CN103571896A - Method for producing arachidonic acid grease by utilizing mortierella alpina mutant strain, and arachidonic acid grease produced by method - Google Patents
Method for producing arachidonic acid grease by utilizing mortierella alpina mutant strain, and arachidonic acid grease produced by method Download PDFInfo
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- CN103571896A CN103571896A CN201310577448.5A CN201310577448A CN103571896A CN 103571896 A CN103571896 A CN 103571896A CN 201310577448 A CN201310577448 A CN 201310577448A CN 103571896 A CN103571896 A CN 103571896A
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- arachidonic acid
- mortierella alpina
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- acid oil
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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 189
- 235000021342 arachidonic acid Nutrition 0.000 title claims abstract description 92
- 229940114079 arachidonic acid Drugs 0.000 title claims abstract description 92
- 241000907999 Mortierella alpina Species 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000004519 grease Substances 0.000 title claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 title abstract description 8
- 150000004671 saturated fatty acids Chemical class 0.000 claims abstract description 35
- 238000000855 fermentation Methods 0.000 claims abstract description 33
- 230000004151 fermentation Effects 0.000 claims abstract description 33
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 37
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 35
- 239000001965 potato dextrose agar Substances 0.000 claims description 27
- 235000003441 saturated fatty acids Nutrition 0.000 claims description 25
- 235000021353 Lignoceric acid Nutrition 0.000 claims description 23
- CQXMAMUUWHYSIY-UHFFFAOYSA-N Lignoceric acid Natural products CCCCCCCCCCCCCCCCCCCCCCCC(=O)OCCC1=CC=C(O)C=C1 CQXMAMUUWHYSIY-UHFFFAOYSA-N 0.000 claims description 23
- FARYTWBWLZAXNK-WAYWQWQTSA-N ethyl (z)-3-(methylamino)but-2-enoate Chemical compound CCOC(=O)\C=C(\C)NC FARYTWBWLZAXNK-WAYWQWQTSA-N 0.000 claims description 23
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 19
- 239000000725 suspension Substances 0.000 claims description 19
- 239000001963 growth medium Substances 0.000 claims description 18
- 238000011218 seed culture Methods 0.000 claims description 18
- 238000000605 extraction Methods 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 17
- 241000894006 Bacteria Species 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 12
- 239000002054 inoculum Substances 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 11
- 239000012531 culture fluid Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 8
- 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 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 claims description 7
- 238000012807 shake-flask culturing Methods 0.000 claims description 6
- 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 5
- 229920002472 Starch Polymers 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
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- 235000019698 starch Nutrition 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 238000013341 scale-up Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000007710 freezing Methods 0.000 abstract 1
- 230000008014 freezing Effects 0.000 abstract 1
- UKMSUNONTOPOIO-UHFFFAOYSA-N docosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCC(O)=O UKMSUNONTOPOIO-UHFFFAOYSA-N 0.000 description 32
- VKOBVWXKNCXXDE-UHFFFAOYSA-N icosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCC(O)=O VKOBVWXKNCXXDE-UHFFFAOYSA-N 0.000 description 32
- 230000001580 bacterial effect Effects 0.000 description 23
- QZZGJDVWLFXDLK-UHFFFAOYSA-N tetracosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCCCC(O)=O QZZGJDVWLFXDLK-UHFFFAOYSA-N 0.000 description 21
- 235000021357 Behenic acid Nutrition 0.000 description 11
- 229940116226 behenic acid Drugs 0.000 description 11
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 10
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 10
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 10
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 10
- 230000000813 microbial effect Effects 0.000 description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- 238000010899 nucleation Methods 0.000 description 7
- 239000000284 extract Substances 0.000 description 6
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 6
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 6
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 5
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 5
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 5
- 239000005642 Oleic acid Substances 0.000 description 5
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 5
- 235000021355 Stearic acid Nutrition 0.000 description 5
- AHANXAKGNAKFSK-PDBXOOCHSA-N all-cis-icosa-11,14,17-trienoic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCCCC(O)=O AHANXAKGNAKFSK-PDBXOOCHSA-N 0.000 description 5
- PRHHYVQTPBEDFE-UHFFFAOYSA-N eicosatrienoic acid Natural products CCCCCC=CCC=CCCCCC=CCCCC(O)=O PRHHYVQTPBEDFE-UHFFFAOYSA-N 0.000 description 5
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 description 5
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 5
- 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 description 5
- 229960002733 gamolenic acid Drugs 0.000 description 5
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 5
- 229960004232 linoleic acid Drugs 0.000 description 5
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 5
- 239000008117 stearic acid Substances 0.000 description 5
- 235000014113 dietary fatty acids Nutrition 0.000 description 4
