CN101575623B - Method for coproduction of arachidonic acid and chitosan through microbial fermentation - Google Patents
Method for coproduction of arachidonic acid and chitosan through microbial fermentation Download PDFInfo
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- CN101575623B CN101575623B CN200910033328A CN200910033328A CN101575623B CN 101575623 B CN101575623 B CN 101575623B CN 200910033328 A CN200910033328 A CN 200910033328A CN 200910033328 A CN200910033328 A CN 200910033328A CN 101575623 B CN101575623 B CN 101575623B
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- 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 title claims abstract description 67
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 44
- 235000021342 arachidonic acid Nutrition 0.000 title claims abstract description 34
- 229940114079 arachidonic acid Drugs 0.000 title claims abstract description 33
- 238000000855 fermentation Methods 0.000 title claims abstract description 29
- 230000004151 fermentation Effects 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 20
- 230000000813 microbial effect Effects 0.000 title claims abstract description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 51
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 35
- 238000005406 washing Methods 0.000 claims abstract description 28
- 230000007935 neutral effect Effects 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000000967 suction filtration Methods 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 claims abstract description 7
- 238000004821 distillation Methods 0.000 claims abstract description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 16
- HNBDQABBWNOTRU-UHFFFAOYSA-N thalline Chemical compound C1=CC=[Tl]C=C1 HNBDQABBWNOTRU-UHFFFAOYSA-N 0.000 claims description 12
- 241000907999 Mortierella alpina Species 0.000 claims 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 claims description 9
- 229910019142 PO4 Inorganic materials 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- -1 nitrogen-containing compound Chemical class 0.000 claims description 9
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 8
- 241000306282 Umbelopsis isabellina Species 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 8
- 239000010452 phosphate Substances 0.000 claims description 8
- 239000011591 potassium Substances 0.000 claims description 8
- 229910052700 potassium Inorganic materials 0.000 claims description 8
- 235000010344 sodium nitrate Nutrition 0.000 claims description 8
- 239000004317 sodium nitrate Substances 0.000 claims description 8
- 229940001516 sodium nitrate Drugs 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 241000134363 Umbelopsis ramanniana Species 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 5
- 244000144992 flock Species 0.000 claims description 5
- 229910000041 hydrogen chloride Inorganic materials 0.000 claims description 5
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 150000002978 peroxides Chemical class 0.000 claims description 5
- 239000012286 potassium permanganate Substances 0.000 claims description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 239000005695 Ammonium acetate Substances 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- 229930091371 Fructose Natural products 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 230000001476 alcoholic effect Effects 0.000 claims description 2
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 claims description 2
- 235000019257 ammonium acetate Nutrition 0.000 claims description 2
- 229940043376 ammonium acetate Drugs 0.000 claims description 2
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- 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 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 239000006071 cream Substances 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 235000010333 potassium nitrate Nutrition 0.000 claims description 2
- 239000004323 potassium nitrate Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 235000015099 wheat brans Nutrition 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 9
- 230000008569 process Effects 0.000 abstract description 7
- 238000003912 environmental pollution Methods 0.000 abstract description 6
- 241000233866 Fungi Species 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 abstract 4
- 239000003208 petroleum Substances 0.000 abstract 2
- 241001052560 Thallis Species 0.000 abstract 1
- 238000001035 drying Methods 0.000 abstract 1
- 235000019197 fats Nutrition 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 238000000227 grinding Methods 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 239000002244 precipitate Substances 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000004519 grease Substances 0.000 description 7
- 230000004913 activation Effects 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000004321 preservation Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 229930191978 Gibberellin Natural products 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 239000003448 gibberellin Substances 0.000 description 3
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical class C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000003375 plant hormone Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- GWHCXVQVJPWHRF-KTKRTIGZSA-N (15Z)-tetracosenoic acid Chemical compound CCCCCCCC\C=C/CCCCCCCCCCCCCC(O)=O GWHCXVQVJPWHRF-KTKRTIGZSA-N 0.000 description 2
