CN103571896B - A kind of Mortierella alpina mutant strain that utilizes is produced the method for arachidonic acid oil and the arachidonic acid oil of production thereof - Google Patents
A kind of Mortierella alpina mutant strain that utilizes is produced the method for arachidonic acid oil and the arachidonic acid oil of production thereof Download PDFInfo
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- 235000021342 arachidonic acid Nutrition 0.000 title claims abstract description 82
- 229940114079 arachidonic acid Drugs 0.000 title claims abstract description 82
- 241000907999 Mortierella alpina Species 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000004519 manufacturing process Methods 0.000 title abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 50
- 230000004151 fermentation Effects 0.000 claims abstract description 50
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 37
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- 238000004321 preservation Methods 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 31
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- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 19
- 239000000725 suspension Substances 0.000 claims description 19
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Abstract
The present invention relates to a kind of Mortierella alpina mutant strain that utilizes and produce the method for arachidonic acid oil and the Mortierella alpina of production thereof. The method is by the fermentation of Mortierella alpina mutant strain, collects tunning, and post processing obtains arachidonic acid oil; Described Mortierella alpina mutant strain (Mortierella alpina ZR36, Mortierella? alpine? ZR36) be preserved in Chinese Typical Representative culture collection center (CCTCC) on September 13rd, 2013, preservation address is, China, Wuhan, Wuhan University, does is deposit number CCTCC? NO:M2013419. The particularly total content lower (lower than 15wt%) of the above chain saturated fatty acids of 20 carbon of saturated fatty acid in the arachidonic acid oil that this Mortierella alpina mutant strain is produced, and the freezing point of arachidonic acid oil is lower (can be low to moderate 4 DEG C) also, even if it also can not separate out saturated fatty acid by solidification and crystallization at a lower temperature, can keep limpid transparent, there is 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 unrighted acid, unrighted acid mainly comprises monounsaturated fatty acids and polyunsaturated fatty acid, and they all have very large benefit to health. Polyunsaturated fatty acid can synthesize DHA (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), they have in vivo reducing blood lipid, improve blood circulation, suppress platelet aggregation, prevent the effect such as atherosclerotic plaque and thrombosis, and cardiovascular and cerebrovascular diseases 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 freezing point of grease, particularly saturated fatty acid more than 20 carbon, for example arachidic acid (C20:0), behenic acid (C22:0), lignoceric acid (C24:0) etc., and the freezing point of saturated fatty acid is generally to increase with the increase of alkyl carbon chain length (being carbon number). 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 DEG C.
Chinese invention patent publication number be CN1362522A Patent Application Publication a kind of method of carrying out particle beams mutagenesis taking Mortierella alpina as starting strain obtain the higher bacterial strain of a kind of arachidonic acid yield. 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 that adopts ultraviolet 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% triglycerides, polyunsaturated fatty acid (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 unrighted acid in microbial grease 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 triglycerides 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, post processing obtains arachidonic acid oil; Described Mortierella alpina mutant strain (Mortierella alpina ZR36, MortierellaalpineZR36) be preserved in Chinese Typical Representative culture collection center (CCTCC) on September 13rd, 2013, preservation address is, China, Wuhan, Wuhan University, deposit number is CCTCCNO: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 shaking flask culture medium and cultivates acquisition seed culture fluid;
(3) fermented and cultured: treat that the seed culture fluid that above-mentioned steps (2) jolting is cultivated is inoculated into fermented and cultured in the fermentation flask that fermentation medium is housed;
Or according to the size of final fermentation tank, the seed culture fluid that above-mentioned steps (2) jolting is cultivated expands cultivation step by step, obtains seed scale-up medium, be then inoculated into and in fermentation tank, carry out fermented and cultured;
(4) zymotic fluid is obtained to arachidonic acid oil through fermentation post processing.
Press such scheme, described step (1) is: Mortierella alpina inoculation is arrived on potato dextrose agar (PDA) culture medium flat plate, at 25-28 DEG C, cultivate and generate also ripe to spore in 6-9 days, take off mycelia and spore on culture medium, be mixed with spore suspension with sterilized water.