- 229930195729 fatty acid Natural products 0.000 description 4
- 239000000194 fatty acid Substances 0.000 description 4
- 150000004665 fatty acids Chemical class 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
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- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- DVSZKTAMJJTWFG-UHFFFAOYSA-N docosa-2,4,6,8,10,12-hexaenoic acid Chemical compound CCCCCCCCCC=CC=CC=CC=CC=CC=CC(O)=O DVSZKTAMJJTWFG-UHFFFAOYSA-N 0.000 description 2
- MBMBGCFOFBJSGT-KUBAVDMBSA-N docosahexaenoic acid Natural products CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000020673 eicosapentaenoic acid Nutrition 0.000 description 2
- BHAAPTBBJKJZER-UHFFFAOYSA-N p-anisidine Chemical compound COC1=CC=C(N)C=C1 BHAAPTBBJKJZER-UHFFFAOYSA-N 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 238000007711 solidification Methods 0.000 description 2
- 230000008023 solidification Effects 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000007536 Thrombosis Diseases 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
- 241000223259 Trichoderma Species 0.000 description 1
- 230000009471 action Effects 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
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- 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
- 230000017531 blood circulation Effects 0.000 description 1
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Abstract
The invention relates to a method for producing arachidonic acid grease by utilizing a mortierella alpina mutant strain, and the arachidonic acid grease produced by the method. The method comprises the steps that the mortierella alpina mutant strain is fermented; a fermentation product is collected; aftertreatment is performed; the arachidonic acid grease is obtained. The mortierella alpina mutant strain (mortierella alpina ZR36) is preserved in China Center For Type Culture Collection (CCTCC) on September 13, 2013; the preservation address is Wuhan University, Wuhan City, China; the preservation number is CCTCC NO:M2013419. The total content of saturated fatty acid, particularly above eicosanoic long-chain saturated fatty acid in the arachidonic acid grease produced by the mortierella alpina mutant strain is relatively low (lower than 15wt%); a freezing point of the arachidonic acid grease is relatively low (low to 4 DEG C); the arachidonic acid grease cannot be frozen or crystallized even if at a lower temperature; saturated fatty acid cannot be separated out; the arachidonic acid grease can keep clear and transparent, and has higher quality.
Description
Technical field
The present invention relates to a kind of Mortierella alpina mutant strain that utilizes and produce the method for arachidonic acid oil and the arachidonic acid oil of production thereof.
Background technology
In microbial oil, have multiple unsaturated fatty acids, unsaturated fatty acids mainly comprises monounsaturated fatty acids and polyunsaturated fatty acid, and they all have very large benefit to HUMAN HEALTH.Polyunsaturated fatty acid can synthesize docosahexenoic acid (DHA), timnodonic acid (EPA), arachidonic acid (ARA), they have in vivo reducing blood-fat, improve blood circulation, suppress platelet aggregation, prevent the effects such as atherosclerotic plaque and thrombosis, and cardiovascular and cerebrovascular disease are also had to good prevention effect.
Arachidonic acid oil has entered the suitability for industrialized production stage at present, still, studies at present many and pays close attention to arachidonic content in grease, has but ignored the content of other compositions, particularly chain saturated fatty acids in grease.And the content of chain saturated fatty acids is very large on the quality impact of grease, as the shared ratio in system of chain saturated fatty acids in arachidonic acid oil plays decisive action to the zero pour of grease, saturated fatty acid more than 20 carbon particularly, eicosanoic acid (C20:0) for example, behenic acid (C22:0), lignoceric acid (C24:0) etc., and the zero pour of saturated fatty acid is generally that increase with alkyl carbon chain length (being carbonatoms) increases.At present, in the arachidonic acid oil product that commercially available Mortierella alpina is produced, the content of chain saturated fatty acids more than 20 carbon is in 20wt% left and right.This grease can separate out saturated fatty acid, occur the phenomenon of system muddiness, so just affect the quality of arachidonic acid oil in the time of 12 ℃.
Chinese invention patent publication number be CN1362522A Patent Application Publication a kind ofly take Mortierella alpina and obtain the higher bacterial strain of a kind of arachidonic acid yield as the method that starting strain carries out particle beam mutagenesis.But, the not mentioned content that how to reduce chain saturated fatty acids of the present invention.Chinese invention patent publication No. be CN101709297 Patent Application Publication a kind of method of ultraviolet that adopts Mortierella alpina is carried out to mutagenesis, thereby can produce the arachidonic acid of high yield.But the present invention is the not mentioned content that how to reduce chain saturated fatty acids also.Chinese invention patent publication number is that the patent of CN1662642 relates to a kind of microbial oil, contain at least 90% triglyceride level, PUFA content is at least 40%, its peroxide value (POV) is lower than 1.5 (or 1.0), and/or its anisidine value (AnV) is lower than 15, alternatively, lower than 12.Also just content, peroxide value and the anisidine value etc. of unsaturated fatty acids in microbial oil that the application mainly pays close attention to, and and the content of not mentioned chain saturated fatty acids.