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 2
- 241000235575 Mortierella Species 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 238000009655 industrial fermentation Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 241001480517 Conidiobolus Species 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000035126 Facies Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000235395 Mucor Species 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 241001450872 Thamnidium Species 0.000 description 1
- HXWJFEZDFPRLBG-UHFFFAOYSA-N Timnodonic acid Natural products CCCC=CC=CCC=CCC=CCC=CCCCC(O)=O HXWJFEZDFPRLBG-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229960005135 eicosapentaenoic acid Drugs 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- CASUWPDYGGAUQV-UHFFFAOYSA-M potassium;methanol;hydroxide Chemical compound [OH-].[K+].OC CASUWPDYGGAUQV-UHFFFAOYSA-M 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for coproduction of arachidonic acid and chitosan through microbial fermentation, which comprises the following production steps: connecting mould fungi with a culture medium for cultivation; after fermentation, grinding and crushing the collected thalli, performing extraction by using petroleum ether and ethanol, and performing reduced pressure distillation to remove the petroleum ether and the ethanol so as to obtain arachidonic acid oil and fat; and extracting the chitosan: adding sodium hydroxide solution to the filter residue after the extraction, boilingand washing the mixture until the mixture is neutral, performing suction filtration, adding oxyful to the filter residue, placing the mixture in a water bath and washing the mixture until the mixture is neutral, using the sodium hydroxide solution to adjust the pH of the filter residue so that a chitosan flocculent precipitate is precipitated, filtering the mixture and washing the mixture until t he mixture is neutral, using the ethanol to wash the mixture, and drying the mixture to obtain the chitosan. The method has the advantages of simple process, short cycle, and small power consumption and environmental pollution, and can be applied to industrial production.
Description
One, technical field
The invention belongs to technical field of biochemical industry, relate to a kind of microbial fermentation coproduction of arachidonic acid and methods of chitosan.
Two, background technology
Arachidonic acid (Arachidonic Acid; Be called for short AA) be a kind of 20 carbon pufass; In organism, bringing into play the unique biological function; Therefore at aspects such as food, medicine, makeup huge using value is arranged; The advantage that microbial fermentation yield peanut tetraenoic acid is easy to cultivate because of having mikrobe, growth cycle is short, can large-scale production has caused people's extensive concern gradually.
Analyze both at home and abroad and report on the existing document, the arachidonic bacterial classification of fermentative prodn, most researchs concentrate on the Conidiobolus of phycomycetes, genus mortierella, Mucor, Rhizopus, Thamnidium etc.Especially with moulds such as Mortierella alpina, long mortierella, Mortierella isabellina, mortierella ramannianas for.In the century in the past, people produce arachidonic acid to these microbial fermentations and have carried out system and deep research.Hillside plots in 1987 etc. are separated to many strains AA and produce bacterium from soil; Obtain 1 plant height through seed selection and produce bacterium Mortierella alpina (Mortierellaalpina IS-4); When making carbon source with glucose, it produces 4.3g/L AA (Yamad H; Et al.Agricult.Biol.Chem, 1987,51 (3): 785-790); It is starting strain with Mortierella isabellina (Mortierella isabellina AS 3.3410) that Fujian Normal University's Huang in 1998 is built people such as loyalty; Go out M018 through UV, DES (ethyl sulfate), NTG (nitrosoguanidine) complex mutation breeding; The shake flask fermentation fat content improves 133% (Huang Jianzhong than starting strain; Et al. microbiology circular, 1998, (4): 187-191); Japanese Higashiyama K in 1998 etc. adopt M.alpina IS-4, add an amount of KH
2PO4, Na
2SO4, CaCl
2And MgCl
2, 10,000 L ferment tanks 8 days, the output of AA reaches 10.8g/L, and has studied the influence of dissolved oxygen to this bacterial classification form and AA output, and drawn optimum value (Higashiyama K, et al.J.Am.OilChem.Soc., 1998,12:1815-1819.); 2007; Nanjing University of Technology has applied for a patent, publication number: CN 101109015A (title: preparation method of arachidonic acid oil), adopt Mortierella alpina fermentative prodn arachidonic acid; Advantage is to cultivate through overaging; Reduce the generation of by product, cost is low, is easy to the industrialization utilization; 2007; Maikede Biological Technology Co., Ltd., Wuhan has applied for a patent; Publication number: CN 101153298A (title: fermentative prodn is hanged down the method for the arachidonic acid oil of Selacholeic acid, EPA content) adopts Mortierella isabellina fermentative prodn arachidonic acid, and advantage is that fermenting process is easy to control; Can obtain high-quality arachidonic acid oil, by product Selacholeic acid, timnodonic acid (EPA) content are extremely low.
