycerides Chemical class 0.000 claims 1
- 235000015112 vegetable and seed oil Nutrition 0.000 claims 1
- 239000008158 vegetable oil Substances 0.000 claims 1
- 150000002632 lipids Chemical class 0.000 abstract description 40
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 abstract description 16
- 235000013305 food Nutrition 0.000 abstract description 7
- 241001465754 Metazoa Species 0.000 abstract description 3
- 235000020665 omega-6 fatty acid Nutrition 0.000 abstract description 2
- 229940033080 omega-6 fatty acid Drugs 0.000 abstract description 2
- 238000012262 fermentative production Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 33
- 239000002028 Biomass Substances 0.000 description 32
- 239000002609 medium Substances 0.000 description 28
- 230000007935 neutral effect Effects 0.000 description 13
- 235000008390 olive oil Nutrition 0.000 description 11
- 239000004006 olive oil Substances 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 8
- 239000003921 oil Substances 0.000 description 8
- 235000019198 oils Nutrition 0.000 description 8
- 238000000746 purification Methods 0.000 description 7
- 102100034544 Acyl-CoA 6-desaturase Human genes 0.000 description 6
- 108010037138 Linoleoyl-CoA Desaturase Proteins 0.000 description 6
- 235000010633 broth Nutrition 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000004817 gas chromatography Methods 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 241000223877 Tetrahymena australis Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 150000002066 eicosanoids Chemical class 0.000 description 3
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- HOBAELRKJCKHQD-UHFFFAOYSA-N (8Z,11Z,14Z)-8,11,14-eicosatrienoic acid Natural products CCCCCC=CCC=CCC=CCCCCCCC(O)=O HOBAELRKJCKHQD-UHFFFAOYSA-N 0.000 description 2
- 240000004355 Borago officinalis Species 0.000 description 2
- 235000007689 Borago officinalis Nutrition 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- 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 2
- 108090001060 Lipase Proteins 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 235000004496 Oenothera biennis Nutrition 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 241000248419 Tetrahymena malaccensis Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000001627 detrimental effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- HOBAELRKJCKHQD-QNEBEIHSSA-N dihomo-γ-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCCCC(O)=O HOBAELRKJCKHQD-QNEBEIHSSA-N 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 229960004488 linolenic acid Drugs 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012454 non-polar solvent Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 150000003180 prostaglandins Chemical class 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical group 0.000 description 2
- 102100034542 Acyl-CoA (8-3)-desaturase Human genes 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 240000002900 Arthrospira platensis Species 0.000 description 1
- 235000016425 Arthrospira platensis Nutrition 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 241000252095 Congridae Species 0.000 description 1
- 241001464430 Cyanobacterium Species 0.000 description 1