Therefore,, in view of the content of chain saturated fatty acids in the prepared arachidonic acid oil of existing Mortierella alpina is all higher, provide arachidonic acid oil and production method thereof that a kind of new chain saturated fatty acids content is lower real in necessary.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Mortierella alpina mutant strain that utilizes to produce the method for arachidonic acid oil and the arachidonic acid oil of production thereof.The arachidonic acid oil of its production contains at least triglyceride level of 90wt%, and in grease, arachidonic acid content is at least 35wt%, and chain saturated fatty acids content more than 20 carbon is lower than 15wt%.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
Utilize Mortierella alpina mutant strain to produce a method for arachidonic acid oil, it is characterized in that: by the fermentation of Mortierella alpina mutant strain, collect tunning, aftertreatment obtains arachidonic acid oil; Described Mortierella alpina mutant strain (Mortierella alpina ZR36, Mortierella alpine ZR36) is preserved in Chinese Typical Representative culture collection center (CCTCC) on September 13rd, 2013, and preservation address is, China, Wuhan, Wuhan University, deposit number is CCTCC NO:M2013419.
Press such scheme, the described concrete steps of utilizing above-mentioned Mortierella alpina mutant strain to produce the method for arachidonic acid oil are:
(1) preparation of spore suspension: above-mentioned Mortierella alpina sudden change is inoculated on potato dextrose agar (PDA) culture medium flat plate and is cultured to spore generation ripe, obtain spore suspension;
(2) seed culture: above-mentioned spore suspension is inoculated in to jolting in shake-flask culture base and cultivates acquisition seed culture fluid;
(3) fermentation culture: treat that the seed culture fluid that above-mentioned steps (2) jolting is cultivated is inoculated into fermentation culture in the fermentation flask that fermention medium is housed;
Or according to the size of final fermentor tank, the seed culture fluid that above-mentioned steps (2) jolting is cultivated is enlarged culturing step by step, obtains seed scale-up medium, be then inoculated into and in fermentor tank, carry out fermentation culture;
(4) fermented liquid is obtained to arachidonic acid oil through fermentation aftertreatment.
Press such scheme, described step (1) is: Mortierella alpina inoculation is arrived on potato dextrose agar (PDA) culture medium flat plate, at 25-28 ℃, cultivate 6-9 days to spore generation ripe, take off mycelia and spore on substratum, with sterilized water, be mixed with spore suspension.
Press such scheme, described step (2) is: the spore suspension of step (1) is inoculated in shaking flask and is cultivated, inoculum size is 10-20%(volume ratio), culture temperature 25-30 ℃, incubation time 36-50h, shaking table jolting rotating speed is 120-300 rev/min, and the seed culture medium in described shaking flask is: carbon source 20-50g/l; Nitrogenous source 5-30g/l; PH5.5-8.5.
Press such scheme, described step (3) is: in the time of the dense 15%～30%(volumetric concentration that reaches of seed culture fluid bacterium in step (2)), according to 10%～20%(volume ratio) inoculum size be linked into shaking culture in fermentation flask, the temperature that described jolting is cultivated is 25-30 ℃, incubation time is 8-12 days, shaking table speed is 120-300 rev/min, and described fermention medium is: carbon source 50-80g/l; Nitrogenous source is 10-30g/l; PH5.5-8.5;
Or according to the size of final fermentor tank, by the seed culture fluid of above-mentioned steps (2) shake-flask culture enlarged culturing step by step, the seed culture medium of using in the described process of enlarged culturing is step by step: carbon source 20-50g/l; Nitrogenous source 10-20g/l; PH5.5-8.5, and in the dense 15-30%(of the reaching volumetric concentration of bacterium) time, carry out next step enlarged culturing; Every grade of incubation time is 24-50h, and culture temperature is 25-30 ℃, and air flow is 1-2vvm(L/L.min) be that in every liter of fermented liquid, the needed air intake of per minute is 1-2L, tank pressure 0.05-0.15MPa, stirring velocity is turn/min of 150-300; Then the seed scale-up medium of above-mentioned acquisition is inoculated into and in final fermentor tank, carries out fermentation culture, inoculum size 10-20%(volume ratio), fermentation jar temperature 25-30 ℃, stirring velocity 200-300 rev/min, air flow 1-2vvm(L/L.min) be that in every liter of fermented liquid, the needed air intake of per minute is 1-2L, tank pressure 0.05-0.15Mpa, cultivate 150-180h, and by stream, add carbon source during the fermentation and come that in controlled fermentation liquid, carbon source concentration is at 5-20g/L, the fermention medium in described fermentor tank is: carbon source 30-50g/l; Nitrogenous source 10-20g/l; PH5.5-8.5.
Press such scheme, described carbon source is selected from glucose, sucrose or starch; Nitrogenous source is selected from yeast powder, yeast extract, yeast and soaks powder.
Press such scheme, the aftertreatment of described step (4), for the wet thallus after separation of fermentative broth is dried and obtained dry mycelium, then adds extraction agent to extract.
An arachidonic acid oil of being produced by aforesaid method, described arachidonic acid oil contains at least triglyceride level of 90wt%, and in grease, arachidonic acid content is at least 35wt%, and the above chain saturated fatty acids content of 20 carbon is lower than 15wt%.Chain saturated fatty acids more than 20 described carbon comprises eicosanoic acid, behenic acid, lignoceric acid.