Chitosan is a kind of polysaccharide, is the de-acetyl chitin product, is with a wide range of applications.A kind of important chemical product of chitosan because its binding property is good, become good, nontoxic, the odorlessness of fine film forming properties, is widely used in food preservative technology; Because it has effects such as anti-ageing, wrinkle resistant, beauty treatment and health care, at cosmetic field bigger application is arranged, also obviously reducing blood-fat of chitosan, hypoglycemic, enhancing immunity are widely used at field of medicaments simultaneously; In addition, chitosan also can be applicable to environmental improvement process etc.The raw material that traditional industry is produced chitosan is shrimp, crab shell etc., adopts chemical process to obtain, complex process, and DR is more serious, and its raw material sources are restricted, and causes cost very expensive.In recent years, it is found that chitosan removes and extensively be present in insect that outside the shell of Crustaceans, the still main moity of most fungal cell walls, thereby fungi is expected to become the new resources of producing chitosan.The eighties in 20th century, Japan, the U.S. begin that successively microbial fermentation is produced chitosan to be studied, and the beginning of the nineties, China also began the research of this respect.With shrimp, crab shell facies ratio, from fungal cell wall, producing chitosan has many advantages, for example; Most of fungi can pass through the industrial fermentation technology large scale culturing; Do not receive the restriction of raw material complicacy, seasonality, region etc., quality product and output are easy to control, and production technique is simple; Cycle is short, and power consumption and environmental pollution are little.
In sum, along with utilizing mold fermentation to produce the continuous expansion of arachidonic acid scale, a large amount of residual waste mycelias generally all directly abandon, and certainly will cause environmental pollution so at present, and the wasting of resources is unfavorable for environmental protection.The recycling problem of residual thalline residue in existing document and all not mentioned arachidonic acid production process of patent; Utilization of the present invention not influencing the arachidonic acid synthetic simultaneously, promotes the accumulation of chitosan in the thalline through in arachidonic fermention medium, adding suitable promotor; After the fermentation ends; The depleted thalline is collected the preparation chitosan, when turning waste into wealth, also can reduce environmental pollution, economic benefit and social benefit are fairly obvious.
Three, summary of the invention
Technical problem: the purpose of this invention is to provide a kind of microbial fermentation and produce arachidonic acid coproduction methods of chitosan, thereby realize the recycling of residual thalline residue in the arachidonic acid large-scale production process, effectively alleviate environmental stress.