- 108010073542 Delta-5 Fatty Acid Desaturase Proteins 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 208000032131 Diabetic Neuropathies Diseases 0.000 description 1
- 235000021298 Dihomo-γ-linolenic acid Nutrition 0.000 description 1
- 102100034543 Fatty acid desaturase 3 Human genes 0.000 description 1
- 108010087894 Fatty acid desaturases Proteins 0.000 description 1
- OPGOLNDOMSBSCW-CLNHMMGSSA-N Fursultiamine hydrochloride Chemical compound Cl.C1CCOC1CSSC(\CCO)=C(/C)N(C=O)CC1=CN=C(C)N=C1N OPGOLNDOMSBSCW-CLNHMMGSSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000578774 Homo sapiens MAP kinase-activated protein kinase 5 Proteins 0.000 description 1
- 102100028396 MAP kinase-activated protein kinase 5 Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 208000019695 Migraine disease Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000219925 Oenothera Species 0.000 description 1
- 240000008916 Oenothera biennis Species 0.000 description 1
- 241000222350 Pleurotus Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 240000001890 Ribes hudsonianum Species 0.000 description 1
- 235000016954 Ribes hudsonianum Nutrition 0.000 description 1
- 235000001466 Ribes nigrum Nutrition 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000223879 Tetrahymena borealis Species 0.000 description 1
- 241000223881 Tetrahymena canadensis Species 0.000 description 1
- 241000223889 Tetrahymena furgasoni Species 0.000 description 1
- 241000248389 Tetrahymena setosa Species 0.000 description 1
- 241000248388 Tetrahymena silvana Species 0.000 description 1
- 241000248383 Tetrahymena tropicalis Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 244000093732 Toddalia asiatica Species 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 159000000021 acetate salts Chemical class 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000019728 animal nutrition Nutrition 0.000 description 1
- 235000021342 arachidonic acid Nutrition 0.000 description 1
- 229940114079 arachidonic acid Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 108010011713 delta-15 desaturase Proteins 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- SQNZJJAZBFDUTD-UHFFFAOYSA-N durene Chemical compound CC1=CC(C)=C(C)C=C1C SQNZJJAZBFDUTD-UHFFFAOYSA-N 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 235000004626 essential fatty acids Nutrition 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 229940045761 evening primrose extract Drugs 0.000 description 1
- 235000008524 evening primrose extract Nutrition 0.000 description 1
- 150000002194 fatty esters Chemical class 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 150000002617 leukotrienes Chemical class 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 206010027599 migraine Diseases 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 150000003815 prostacyclins Chemical class 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 229940082787 spirulina Drugs 0.000 description 1
- JIWBIWFOSCKQMA-UHFFFAOYSA-N stearidonic acid Natural products CCC=CCC=CCC=CCC=CCCCCC(O)=O JIWBIWFOSCKQMA-UHFFFAOYSA-N 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000020238 sunflower seed Nutrition 0.000 description 1
- 150000003595 thromboxanes Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