Press such scheme, the content of lignoceric acid in described arachidonic acid oil (C24:0) is less than 8wt%.
Press such scheme, the zero pour of described arachidonic acid oil is 4 ℃-11 ℃.
Above-mentioned bacterium is dense is to be about to the ratio that the volume of lower floor's thalline after medium centrifugal accounts for total system volume.
Beneficial effect of the present invention:
The saturated fatty acid total content lower (lower than 15wt%) of the above chain saturated fatty acids of 20 carbon particularly in the arachidonic acid oil that the method for utilizing the described Mortierella alpina mutant strain of this invention to produce arachidonic acid oil is produced, and the zero pour of arachidonic acid oil is lower (can be low to moderate 4 ℃) also, even if it can not separate out saturated fatty acid by solidification and crystallization at a lower temperature yet, can keep limpid transparent, there is higher quality.Especially, the content lower (being less than 8wt%) of the saturated fatty acid lignoceric acid (C24:0) of long-chain, can more effectively reduce the zero pour of arachidonic acid oil thus, guarantees better the quality of arachidonic acid oil.
Embodiment
Following examples are used for describing in detail particular content of the present invention, but the present invention is not limited to the content of following examples.
The selection of embodiment 1. Mortierella alpina mutant strains
(1) getting commercially available Mortierella alpina is starting strain.
(2) bacterial classification is inoculated on potato dextrose agar (PDA) substratum and under 28 ℃ of constant temperatures, cultivates 8 days to spore maturation.
(3) by gauze or filter paper filtering, obtain pure spore liquid, spore liquid is injected into process ultra violet lamp in sterile petri dish, ultra violet lamp distance is 30 centimetres, and irradiation time is 60 seconds, and the power of ultraviolet lamp is 30 watts.
(4) spore liquid after ultraviolet mutagenesis is air-dry through sterile wind, becomes bacterial plaque.By containing in the aseptic immigration high energy particle of the culture dish beam implanter of bacterial plaque, through high energy ion beam, inject mutagenesis.
(5) bacterial plaque is used to sterilized water wash-out, dilution, is applied on potato dextrose agar (PDA) culture medium flat plate and cultivates, random 10 single bacterium colonies of picking.
(6) single bacterium colony of above-mentioned picking is carried out to liquid shaking bottle cultivation, according to the fatty acid distribution of the microorganism grease of each bacterium acquisition, choose bacterium and the highest bacterium (higher than 50wt%) of arachidonic acid content of chain saturated fatty acids eicosanoic acid (C20:0), behenic acid (C22:0), lignoceric acid (C24:0) total content minimum (lower than 10wt%).Two strain bacterium of screening are thus received respectively on potato dextrose agar (PDA) culture medium flat plate, in 25 ℃, the constant incubator of humidity 50% is cultivated 5 days, calmodulin binding domain CaM picking mycelia or spore two strain bacterium, be transferred on fresh potato dextrose agar (PDA) culture medium flat plate and cultivate, obtain Mortierella alpina mutant strain (Mortierella alpina ZR36 of the present invention, Mortierella alpine ZR36), this bacterial strain is preserved in Chinese Typical Representative culture collection center (CCTCC) on September 13rd, 2013, preservation address is, China, Wuhan, Wuhan University, deposit number is CCTCC NO:M2013419.
Above-mentioned Mortierella alpine trichoderma strain CCTCC NO:M2013419 has following physio-biochemical characteristics:
(1) grow in PDA slant medium, grow after 2 days at 28 ℃, in media surface, can form white colony, after 3 days, grow into logarithmic phase, aerial hyphae color is snow-white, and after 6 days, mycelia is covered with whole inclined-plane;
(2) the sporulation phase very late, after 7 days, aerial hyphae top starts flavescence, starts to be afterwards divided into yellow sporocyst;
(2) optimum culture temperature is 28 ℃;
(4) for the available carbon source of substratum of cultivating, be: glucose, sucrose, starch, do not utilize or seldom utilize glycerine; Available nitrogenous source is mainly yeast powder, yeast extract, yeast and soaks powder.
Embodiment 2 utilizes Mortierella alpina mutant strain to produce arachidonic acid
A) spore suspension is prepared: get respectively commercially available Mortierella alpina and deposit number of the present invention: the Mortierella alpina mutant strain of CCTCC M2013419 is inoculated on potato dextrose agar (PDA) culture medium flat plate, cultivate 7 days to spore maturation for 25-28 ℃, after the spore on potato dextrose agar (PDA) culture medium flat plate and mycelia are scraped 10 ml sterile waters are housed, concussion obtains spore suspension.
B) seed shake-flask culture: the spore suspension of step (1) is inoculated in the seed bottle that is placed with substratum to inoculum size 10%(volume ratio), be placed in 25 ℃, cultivate 36 hours on the shaking table of 120 revs/min, described substratum is: carbon source glucose 20g/l; Nitrogenous source yeast powder 5g/l; PH5.5.