Technical scheme: a kind of microbial fermentation coproduction of arachidonic acid and methods of chitosan; Production stage is: the inoculum size according to culture volume 10% inserts mould in substratum; It is that 25～28 ℃, rotating speed are the shaking table fermentation culture of 120～140rpm that postvaccinal substratum places temperature, cultivates 6～8 days; After the fermentation ends,, the thalline of collecting dried to constant weight at 40～60 ℃ grind,, sherwood oil and ethanol are removed through 0.01MPa/45 ℃ of underpressure distillation can be obtained arachidonic acid oil again with sherwood oil and ethanol extracting to the fermented liquid suction filtration; Said sherwood oil and alcoholic acid volume ratio are 3: 1, and wherein the thalline of 1 volume carries out extracting with the sherwood oil and the ethanol of 5～8 volumes; The extraction of chitosan: add the sodium hydroxide solution of 2～10wt% in the filter residue after extracting, the ratio of filter residue quality and sodium hydroxide solution volume is 1: 3～1: 6w/v, and boil 2～4 hours deproteinated, washing is to neutral; Suction filtration adds ydrogen peroxide 50 in filter residue, in boiling water bath, placed 0.5～1 hour; Washing is drained to neutral, filter residue is added soak decolouring in 1～2.5 hour in the 0.5wt% potassium permanganate solution; After the decolouring filter residue adding 2～10wt% Hydrogen chloride or acetum were soaked 1～4 hour, filter, washing is to neutrality; Transfer filter residue pH to 8～10 with 10～20wt% sodium hydroxide solution, the chitosan flocks is separated out, filter; Washing is used washing with alcohol to neutral, the dry chitosan that gets.
Add Plant hormones regulators,gibberellins in the said fermenting process, be earlier fermentation 0～12h interpolation opportunity of Plant hormones regulators,gibberellins, and the starting point concentration of Plant hormones regulators,gibberellins is 0.05mg/L.
Said mould is Mortierella alpina, Mortierella isabellina or mortierella ramanniana.
Carbon source is one or more in glucose, fructose, sucrose or the seminose in the substratum.
Nitrogenous source comprises inorganic nitrogen-containing compound and nitrogen-containing organic compound in the substratum; Wherein inorganic nitrogen-containing compound is one or more in ammonium acetate, ammonium chloride, saltpetre or the SODIUMNITRATE, and nitrogen-containing organic compound is one or more in peptone, yeast extract paste, steeping water, wheat bran hydrolyzed solution or the Carnis Bovis seu Bubali cream.
Inorganic salt are one or more in potassium hydrogenphosphate, potassium primary phosphate, sal epsom or the calcium chloride in the substratum.
Beneficial effect: the present invention can realize the coproduction of two kinds of high added value chemical arachidonic acids and chitosan, has the following advantages: a. raw material sources are abundant, and bacterial classification can pass through the industrial fermentation technology large scale culturing, does not receive the restriction in region, season; B. comprehensive utilization reduces cost, and in Industrial processes, adds suitable nutritive substance, and the discarded mycelium in the industry is changed into chitosan fully, also can reduce environmental pollution when turning waste into wealth, and economic benefit clearly; C. technology is simple, and the cycle is short, and power consumption and environmental pollution are little, and can be applied to suitability for industrialized production.
Four, embodiment
Following embodiment elaborates to the present invention, but to not restriction of the present invention.
General explanation:
The process for extracting of arachidonic acid oil: with the microorganism collection after the fermentation ends, 60 ℃ of oven dry, grind; With sherwood oil and ethanol (volume ratio is 3: 1) extraction 12 hours, sherwood oil and ethanol are removed through underpressure distillation can be obtained arachidonic acid oil again; Wherein, the thalline of 1 volume extracts with the sherwood oil and the ethanol of 5～8 volumes.
Arachidonic detection method:
(1) preparation of mixed methyl aliphatic ester: get grease 0.1g (or dry mycelium 0.2g) in the 10mL volumetric flask, add 5%KOH-methanol solution (mass volume ratio) 1mL, 60 ℃ of water-bath 10min.Take out volumetric flask, room temperature cooling 5min adds methyl alcohol 2mL, and boron trifluoride-diethyl ether solution 1.5mL fully shakes mixing, 60 ℃ of water-bath 10min.Take out volumetric flask, room temperature cooling 5min adds normal hexane 2mL, fully shakes mixing, leaves standstill 10min.Get supernatant liquid 0.1mL and in the 1.5mL centrifuge tube, add the 0.8mL normal hexane, add a little anhydrous Na again
2SO
4(being used for suction), get 0.6 μ L sample introduction.