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
- 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
- C12N1/10—Protozoa; Culture media therefor
- C12N1/105—Protozoal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/90—Protozoa ; Processes using protozoa
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
(i)発酵ブロスを0〜10℃に冷却し、次いで冷却した培養ブロスを遠心分離または濾過にかける段階、
(ii)得られたバイオマスを乾燥させる段階、
(iii)得られた脂質を、
(a)(ii)から得たバイオマスDM(BDM)を、全脂質を抽出できる極性溶媒、好ましくはエタノールでの抽出にかけ、
(b)その代わりに、非極性溶媒、好ましくはn−ヘキサンでBDMから中性脂質を抽出し、および/または
(c)(a)から得た全脂質を非極性溶媒で処理して、中性脂質と極性脂質、たとえばリン脂質を分離することによって抽出する段階
を踏めばよい。
(i)上述の方法の段階(ii)または(iii)で得た生産物をけん化またはエステル化して、遊離脂肪酸またはエステルを生成させる段階、
(ii)沈殿(たとえば尿素沈殿)法および/またはクロマトグラフィ法によって、GLAを濃縮する段階
を踏めばよい。
(i)発酵ブロスを0〜10℃に冷却し、次いで遠心分離または濾過にかける(その後希釈して濾過性を確認する)段階、
(ii)内在するテトラヒメナ(Tetrahymena)リパーゼによって、または他のリパーゼを加えることによって、遊離した脂質を加水分解する段階、
(iii)沈殿(たとえば尿素沈殿)法および/またはクロマトグラフィ法によってGLAを濃縮する段階
を含めばよい。
標準培養物
100ml容コニカルフラスコに20mlのMM−Var培地(表1を参照されたい)を装入し、またはバッフルを備えた500ml容コニカルフラスコに100mlのMM−Var培地を装入し、セルロース/綿製の栓で閉め、121℃で20分間滅菌した。
予備培養物については、20mlの培地を含む100ml容フラスコに、細胞密度が1mlあたり細胞約5×104〜1×105個のMM−Var培地(表1を参照されたい)を接種し、25℃、60rpmで2日間かけて培養した。細胞数を、1mlあたり細胞1×106個と3×106個の間の値に到達させた。
100mlの培地を含む500ml容コニカルフラスコ中で振とうフラスコ実験を行った。実験用フラスコに1mlあたり約5×104〜1×105個の細胞密度を接種し、25℃、100rpmで2日間かけて培養した。
1l容発酵槽(700mlの稼動体積)、5l容発酵槽(3lの稼動体積)、および13l容発酵槽(10lの稼動体積)中で発酵を行った。発酵槽に、pH電極、pO2電極、通気環、円板型攪拌器を備えた攪拌軸、酸/アルカリ用供給ライン、フィード供給用供給ライン、接種用フィッティング、およびサンプリングを標準装備した。
バイオマス乾燥物を定量するために、5mlの細胞培養物を4℃、3400gで10分間遠心分離し、上清をデカントし、細胞ペレットを−80℃で凍らせた。凍結したバイオマスを凍結乾燥させ、バイオマス乾燥物の重量を決定した。
CASY 1 Cell Counter+Analyser System、TTC型、Scharfe Systemを使用して、細胞密度を決定した。測定については、細胞を弱いCASY電解質溶液中に希釈し(1:500)、直径150μmの毛細管を通して輸送した。粒径が15〜50μmの範囲の細胞をカウントした。
n−ヘキサンを用いてバイオマス乾燥物から非極性脂質成分(中性脂質)を抽出した。1バッチについて、約500mgのバイオマス乾燥物を、5mlのn−ヘキサン中、60℃で1時間攪拌し、次いで2700gで2分間遠心分離し、懸濁した物体を濾別することによって上清を精製した。濾過については、詰める高さを約1cmとして脱脂綿を詰めたパスツールピペットを使用した。最初の抽出の細胞残渣を5mlの溶媒でもう一度抽出し、バイオマスを遠心分離によって除き、上清を濾過した。2回の抽出の精製された有機相を合わせ、回転蒸発装置で溶媒を完全に蒸発させ、抽出された脂質の量を秤量によって決定した。
Iatroscan TM new MK−5 Analyserを使用して、抽出した脂質を質的かつ量的に分析した。クロマトグラフィ材料には、表面材料として耐熱性シリカゲルが使用されている分離棒(クロマロッド)を使用した。分離棒をデシケーター中に保管し、それぞれの分離操作の前にか焼して、妨害物質を除去した。FIDの測定条件は、走査速度:30秒/スキャン、O2流量:2l/分、およびH2流量:160ml/分となるように選択した。
水素炎イオン化検出器(ヒューレットパッカード社、米ウィルミントン)を装備したガスクロマトグラフィ装置(HP GC 6890)によって、脂肪酸スペクトルを決定した。
アザミ油(リノレン酸供給源)がGLA生成に及ぼす影響
1ml/lのアザミ油を、滅菌する前に、100mlのMM−Var培地を含む2本の500ml容フラスコそれぞれに加えた。100mlのMM−Var培地を含む2本の500ml容フラスコを基準として使用した。アザミ油は、表6に報告された組成を有していた。脂肪酸は、トリグリセリドの形であった。
オリーブ油(オレイン酸供給源)がGLA生成に及ぼす影響
1ml/lのオリーブ油を、滅菌する前に、100mlのMM−Var培地を含む2本の500ml容フラスコそれぞれに加えた。100mlのMM−Var培地を含む2本の500ml容フラスコを基準とした。オリーブ油の組成は、表6(上)に示したものであった。脂肪酸は、トリグリセリドの形であった。結果は、図2〜4に示す。これを加えることによって、バイオマスが約8.3g/lから約9.1g/lに増加した(図2)。バイオマス中のGLA含有量は、有意に増加し(2.1%から3%)(図3)、バイオマス濃度も高くなり、GLAの体積濃度は、53%(180mg/lから275mg/lに)増大した。オリーブ油を加えることによって、全脂肪酸中のGLAの比率は、33%から24%に低下した(図5)。
アセタートがGLA生成に及ぼす影響(基本培地中のアセタート)
700mlのMM−Var培地を含む1l容発酵槽において発酵を行った。1つの発酵物を基準とし、他の2つには、滅菌前に、その培地に0.5および1.0g/lの酢酸ナトリウム(NaAc)を加えた。
アセタートがGLA生成に及ぼす影響(24時間後に培養物に加えたアセタート)
700mlのMM−Var培地を含む1l容発酵槽において発酵を行った。1つの発酵物を基準とし、他の発酵物には、発酵29時間後に滅菌した(121℃で20分間)10%強度溶液の形で、0.2、0.5、および1.0g/lの酢酸ナトリウム(NaAc)を加えた。29時間後に0.2および0.5g/lのNaAcを供給した発酵物では、発酵54時間後に再度同じ量を加えた。
酢酸ナトリウムとアザミ油を加える
700mlのMM−Var培地を含む1l容発酵槽において発酵を行った。1つの発酵物を基準とし、1つには滅菌前に0.5g/lのNaAcおよび1ml/lのアザミ油を、1つには0.5g/lのNaAcおよび1ml/lのアザミ油を、対数期の終わりに向けて(28時間後まで)加えた。