C) fermentation flask is cultivated: treat the dense 15%(of the reaching volume ratio of bacterium in seed bottle), be linked in the fermentation flask that is placed with fermention medium inoculum size 10%(volume ratio), be placed in 25 ℃, on the shaking table of 120 revs/min, cultivate 9 days.Described fermention medium is: carbon source glucose 50g/l; Nitrogenous source yeast powder 10g/l; PH5.5.
D) aftertreatment: the separation of fermentative broth that fermentation culture is obtained, obtain wet thallus, dry and obtain dry mycelium 30g.In dry mycelium, add extraction agent normal hexane to extract, after extraction, the solid formation that obtains of separation proceeds to and in extraction container, carries out re-extract, so, until finish extraction process without when oil in extraction liquid, while extracting for the first time, add 200 ml n-hexanes, add afterwards 150 ml n-hexanes at every turn, will extract and fill the mixing oil that rear filtering separation obtains at every turn, precipitation, obtains microorganism grease.
E) this microbial oil is carried out to gas chromatographic analysis, carry out setting-point test, the main unsaturated fatty acids in this microbial oil and the content of chain saturated fatty acids comprise that palmitinic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linolic acid (C18:2), gamma-linolenic acid (C18:3), eicosanoic acid (C20:0), eicosatrienoic acid (C20:3), arachidonic acid (C20:4), behenic acid (C22:0), lignoceric acid (C24:0) see the following form simultaneously:
Each fatty acid content in arachidonic acid oil in table 1 embodiment 2
| ? | Commercially available bacterial classification (wt%) | Bacterial classification for the present invention (wt%) |
| Palmitinic acid (C16:0) | 7.99 | 9.19 |
| Stearic acid (C18:0) | 6.52 | 10.2 |
| Oleic acid (C18:1) | 9.52 | 9.21 |
| Linolic acid (C18:2) | 8.22 | 8.45 |
| Gamma-linolenic acid (C18:3) | 5.21 | 6.33 |
| Eicosatrienoic acid (C20:3) | 8.09 | 5.46 |
| Arachidonic acid (C20:4) | 35.3 | 35.6 |
| Eicosanoic acid (C20:0) | 3.28 | 3.43 |
| Behenic acid (C22:0) | 4.1 | 4.12 |
| Lignoceric acid (C24:0) | 11.78 | 7.26 |
The present embodiment is used in the prepared arachidonic acid oil of Mortierella alpina mutant strain bacterial classification, the content of triglyceride is 91wt%, another associative list 1 is known: in this arachidonic acid oil, the content of arachidonic acid oil is 35.3wt%, the total content that three kinds of chain saturated fatty acids are eicosanoic acid (C20:0), behenic acid (C22:0), lignoceric acid (C24:0) is 14.81wt%, and wherein the content of lignoceric acid (C24:0) is 7.26wt%.Be starkly lower than the total content (19.16wt%) of three kinds of chain saturated fatty acids in the prepared arachidonic acid oil of commercially available bacterial classification, and the content of lignoceric acid (C24:0) is also starkly lower than the content (11.78wt%) of lignoceric acid (C24:0) in the prepared arachidonic acid oil of commercially available bacterial classification.Separately, through cold test: the present invention is 9 ℃ by the prepared arachidonic acid oil zero pour of Mortierella alpina mutant strain bacterial classification, the zero pour of the arachidonic acid oil that it is prepared far below commercially available bacterial classification (18 ℃).
Embodiment 3 utilizes Mortierella alpina mutant strain to produce arachidonic acid
A) spore suspension is prepared: get respectively commercially available Mortierella alpina and Mortierella alpina used in the present invention (deposit number: CCTCC M2013419) be inoculated on potato dextrose agar (PDA) culture medium flat plate, cultivate 8 days to spore maturation for 25-28 ℃, after the spore on potato dextrose agar (PDA) culture medium flat plate and mycelia are scraped 20 ml sterile waters are housed, concussion obtains spore suspension.
B) shake-flask seed is cultivated: the spore suspension of step (1) is inoculated in the seed bottle that is placed with substratum to inoculum size 15%(volume ratio), be placed in 28 ℃, on the shaking table of 220 revs/min, to cultivate 48 hours, described substratum is: carbon source sucrose 35g/l; Nitrogenous source yeast soaks powder 12g/l; PH7.
C) seed enlarged culturing: final fermentor cultivation adopts the volume of 50L, so seeding tank selects 10L seed to expand fermentor tank.The shake-flask seed fermented liquid of step (2) is inoculated into and in seeding tank, carries out seed enlarged culturing, seed culture medium carbon source sucrose 35g/l wherein; Nitrogenous source yeast soaks powder 12g/l, controls pH7, and leavening temperature is 28 ℃, 220 revs/min of stirring velocitys, air flow 1vvm(L/L.min), tank pressure 0.1Mpa, cultivates 42h.