(2) GC-MS condition: measure bacterium oil with Thermo finnigan trace GC2000 DSQ GC-MS and form.DB-5MS quartz capillary column (30m * 0.25mm * 0.25 μ m).250 ℃ of Sample Room temperature, carrier gas are helium, flow rate of carrier gas 1mL/Min.Temperature programming: 80 ℃ of initial temperature are warmed up to 200 ℃ with 40 ℃/Min, 10 ℃ then/Min to 300 ℃..250 ℃ of transmission line temperature, ionization mode EI, 70Ev, sweep limit 50-600aum.
Embodiment 1
Step 1: with the Mortierella alpina of preservation (from application number be 200710025004.5, publication number is the bacterial classification that CN101113140A, name are called preservation in the patented claim document of " a kind of Mortierella alpina and application thereof "; This bacterial classification is at China typical culture collection center; Deposit number is CCTCC NO:M207067) mycelium connects a square tiles on the PDA slant medium, cultivated 5～7 days in constant incubator (25～28 ℃).
Step 2: cultured mycelium is washed in the seed activation substratum with the 5mL sterilized water, cultivated 2～3 days.Said substratum quality volume percent consists of: glucose 2%～6%, and yeast extract paste 0.3%～0.6%, potassium primary phosphate 0.2%～0.5%, SODIUMNITRATE 0.2%～0.5%, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.03%～0.1%, all the other are water, pH6.0～8.5.
Step 3: insert shaking in the bottle of 250mL that the 50mL fermention medium has been housed with the inoculum size of culture volume 10% seed liquor after with activation, placing temperature is that 25 ℃, rotating speed are the shaking table fermentation culture of 135rpm, cultivates 6～8 days.Said substratum quality volume percent consists of: glucose 7%～15%, and yeast extract paste 0.6%～1.2%, potassium primary phosphate 0.2%～0.6%, SODIUMNITRATE 0.2%～0.6%, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.05%～0.1%, all the other are water.
Step 4: fermentation ends, suction filtration is collected thalline and is dried to constant weight for 40～60 ℃, grinds sherwood oil and ethanol extracting grease.Arachidonic acid content in the gas chromatography determination grease.
Step 5: the extraction of chitosan: in the filter residue of step 4 gained, add the sodium hydroxide solution of 2～10wt%, the ratio of filter residue quality and sodium hydroxide solution volume is 1: 3, and boils 2～4 hours deproteinated, and washing is to neutral; Suction filtration adds ydrogen peroxide 50 in filter residue, in boiling water bath, placed 0.5～1 hour; Washing is drained to neutral, filter residue is added soak decolouring in 1～2.5 hour in the 0.5wt% potassium permanganate solution; After the decolouring filter residue adding 2～10wt% Hydrogen chloride or acetum were soaked 1～4 hour, filter, washing is to neutrality; Transfer filter residue pH to 8～10 with 10～20wt% sodium hydroxide solution, the chitosan flocks is separated out, filter; Washing is to neutral, with washing with alcohol 3 times, dry must chitosan.
Step 6: result: arachidonic acid content reaches as high as 10g/L, and chitosan content can reach 5g/L.
Embodiment 2
Step 1: the Mortierella isabellina of preservation (from Chinese common micro-organisms culture presevation administrative center, bacterium numbering is AS 3.3410) bacterial classification inoculation was cultivated 5～7 days in constant incubator (25～28 ℃) on the PDA slant medium.
Step 2: cultured spore is washed in the seed activation substratum with 5 mL sterilized waters, cultivated 2～3 days.Said substratum quality volume percent consists of: glucose 2%～6%, and yeast extract paste 0.3%～0.6%, potassium primary phosphate 0.2%～0.5%, SODIUMNITRATE 0.2%～0.5%, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.03%～0.1%, all the other are water, pH6.0～8.5.