Press such scheme, described step (2) is: the spore suspension of step (1) is inoculated in shaking flask and is cultivated, inoculum concentration is 10-20% (volume ratio), cultivation temperature 25-30 DEG C, 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: seed culture fluid bacterium is dense in the step (2) while reaching 15%～30% (volumetric concentration), be linked into shaken cultivation in fermentation flask according to the inoculum concentration of 10%～20% (volume ratio), the temperature that described jolting is cultivated is 25-30 DEG C, incubation time is 8-12 days, shaking table speed is 120-300 rev/min, and described fermentation medium is: carbon source 50-80g/l; Nitrogenous source is 10-30g/l; PH5.5-8.5;
Or according to the size of final fermentation tank, the seed culture fluid that above-mentioned steps (2) shaking flask is cultivated expands cultivation step by step, the described seed culture medium of using in incubation that expands is step by step: carbon source 20-50g/l; Nitrogenous source 10-20g/l; PH5.5-8.5, and in the time of the dense 15-30% of reaching of bacterium (volumetric concentration), carry out next step expansion and cultivate; Every grade of incubation time is 24-50h, and cultivation temperature is 25-30 DEG C, and throughput is that 1-2vvm (L/L.min) is that in every liter of zymotic fluid, needed air intake per minute is 1-2L, tank pressure 0.05-0.15MPa, and mixing speed is turn/min of 150-300; Then the seed scale-up medium of above-mentioned acquisition is inoculated into and in final fermentation tank, carries out fermented and cultured, inoculum concentration 10-20% (volume ratio), fermentation jar temperature 25-30 DEG C, mixing speed 200-300 rev/min, throughput 1-2vvm (L/L.min) is that in every liter of zymotic fluid, needed air intake per minute is 1-2L, tank pressure 0.05-0.15Mpa, cultivate 150-180h, and add carbon source by stream during the fermentation and come that in controlled fermentation liquid, carbon source concentration is at 5-20g/L, the fermentation medium in described fermentation 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 dusty yeast, yeast extract, yeast and soaks powder.
Press such scheme, the post processing of described step (4) is that the wet thallus after separation of fermentative broth is dried and obtained dry mycelium, then adds extractant to extract.
An arachidonic acid oil of being produced by said method, described arachidonic acid oil contains at least triglycerides 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 arachidic 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 freezing point of described arachidonic acid oil is 4 DEG C-11 DEG C.
Above-mentioned bacterium is dense is the ratio that accounts for total system volume by the volume of lower floor's thalline after medium centrifugal.
Beneficial effect of the present invention:
The particularly total content lower (lower than 15wt%) of the above chain saturated fatty acids of 20 carbon of saturated fatty acid in the arachidonic acid oil that the method for utilizing Mortierella alpina mutant strain described in this invention to produce arachidonic acid oil is produced, and the freezing point of arachidonic acid oil is lower (can be low to moderate 4 DEG C) also, even if it also can not separate out saturated fatty acid by solidification and crystallization at a lower temperature, 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 freezing point of arachidonic acid oil thus, ensures better the quality of arachidonic acid oil.
Detailed description of the invention
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) culture medium and cultivates 8 days to spore maturation under 28 DEG C of constant temperatures.
(3) obtain pure spore liquid by gauze or Filter paper filtering, 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 uviol 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, inject mutagenesis through high energy ion beam.
(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 profile 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 arachidic 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 DEG C, the constant incubator of humidity 50% is cultivated 5 days, at calmodulin binding domain CaM picking mycelia or the spore of 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, MortierellaalpineZR36), 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 CCTCCNO:M2013419.
Above-mentioned Mortierella alpine trichoderma strain CCTCCNO:M2013419 has following physio-biochemical characteristics:
(1) grow in PDA slant medium, grow after 2 days at 28 DEG C, can form white colony in media surface, after 3 days, grow into exponential phase, aerial hyphae color is snow-white, and after 6 days, mycelia is covered with whole inclined-plane;
(2) the sporogenesis phase very late, after 7 days, aerial hyphae top starts flavescence, starts to be afterwards divided into yellow sporangium;
(2) optimum cultivation temperature is 28 DEG C;
(4) for the available carbon source of culture medium of cultivating be: glucose, sucrose, starch, do not utilize or seldom utilize glycerine; Available nitrogenous source is mainly dusty yeast, yeast extract, yeast and soaks powder.
Embodiment 2 utilizes Mortierella alpina mutant strain to produce arachidonic acid
A) spore suspension is prepared: the Mortierella alpina mutant strain of getting respectively commercially available Mortierella alpina and deposit number of the present invention: CCTCCM2013419 is inoculated on potato dextrose agar (PDA) culture medium flat plate, cultivate 7 days to spore maturation for 25-28 DEG C, 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 shaking flask is cultivated: the spore suspension of step (1) is inoculated in the seed bottle that is placed with culture medium, inoculum concentration 10% (volume ratio), be placed in 25 DEG C, cultivate 36 hours on the shaking table of 120 revs/min, described culture medium is: carbon source glucose 20g/l; Nitrogenous source dusty yeast 5g/l; PH5.5.
C) fermentation flask is cultivated: treat dense 15% (volume ratio) that reach of bacterium in seed bottle, be linked in the fermentation flask that is placed with fermentation medium, inoculum concentration 10% (volume ratio), is placed in 25 DEG C, cultivates 9 days on the shaking table of 120 revs/min. Described fermentation medium is: carbon source glucose 50g/l; Nitrogenous source dusty yeast 10g/l; PH5.5.