オリーブ油とアセタートの供給の組合せ
オリーブ油を使用しても同様に良好な結果が得られた。
Claims (10)
- γ−リノレン酸を発酵によって製造する方法であって、
γ−リノレン酸前駆体分子を加えた培地でテトラヒメナ種(Tetrahymena sp.)を培養し、培養開始の20〜39時間後に最初の添加を行い培養開始の40〜70時間後に次の添加を行って2段階でアセタートを加え、培養を終えた後、テトラヒメナ種(Tetrahymena sp.)からγ−リノレン酸を単離し、
前記γ−リノレン酸前駆体分子が、パルミチン酸(C16:0)、ステアリン酸(C18:0)、オレイン酸(C18:1)、若しくはリノール酸(C18:2)、又は、パルミチン酸(C16:0)、ステアリン酸(C18:0)、オレイン酸(C18:1)、及びリノール酸(C18:2)のうち何れか一つの脂肪酸のトリグリセリド、ジグリセリド、モノグリセリド、リン脂質、エステル、若しくは塩であることを特徴とするγ−リノレン酸の製造方法。 - テトラヒメナ サーモフィラ(Tetrahymena thermophila)を使用することを特徴とする請求項1に記載の方法。
- テトラヒメナ ピリフォーミス(Tetrahymena pyriformis)を使用することを特徴とする請求項1に記載の方法。
- テトラヒメナ ロストラタ(Tetrahymena rostrata)を使用することを特徴とする請求項1に記載の方法。
- 培養開始の25〜35時間後に最初のアセタートを加えることを特徴とする請求項1〜4のいずれか一項に記載の方法。
- 培養開始の25〜35時間後に最初の添加を行い、培養開始の50〜60時間後に次の添加を行って、2段階でアセタートを加えることを特徴とする請求項1〜4のいずれか一項に記載の方法。
- 酢酸の形、または酢酸塩としてのアセタートを培養物に加えることを特徴とする請求項1〜6のいずれか一項に記載の方法。
- 培地に、最終濃度を0.1ml/l〜10ml/lとして植物油を加えることを特徴とする請求項1〜7のいずれか一項に記載の方法。
- 濃度が0.1g/l〜1g/lの酢酸ナトリウムとしてのアセタートを加えることを特徴とする請求項1〜8のいずれか一項に記載の方法。
- 前記γ−リノレン酸前駆体分子がアザミ油であることを特徴とする請求項1〜9のいずれか一項に記載の方法。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10106660A DE10106660A1 (de) | 2001-02-12 | 2001-02-12 | Verfahren zur Herstellung von gamma-Linolensäure aus einer Ciliaten-Kultur durch Zusatz geeigneter Vorläufermoleküle zu dem Kulturmedium |
PCT/EP2002/001302 WO2002064807A2 (de) | 2001-02-12 | 2002-02-08 | Verfahren zur herstellung von η-linolensäure aus einer ciliaten-kultur durch zusatz geeigneter vorläufermoleküle zu dem kulturmedium |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2004519231A JP2004519231A (ja) | 2004-07-02 |
JP4225783B2 true JP4225783B2 (ja) | 2009-02-18 |
Family
ID=7673897
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2002565119A Expired - Fee Related JP4225783B2 (ja) | 2001-02-12 | 2002-02-08 | 培養培地に適切な前駆体分子を加えることによる、繊毛虫培養物からのγ−リノレン酸の製造方法 |
Country Status (6)
Country | Link |
---|---|
US (2) | US7081356B2 (ja) |
EP (1) | EP1402045B1 (ja) |
JP (1) | JP4225783B2 (ja) |
AT (1) | ATE473285T1 (ja) |
DE (2) | DE10106660A1 (ja) |
WO (1) | WO2002064807A2 (ja) |
Families Citing this family (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA3056110C (en) | 2004-04-22 | 2020-07-14 | Surinder Pal Singh | Synthesis of long-chain polyunsaturated fatty acids by recombinant cells |
CN103451246B (zh) | 2004-04-22 | 2018-02-16 | 联邦科学技术研究组织 | 用重组细胞合成长链多不饱和脂肪酸 |
BRPI0716075A2 (pt) | 2006-08-29 | 2013-08-06 | Commw Scient Ind Res Org | sÍntese de Ácidos graxos |
US8877239B2 (en) | 2010-08-12 | 2014-11-04 | Nutritional Therapeutics, Inc. | Lipid supplements for maintaining health and treatment of acute and chronic disorders |
US9468668B2 (en) | 2011-08-11 | 2016-10-18 | Allergy Research Group, Llc | Flavored chewable lipid supplements for maintaining health and the treatment of acute and chronic disorders |
WO2009102558A2 (en) * | 2008-02-11 | 2009-08-20 | Monsanto Technology Llc | Aquaculture feed, products, and methods comprising beneficial fatty acids |
BR122021003836B1 (pt) | 2008-11-18 | 2022-02-01 | Commonwealth Scientific And Industrial Research Organisation | Polinucleotídeo isolado e/ou exógeno que não ocorre de forma natural, vetor e método para produzir óleo contendo ácidos graxos insaturados |