Step 3: insert shaking in the bottle of 250mL that the 50mL fermention medium has been housed with the inoculum size of culture volume 10% seed liquor after with activation, placing temperature is that 25 ℃, rotating speed are the shaking table fermentation culture of 135rpm, cultivates 6～8 days.Said substratum quality volume percent consists of: glucose 7%～15%, and yeast extract paste 0.6%～1.2%, potassium primary phosphate 0.2%～0.6%, SODIUMNITRATE 0.2%～0.6%, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.05%～0.1%, all the other are water.
Step 4: fermentation ends, suction filtration is collected thalline and is dried to constant weight for 40～60 ℃, grinds sherwood oil and ethanol extracting grease.Arachidonic acid content in the gas chromatography determination grease.
Step 5: the extraction of chitosan: in the filter residue of step 4 gained, add the sodium hydroxide solution of 2～10wt%, the ratio of filter residue quality and sodium hydroxide solution volume is 1: 6 w/v, and boils 2～4 hours deproteinated, and washing is to neutral; Suction filtration adds ydrogen peroxide 50 in filter residue, in boiling water bath, placed 0.5～1 hour; Washing is drained to neutral, filter residue is added soak decolouring in 1～2.5 hour in the 0.5wt% potassium permanganate solution; After the decolouring filter residue adding 2～10wt% Hydrogen chloride or acetum were soaked 1～4 hour, filter, washing is to neutrality; Transfer filter residue pH to 8～10 with 10～20wt% sodium hydroxide solution, the chitosan flocks is separated out, filter; Washing is to neutral, with washing with alcohol 3 times, dry must chitosan.
Step 6: result: arachidonic acid content reaches as high as 8g/L, and chitosan content can reach 4g/L.
Embodiment 3
Step 1: the mortierella ramanniana of preservation (from Chinese common micro-organisms culture presevation administrative center, bacterium numbering is AS 3.3413) bacterial classification inoculation was cultivated 5～7 days in constant incubator (25～28 ℃) on the PDA slant medium.
Step 2: cultured spore is washed in the seed activation substratum with the 5mL sterilized water, cultivated 2～3 days for 25～28 ℃.Said substratum quality volume percent consists of: glucose 2%～6%, and yeast extract paste 0.3%～0.6%, potassium primary phosphate 0.2%～0.5%, SODIUMNITRATE 0.2%～0.5%, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.03%～0.1%, all the other are water, pH6.0～8.5.
Step 3: insert shaking in the bottle of 250mL that the 50mL fermention medium has been housed with the inoculum size of culture volume 10% seed liquor after with activation, placing temperature is that 25 ℃, rotating speed are the shaking table fermentation culture of 135 rpm, cultivates 6～8 days.Said substratum quality volume percent consists of: glucose 7%～15%, and yeast extract paste 0.6%～1.2%, potassium primary phosphate 0.2%～0.6%, SODIUMNITRATE 0.2%～0.6%, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.05%～0.1%, all the other are water.
Step 4: fermentation ends, suction filtration is collected thalline and is dried to constant weight for 40～60 ℃, grinds sherwood oil and ethanol extracting grease.Arachidonic acid content in the gas chromatography determination grease.
Step 5: the extraction of chitosan: in the filter residue of step 4 gained, add the sodium hydroxide solution of 2～10wt%, the ratio of filter residue quality and sodium hydroxide solution volume is 1: 5 w/v, and boils 2～4 hours deproteinated, and washing is to neutral; Suction filtration adds ydrogen peroxide 50 in filter residue, in boiling water bath, placed 0.5～1 hour; Washing is drained to neutral, filter residue is added soak decolouring in 1～2.5 hour in the 0.5wt% potassium permanganate solution; After the decolouring filter residue adding 2～10wt% Hydrogen chloride or acetum were soaked 1～4 hour, filter, washing is to neutrality; Transfer filter residue pH to 8～10 with 10～20wt% sodium hydroxide solution, the chitosan flocks is separated out, filter; Washing is to neutral, with washing with alcohol 3 times, dry must chitosan.