D) post processing: the separation of fermentative broth that fermented and cultured is obtained, obtain wet thallus, dry and obtain dry mycelium 30g. In dry mycelium, add extractant n-hexane to extract, after extraction, separating the solid formation that obtains proceeds to and in extraction container, carries out re-extract, so, until finish extraction process without when oil in extract, while extraction for the first time, add 200 ml n-hexanes, add afterwards 150 ml n-hexanes at every turn, will extract and fill the miscella that rear isolated by filtration obtains at every turn, precipitation, obtains microorganism grease.
E) this microbial grease is carried out to gas chromatographic analysis, carry out setting-point test, the main unrighted acid in this microbial grease and the content of chain saturated fatty acids comprise that palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), gamma-Linolenic acid (C18:3), arachidic 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 content of fatty acid in arachidonic acid oil in table 1 embodiment 2
| Commercially available bacterial classification (wt%) | Bacterial classification for the present invention (wt%) | |
| Palmitic acid (C16:0) | 7.99 | 9.19 |
| Stearic acid (C18:0) | 6.52 | 10.2 |
| Oleic acid (C18:1) | 9.52 | 9.21 |
| Linoleic 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 |
| Arachidic 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 arachidic 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 kryoscopy: the present invention is 9 DEG C by the prepared arachidonic acid oil freezing point of Mortierella alpina mutant strain bacterial classification, the freezing point (18 DEG C) of its arachidonic acid oil prepared far below commercially available bacterial classification.
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: CCTCCM2013419) and be inoculated on potato dextrose agar (PDA) culture medium flat plate, cultivate 8 days to spore maturation for 25-28 DEG C, 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 culture medium, inoculum concentration 15% (volume ratio), be placed in 28 DEG C, cultivate 48 hours on the shaking table of 220 revs/min, described culture medium is: carbon source sucrose 35g/l; Nitrogenous source yeast soaks powder 12g/l; PH7.
C) seed expands cultivation: final fermentation tank culture adopts the volume of 50L, and therefore seeding tank selects 10L seed to expand fermentation tank. The shake-flask seed zymotic fluid of step (2) is inoculated into and in seeding tank, carries out seed and expand and cultivate, seed culture medium carbon source sucrose 35g/l wherein; Nitrogenous source yeast soaks powder 12g/l, controls pH7, and fermentation temperature is 28 DEG C, 220 revs/min of mixing speeds, and throughput 1vvm (L/L.min), tank pressure 0.1Mpa, cultivates 42h.
D) fermented and cultured: in seeding tank, bacterium is dense reach 20% after, be linked in the 50L fermentation tank that 30L fermentation medium is housed and cultivate by culture transferring pipeline, inoculum concentration 15% (volume ratio), 28 DEG C of fermentation tank control temperature, 220 revs/min of mixing speeds, throughput 1vvm (L/L.min), tank pressure 0.1Mpa, cultivates 170h. In sweat, add carbon source by stream and come that in controlled fermentation liquid, carbon source concentration is at 10g/L, the fermentation medium in described fermentation tank is: carbon source sucrose 35g/l; Nitrogenous source yeast soaks powder 12g/l; PH7.
E) post processing: the separation of fermentative broth that fermented and cultured is obtained, obtain wet thallus, dry and obtain dry mycelium 40g. In dry mycelium, add extractant n-hexane to extract, after extraction, separating the solid formation that obtains proceeds to and in extraction container, carries out re-extract, so, until finish extraction process without when oil in extract, while extraction for the first time, add 200 ml n-hexanes, add afterwards 150 ml n-hexanes at every turn, will extract and fill the miscella that rear isolated by filtration obtains at every turn, precipitation, obtains microorganism grease.
F) this microbial grease is carried out to gas chromatographic analysis, carry out setting-point test, the main unrighted acid in this microbial grease and the content of chain saturated fatty acids comprise palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), gamma-Linolenic acid (C18:3), arachidic 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 content of fatty acid 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 arachidic 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 kryoscopy: the arachidonic acid oil freezing point that the present invention produces is 6 DEG C. the freezing point (15 DEG C) of its arachidonic acid oil prepared far below commercially available bacterial classification.
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 (deposit number: CCTCCM2013419) and be inoculated on potato dextrose agar (PDA) culture medium flat plate, cultivate 8 days to spore maturation, 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 culture medium, inoculum concentration 20% (volume ratio), be placed in 30 DEG C, cultivate 50 hours on the shaking table of 300 revs/min, described culture medium is: carbon source sucrose 50g/l; Nitrogenous source yeast soaks powder 12g/l; PH8.5.
C) seed expands cultivation: the volume of final fermentation tank is 200m3, selecting successively volume is 10L, 50L, 5m3，50m3Seeding tank expand cultivate seed liquor, in seeding tank, culture medium loading amount is 60% (volume ratio), incubation technology controlling and process is: 30 DEG C of temperature, 300 revs/min of mixing speeds, throughput 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, expands cultivation step by step by the shaking flask nutrient solution of above-mentioned steps.