ES2345241B1 (es) * | 2009-03-16 | 2011-09-08 | Lipopharma Therapeutics | Uso de 2-hidroxiderivados de acidos grasos poliinsaturados como medicamentos. |
US11253531B2 (en) | 2011-08-11 | 2022-02-22 | Nutritional Therapeutics, Inc. | Lipid supplements for reducing nerve action potentials |
US10117885B2 (en) | 2011-08-11 | 2018-11-06 | Allergy Research Group, Llc | Chewable lipid supplements for treating pain and fibromyalgia |
HUE033766T2 (en) | 2012-06-15 | 2018-01-29 | Commw Scient Ind Res Org | Preparation of long chain polyunsaturated fatty acids in plant cells |
EP2780032B1 (en) | 2013-01-10 | 2018-03-28 | Allergy Research Group, LLC | Chewable wafers containing lipid supplements for maintaining health and the treatment of acute and chronic disorders |
CA3241340A1 (en) | 2013-12-18 | 2015-06-25 | Grains Research And Development Corporation | Lipid comprising long chain polyunsaturated fatty acids |
SG11201610596PA (en) | 2014-06-27 | 2017-01-27 | Commw Scient Ind Res Org | Lipid comprising docosapentaenoic acid |
GB201501081D0 (en) * | 2015-01-22 | 2015-03-11 | Cilian Ag | Use of enzymes with a wide pH activity range as medicaments for promoting digestion |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2574089B1 (fr) | 1984-12-03 | 1987-04-10 | Inst Nat Sante Rech Med | Procede de production d'acides gras, en particulier d'acide g-linoleique a partir de tetrahymena, produits obtenus et preparation medicamenteuse ou alimentaire contenant de l'acide g-linolenique ou ses derives en tant qu'agent anti-agregation plaquettaire |
ES2052564T3 (es) | 1986-07-08 | 1994-07-16 | Suntory Ltd | Procedimiento para la produccion de acido bishomo-gamma-linolenico y acido eicosapanteonico. |
DE19741489A1 (de) * | 1997-09-19 | 1999-03-25 | Hoechst Ag | Fermentationsverfahren mit kontinuierlicher Massenkultivierung von Ciliaten (Protozoa) zur Produktion biogener Wertstoffe |
DE19903095C2 (de) * | 1999-01-27 | 2003-05-22 | Nutrinova Gmbh | Gewinnung von gamma-Linolensäure aus Protozoen der Gattung Colpidium |
-
2001
- 2001-02-12 DE DE10106660A patent/DE10106660A1/de not_active Withdrawn
-
2002
- 2002-02-08 WO PCT/EP2002/001302 patent/WO2002064807A2/de active Application Filing
- 2002-02-08 JP JP2002565119A patent/JP4225783B2/ja not_active Expired - Fee Related
- 2002-02-08 EP EP02716743A patent/EP1402045B1/de not_active Expired - Lifetime
- 2002-02-08 DE DE50214524T patent/DE50214524D1/de not_active Expired - Lifetime
- 2002-02-08 AT AT02716743T patent/ATE473285T1/de active
- 2002-02-08 US US10/468,259 patent/US7081356B2/en not_active Expired - Fee Related
-
2006
- 2006-05-18 US US11/436,416 patent/US7476522B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
US7476522B2 (en) | 2009-01-13 |
US20040180414A1 (en) | 2004-09-16 |
DE10106660A1 (de) | 2002-08-29 |
US7081356B2 (en) | 2006-07-25 |
WO2002064807A2 (de) | 2002-08-22 |
DE50214524D1 (de) | 2010-08-19 |
ATE473285T1 (de) | 2010-07-15 |
WO2002064807A3 (de) | 2003-03-20 |
EP1402045B1 (de) | 2010-07-07 |
JP2004519231A (ja) | 2004-07-02 |
EP1402045A2 (de) | 2004-03-31 |
US20060205047A1 (en) | 2006-09-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7476522B2 (en) | Method for producing γ-linolenic acids from a ciliate culture by adding suitable precursor molecules to said culture medium | |