D) fermentation culture: in seeding tank, bacterium is dense reach 20% after, by culture transferring pipeline, be linked in the 50L fermentor tank that 30L fermention medium is housed and cultivate, inoculum size 15%(volume ratio), fermentor tank is controlled 28 ℃ of temperature, 220 revs/min of stirring velocitys, air flow 1vvm(L/L.min), tank pressure 0.1Mpa, cultivates 170h.In fermenting process, by stream, add carbon source and come that in controlled fermentation liquid, carbon source concentration is at 10g/L, the fermention medium in described fermentor tank is: carbon source sucrose 35g/l; Nitrogenous source yeast soaks powder 12g/l; PH7.
E) aftertreatment: the separation of fermentative broth that fermentation culture is obtained, obtain wet thallus, dry and obtain dry mycelium 40g.In dry mycelium, add extraction agent normal hexane to extract, after extraction, the solid formation that obtains of separation proceeds to and in extraction container, carries out re-extract, so, until finish extraction process without when oil in extraction liquid, while extracting for the first time, add 200 ml n-hexanes, add afterwards 150 ml n-hexanes at every turn, will extract and fill the mixing oil that rear filtering separation obtains at every turn, precipitation, obtains microorganism grease.
F) this microbial oil is carried out to gas chromatographic analysis, carry out setting-point test, the main unsaturated fatty acids in this microbial oil and the content of chain saturated fatty acids comprise palmitinic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linolic acid (C18:2), gamma-linolenic acid (C18:3), eicosanoic acid (C20:0), eicosatrienoic acid (C20:3), arachidonic acid (C20:4), behenic acid (C22:0), (C24:0) is as follows for lignoceric acid simultaneously:
Each fatty acid content in arachidonic acid oil in table 2 embodiment 3
The present embodiment is used in the prepared arachidonic acid oil of Mortierella alpina mutant strain bacterial classification, the content of triglyceride is 93wt%, another associative list 2 is known: in this arachidonic acid oil, the content of arachidonic acid oil is 40.8wt%, three kinds of chain saturated fatty acids, be eicosanoic acid (C20:0), behenic acid (C22:0), the total content of lignoceric acid (C24:0) is 10.3wt%, wherein the content of lignoceric acid (C24:0) is 3.2wt%, be starkly lower than the total content (18.73wt%) of three kinds of chain saturated fatty acids in the prepared arachidonic acid oil of commercially available bacterial classification, the content of another lignoceric acid (C24:0) is also starkly lower than the content (11.23wt%) of lignoceric acid (C24:0) in the prepared arachidonic acid oil of commercially available bacterial classification.Separately, through cold test: the arachidonic acid oil zero pour that the present invention produces is 6 ℃.The zero pour of the arachidonic acid oil that it is prepared far below commercially available bacterial classification (15 ℃).
Embodiment 4 utilizes Mortierella alpina mutant strain to produce arachidonic acid
A) spore suspension is prepared: get respectively commercially available Mortierella alpina and Mortierella alpina mutant strain used in the present invention (preservation
Numbering: CCTCC M2013419) be inoculated on potato dextrose agar (PDA) culture medium flat plate;?-? ℃ cultivate 8 days ripe to spore, after the spore on potato dextrose agar (PDA) culture medium flat plate and mycelia are scraped 30 ml sterile waters are housed, concussion obtains spore suspension.
B) shake-flask seed is cultivated: the spore suspension of step (1) is inoculated in the seed bottle that is placed with substratum to inoculum size 20%(volume ratio), be placed in 30 ℃, on the shaking table of 300 revs/min, to cultivate 50 hours, described substratum is: carbon source sucrose 50g/l; Nitrogenous source yeast soaks powder 12g/l; PH8.5.
C) seed enlarged culturing: the volume of final fermentor tank is 200m
3, selecting successively volume is 10L, 50L, 5m
3, 50m
3seeding tank enlarged culturing seed liquor, in seeding tank, substratum loading amount is 60%(volume ratio), culturing process technology controlling and process is: 30 ℃ of temperature, 300 revs/min of stirring velocitys, air flow 1.5vvm(L/L.min), incubation time 48h.Seed culture medium in described seeding tank is: carbon source starch 50g/l; Nitrogenous source yeast extract 20g/l; PH8.5, by the shake-flask culture liquid of above-mentioned steps enlarged culturing step by step.
D) fermentation culture: treat 50m
3the dense 30%(volume ratio that reaches of bacterium in seeding tank), after, by culture transferring pipeline, be linked into 130m is housed
3the 200m of fermention medium
3in fermentor tank, cultivate inoculum size 20%(volume ratio), fermentor tank is controlled 30 ℃ of temperature, 300 revs/min of stirring velocitys, air flow 1.5vvm(L/L.min), tank pressure 0.15Mpa, cultivates 180h.In fermenting process, by stream, add carbon source and come that in controlled fermentation liquid, carbon source concentration is at 20g/L, described fermentation tank culture medium is: carbon source starch 50g/l; Nitrogenous source yeast extract 20g/l; PH8.5.