Step 6: result: arachidonic acid content reaches as high as 7g/L, and chitosan content can reach 4g/L.
Claims (4)
1. microbial fermentation coproduction of arachidonic acid and methods of chitosan is characterized in that production stage is:
A. the inoculum size according to culture volume 10% inserts mould in substratum, and it is that 25～28 ℃, rotating speed are the shaking table fermentation culture of 120～140rpm that postvaccinal substratum places temperature, cultivates 6～8 days; Said mould is Mortierella alpina (Mortierella alpina) CCTCC NO:M207067, Mortierella isabellina (Mortierella isabellina) AS3.3410 or mortierella ramanniana (Mortierella ramanniana) AS3.3413;
B. after the fermentation ends,, the thalline of collecting dried to constant weight at 40～60 ℃ grind,, sherwood oil and ethanol are removed through 0.01MPa/45 ℃ of underpressure distillation can be obtained arachidonic acid oil again with sherwood oil and ethanol extracting to the fermented liquid suction filtration; Said sherwood oil and alcoholic acid volume ratio are 3: 1, and wherein the thalline of 1 volume carries out extracting with the sherwood oil and the ethanol of 5～8 volumes;
C. the extraction of chitosan: add the sodium hydroxide solution of 2～10wt% in the filter residue after the extracting of step b gained, the ratio of filter residue quality and sodium hydroxide solution volume is 1: 3～1: 6w/v, and boil 2～4 hours deproteinated, washing is to neutral; Suction filtration adds ydrogen peroxide 50 in filter residue, in boiling water bath, placed 0.5～1 hour; Washing is drained to neutral, filter residue is added soak decolouring in 1～2.5 hour in the 0.5wt% potassium permanganate solution; After the decolouring filter residue adding 2～10wt% Hydrogen chloride or acetum were soaked 1～4 hour, filter, washing is to neutrality; Transfer filter residue pH to 8～10 with 10～20wt% sodium hydroxide solution, the chitosan flocks is separated out, filter; Washing is used washing with alcohol to neutral, the dry chitosan that gets.
2. microbial fermentation coproduction of arachidonic acid according to claim 1 and methods of chitosan is characterized in that carbon source in the said substratum is one or more in glucose, fructose, sucrose or the seminose.
3. microbial fermentation coproduction of arachidonic acid according to claim 1 and methods of chitosan; It is characterized in that nitrogenous source comprises inorganic nitrogen-containing compound and nitrogen-containing organic compound in the said substratum; Wherein inorganic nitrogen-containing compound is one or more in ammonium acetate, ammonium chloride, saltpetre or the SODIUMNITRATE, and nitrogen-containing organic compound is one or more in peptone, yeast extract paste, steeping water, wheat bran hydrolyzed solution or the Carnis Bovis seu Bubali cream.
4. microbial fermentation coproduction of arachidonic acid according to claim 1 and methods of chitosan is characterized in that inorganic salt in the said substratum are one or more in potassium hydrogenphosphate, potassium primary phosphate, sal epsom or the calcium chloride.
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| CN101109015A (en) * | 2007-07-09 | 2008-01-23 | 南京工业大学 | Method of preparing arachidonic acid oil and fat |
| CN101113410A (en) * | 2007-07-09 | 2008-01-30 | 南京工业大学 | Mortierella alpina and uses thereof |
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| CN101109015A (en) * | 2007-07-09 | 2008-01-23 | 南京工业大学 | Method of preparing arachidonic acid oil and fat |
| CN101113410A (en) * | 2007-07-09 | 2008-01-30 | 南京工业大学 | Mortierella alpina and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| PRAMOD, K. et al..Arachidonic Acid Production by Fungi.《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》.1991,第57卷(第4期),1255-1258. * |
| 王啸等.花生四烯酸产生菌的选育.《贵州工业大学学报(自然科学版)》.2005,第34卷(第1期),56-59. * |
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