JP6226601B2 (ja) | 微生物油の調製 | |
JP3538418B2 (ja) | ドコサヘキサエン酸およびドコサペンタエン酸の製造方法 | |
Cheng et al. | Fungal production of eicosapentaenoic and arachidonic acids from industrial waste streams and crude soybean oil | |
Kennedy et al. | Fatty acid production characteristics of fungi with particular emphasis on gamma linolenic acid production | |
DK2895628T3 (da) | Olie beriget på arachidonsyre fra mikroorganismer (encellet mortierella alpina-svamp) og fremgangsmåde til fremstilling heraf | |
US20080269329A1 (en) | Process for Production of Microbial Fat/Oil Containing Discretional Amount of Diacylglycerol and Said Fat/Oil | |
KR102202287B1 (ko) | 미세조류 시조카이트리움 만그로베이의 바이오매스 및 이의 제조 방법 | |
He et al. | Rationale behind the near-ideal catalysis of Candida antarctica lipase A (CAL-A) for highly concentrating ω-3 polyunsaturated fatty acids into monoacylglycerols | |
Silva et al. | Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production | |
Grygier et al. | Galactomyces geotrichum mold isolated from a traditional fried cottage cheese produced omega-3 fatty acids | |
JP4510203B2 (ja) | 原生動物Colpidium属からのγ−リノレン酸の獲得 | |
KR100524198B1 (ko) | 오메가-9계의고도의불포화지방산및이를함유하는지질의제조방법및당해방법에사용된미생물 | |
JP4748907B2 (ja) | 脂質を菌体外に分泌する微生物ならびに当該微生物を用いた脂質および脂質を内包する油滴小胞の製造方法 | |
Yamada et al. | Biotechnological processes for production of poly-unsaturated fatty acids | |
Chtourou et al. | Dunaliella sp. a wild algal strain isolated from the Sfax-Tunisia solar evaporating salt-ponds, a high potential for biofuel production purposes | |
Irmak et al. | Determination of the fatty-acid composition of four native microalgae species | |
Bajpai et al. | Eicosapentaenoic acid (EPA) formation: comparative studies with Mortierella strains and production by Mortierella elongata | |
CA2904038A1 (en) | Production of omega-3 fatty acids from pythium species | |
Shimizu et al. | Mortierella species (fungi): production of C20 polyunsaturated fatty acids | |
JP4079494B2 (ja) | アラキドン酸及び/又はエイコサペンタエン酸含有油脂の製造方法 | |
Streekstra | Arachidonic acid: fermentative production by Mortierella fungi | |
Sharma et al. | Omega 3 polyunsaturated fatty acids: sources, synthesis and benefits | |
Zarea | ISOLATION AND CHARACTERIZATION OF OLEAGINOUS MARINE YEAST PRODUCING OF FATTY ACIDS | |
Shimizu et al. | Microbial production of polyunsaturated fatty acids (vitamin-F group) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20050202 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20070919 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20071219 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20071227 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20080314 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080409 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20080704 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20080711 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20081009 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20081105 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20081125 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111205 Year of fee payment: 3 |
|
R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121205 Year of fee payment: 4 |
|
LAPS | Cancellation because of no payment of annual fees |