E) aftertreatment: the separation of fermentative broth that fermentation culture is obtained, obtain wet thallus, dry and obtain dry mycelium 50g.In dry mycelium, add extraction agent normal hexane to extract, after extraction, the solid formation that obtains of separation proceeds to and in extraction container, carries out re-extract, so, until finish extraction process without when oil in extraction liquid, while extracting for the first time, add 200 ml n-hexanes, add afterwards 150 ml n-hexanes at every turn, will extract and fill the mixing oil that rear filtering separation obtains at every turn, precipitation, obtains microorganism grease.
F) this microbial oil is carried out to gas chromatographic analysis, carry out setting-point test, the main unsaturated fatty acids in this microbial oil and the content of chain saturated fatty acids comprise palmitinic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linolic acid (C18:2), gamma-linolenic acid (C18:3), eicosanoic acid (C20:0), eicosatrienoic acid (C20:3), arachidonic acid (C20:4), behenic acid (C22:0), (C24:0) is as follows for lignoceric acid simultaneously:
Each fatty acid content in arachidonic acid oil in table 3 embodiment 4
| ? | Contrast bacterial classification (wt%) | The present invention's bacterial classification used (wt%) |
| Palmitinic acid (C16:0) | 7.99 | 9.78 |
| Stearic acid (C18:0) | 7.31 | 10.52 |
| Oleic acid (C18:1) | 6.77 | 9.32 |
| Linolic acid (C18:2) | 7.36 | 8.37 |
| Gamma-linolenic acid (C18:3) | 2.65 | 3.1 |
| Eicosatrienoic acid (C20:3) | 4.63 | 6.27 |
| Arachidonic acid (C20:4) | 44.97 | 44.56 |
| Eicosanoic acid (C20:0) | 3.1 | 1.5 |
| Behenic acid (C22:0) | 4 | 3.08 |
| Lignoceric acid (C24:0) | 12 | 2.92 |
The present embodiment is used in the prepared arachidonic acid oil of Mortierella alpina mutant strain bacterial classification, the content of triglyceride is 95wt%, another associative list 2 is known: in this arachidonic acid oil, the content of arachidonic acid oil is 44.97wt%, three kinds of chain saturated fatty acids, be eicosanoic acid (C20:0), behenic acid (C22:0), the total content of lignoceric acid (C24:0) is 7.5wt%, wherein the content of lignoceric acid (C24:0) is 2.92wt%, be starkly lower than the total content (19.1wt%) of three kinds of chain saturated fatty acids in the prepared arachidonic acid oil of commercially available bacterial classification, the content of another lignoceric acid (C24:0) is also starkly lower than the content (12wt%) of lignoceric acid (C24:0) in the prepared arachidonic acid oil of commercially available bacterial classification.Separately, through cold test: the arachidonic acid oil zero pour that the present invention produces is 4 ℃, the zero pour of the arachidonic acid oil that it is prepared far below commercially available bacterial classification (15 ℃).
Comprehensive above embodiment can find out, the arachidonic acid oil that utilizes Mortierella alpina mutant strain of the present invention to produce, its arachidonic content and commercially available prod difference are little, but the total content of the above chain saturated fatty acids of 20 carbon is obviously lower, particularly the content of tetracosa carbon saturated fatty acid is obviously lower, the zero pour that makes thus arachidonic acid oil is lower (minimum be low to moderate 4 ℃) also, even if it can not separate out saturated fatty acid by solidification and crystallization at a lower temperature yet, can keep limpid transparent, there is higher quality.
Claims (10)
1. utilize Mortierella alpina mutant strain to produce a method for arachidonic acid oil, it is characterized in that: by the fermentation of Mortierella alpina mutant strain, collect tunning, aftertreatment obtains arachidonic acid oil; Described Mortierella alpina mutant strain is preserved in Chinese Typical Representative culture collection center (CCTCC), and preservation address is, China, and Wuhan, Wuhan University, deposit number is CCTCC NO:M2013419.
2. the method for utilizing Mortierella alpina mutant strain to produce arachidonic acid oil according to claim 1, is characterized in that: its concrete steps are:
(1) preparation of spore suspension: above-mentioned Mortierella alpina sudden change is inoculated on potato dextrose agar flat board and is cultured to spore generation ripe, obtain spore suspension;
(2) seed culture: above-mentioned spore suspension is inoculated in to jolting in shake-flask culture base and cultivates acquisition seed culture fluid;
(3) fermentation culture: treat that the seed culture fluid that above-mentioned steps (2) jolting is cultivated is inoculated into fermentation culture in the fermentation flask that fermention medium is housed;
Or according to the size of final fermentor tank, the seed culture fluid that above-mentioned steps (2) jolting is cultivated is enlarged culturing step by step, obtains seed scale-up medium, be then inoculated into and in fermentor tank, carry out fermentation culture;
(4) fermented liquid is obtained to arachidonic acid oil through fermentation aftertreatment.
3. the method for utilizing Mortierella alpina mutant strain to produce arachidonic acid oil according to claim 2, it is characterized in that: described step (1) is: by Mortierella alpina inoculation to potato dextrose agar flat board, at 25-28 ℃, cultivate and to spore, generate also ripe in 6-9 days, take off mycelia and spore on substratum, with sterilized water, be mixed with spore suspension.
4. the method for utilizing Mortierella alpina mutant strain to produce arachidonic acid oil according to claim 2, it is characterized in that: described step (2) is: the spore suspension of step (1) is inoculated in shaking flask and is cultivated, inoculum size is 10-20%(volume ratio), culture temperature 25-30 ℃, incubation time 36-50h, shaking table jolting rotating speed is 120-300 rev/min, and the seed culture medium in described shaking flask is: carbon source 20-50g/l; Nitrogenous source 5-30g/l; PH5.5-8.5.
5. the method for utilizing Mortierella alpina mutant strain to produce arachidonic acid oil according to claim 2, it is characterized in that: described step (3) is: in the time of the dense 15%～30%(volumetric concentration that reaches of seed culture fluid bacterium in step (2)), according to 10%～20%(volume ratio) inoculum size be linked into shaking culture in fermentation flask, the temperature that described jolting is cultivated is 25-30 ℃, incubation time is 8-12 days, shaking table speed is 120-300 rev/min, and described fermention medium is: carbon source 50-80g/l; Nitrogenous source is 10-30g/l; PH5.5-8.5;
Or according to the size of final fermentor tank, by the seed culture fluid of above-mentioned steps (2) shake-flask culture enlarged culturing step by step, the seed culture medium of using in the described process of enlarged culturing is step by step: carbon source 20-50g/l; Nitrogenous source 10-20g/l; PH5.5-8.5, and in the dense 15-30%(of the reaching volumetric concentration of bacterium) time, carry out next step enlarged culturing; Every grade of incubation time is 24-50h, and culture temperature is 25-30 ℃, and air flow is 1-2vvm(L/L.min), tank pressure 0.05-0.15MPa, stirring velocity is turn/min of 150-300; Then the seed scale-up medium of above-mentioned acquisition is inoculated into and in final fermentor tank, carries out fermentation culture, inoculum size 10-20%(volume ratio), fermentation jar temperature 25-30 ℃, stirring velocity 200-300 rev/min, air flow 1-2vvm(L/L.min), tank pressure 0.05-0.15Mpa, cultivates 150-180h, and by stream, add carbon source during the fermentation and come that in controlled fermentation liquid, carbon source concentration is at 5-20g/L, the fermention medium in described fermentor tank is: carbon source 30-50g/l; Nitrogenous source 10-20g/l; PH5.5-8.5.
6. according to the Mortierella alpina mutant strain that utilizes described in claim 4 or 5, produce the method for arachidonic acid oil, it is characterized in that: described carbon source is selected from glucose, sucrose or starch; Nitrogenous source is selected from yeast powder, yeast extract, yeast and soaks powder.
7. the method for utilizing Mortierella alpina mutant strain to produce arachidonic acid oil according to claim 2, is characterized in that: the aftertreatment of described step (4), for the wet thallus after separation of fermentative broth is dried and obtained dry mycelium, then adds extraction agent to extract.
8. the arachidonic acid oil that method claimed in claim 1 is produced, it is characterized in that: described arachidonic acid oil contains at least triglyceride level of 90wt%, in grease, arachidonic acid content is at least 35wt%, and the above chain saturated fatty acids content of 20 carbon is lower than 15wt%.
9. arachidonic acid oil according to claim 8, is characterized in that: in described arachidonic acid oil, the content of lignoceric acid is less than 8wt%.
10. arachidonic acid oil according to claim 8, is characterized in that: the zero pour of described arachidonic acid oil is 4 ℃-11 ℃.
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| CN105861339A (en) * | 2016-06-16 | 2016-08-17 | 江南大学 | Recombination mortierella alpine of overexpression GTP ring type hydrolytic enzyme gene and construction method and application of recombination mortierella alpine |
| CN106636236A (en) * | 2017-01-13 | 2017-05-10 | 江南大学 | Fermentation medium for promoting mortierella alpina to produce EPA (eicosapentaenoic acid) and application thereof |
| CN107418982A (en) * | 2017-09-25 | 2017-12-01 | 嘉必优生物技术（武汉）股份有限公司 | A kind of low chloropropyl alcohol microbial grease and preparation method thereof |
| CN108410916A (en) * | 2018-05-30 | 2018-08-17 | 湖北福星生物科技有限公司 | The production method of arachidonic acid oil |
| CN108517338A (en) * | 2018-03-15 | 2018-09-11 | 南京工业大学 | A method of based on active oxygen regulation and control Mortierella alpina fermentation producing arachidonic acid oil |
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| CN108410916A (en) * | 2018-05-30 | 2018-08-17 | 湖北福星生物科技有限公司 | The production method of arachidonic acid oil |
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