D) fermented and cultured: treat 50m3Dense the reaching after 30% (volume ratio) of bacterium in seeding tank, is linked into 130m is housed by culture transferring pipeline3The 200m of fermentation medium3In fermentation tank, cultivate, inoculum concentration 20% (volume ratio), 30 DEG C of fermentation tank control temperature, 300 revs/min of mixing speeds, throughput 1.5vvm (L/L.min), tank pressure 0.15Mpa, cultivates 180h. In sweat, add carbon source by stream 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) post processing: the separation of fermentative broth that fermented and cultured is obtained, obtain wet thallus, dry and obtain dry mycelium 50g. In dry mycelium, add extractant n-hexane to extract, after extraction, separating the solid formation that obtains proceeds to and in extraction container, carries out re-extract, so, until finish extraction process without when oil in extract, while extraction for the first time, add 200 ml n-hexanes, add afterwards 150 ml n-hexanes at every turn, will extract and fill the miscella that rear isolated by filtration obtains at every turn, precipitation, obtains microorganism grease.
F) this microbial grease is carried out to gas chromatographic analysis, carry out setting-point test, the main unrighted acid in this microbial grease and the content of chain saturated fatty acids comprise palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), gamma-Linolenic acid (C18:3), arachidic 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 content of fatty acid in arachidonic acid oil in table 3 embodiment 4
| Contrast bacterial classification (wt%) | The present invention's bacterial classification used (wt%) | |
| Palmitic acid (C16:0) | 7.99 | 9.78 |
| Stearic acid (C18:0) | 7.31 | 10.52 |
| Oleic acid (C18:1) | 6.77 | 9.32 |
| Linoleic 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 |
| Arachidic 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 arachidic 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 kryoscopy: the arachidonic acid oil freezing point that the present invention produces is 4 DEG C, the freezing point (15 DEG C) of its arachidonic acid oil prepared far below commercially available bacterial classification.
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 freezing point that makes thus arachidonic acid oil is lower (minimum be low to moderate 4 DEG C) also, even if it also can not separate out saturated fatty acid by solidification and crystallization at a lower temperature, can keep limpid transparent, there is higher quality.
Claims (7)
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, post processing 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 CCTCCNO: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: Mortierella alpina mutant strain claimed in claim 1 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 shaking flask culture medium and cultivates acquisition seed culture fluid;
(3) fermented and cultured: treat that the seed culture fluid that above-mentioned steps (2) jolting is cultivated is inoculated into fermented and cultured in the fermentation flask that fermentation medium is housed;
Or according to the size of final fermentation tank, the seed culture fluid that above-mentioned steps (2) jolting is cultivated expands cultivation step by step, obtains seed scale-up medium, be then inoculated into and in fermentation tank, carry out fermented and cultured;
(4) zymotic fluid is obtained to arachidonic acid oil through fermentation post processing.
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 DEG C, cultivate and generate also ripe to spore in 6-9 days, take off mycelia and spore on culture medium, be mixed with spore suspension with sterilized water.
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 concentration is 10-20%(volume ratio), cultivation temperature 25-30 DEG C, 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: when the dense 15% ~ 30%(volumetric concentration that reaches of seed culture fluid bacterium in step (2)) time, according to 10% ~ 20%(volume ratio) inoculum concentration be linked in fermentation flask jolting and cultivate, the temperature that described jolting is cultivated is 25-30 DEG C, incubation time is 8-12 days, shaking table speed is 120-300 rev/min, and described fermentation medium is: carbon source 50-80g/l; Nitrogenous source is 10-30g/l; PH5.5-8.5;
Or according to the size of final fermentation tank, the seed culture fluid that above-mentioned steps (2) jolting is cultivated expands cultivation step by step, the described seed culture medium of using in incubation that expands 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 carries out next step and expands and cultivate; Every grade of incubation time is 24-50h, and cultivation temperature is 25-30 DEG C, and throughput is 1-2vvm(L/L.min), tank pressure 0.05-0.15MPa, mixing speed is turn/min of 150-300; Then the seed scale-up medium of above-mentioned acquisition is inoculated into and in final fermentation tank, carries out fermented and cultured, inoculum concentration 10-20%(volume ratio), fermentation jar temperature 25-30 DEG C, mixing speed 200-300 rev/min, throughput 1-2vvm(L/L.min), tank pressure 0.05-0.15Mpa, cultivates 150-180h, and add carbon source by stream during the fermentation and come that in controlled fermentation liquid, carbon source concentration is at 5-20g/L, the fermentation medium in described fermentation tank is: carbon source 30-50g/l; Nitrogenous source 10-20g/l; PH5.5-8.5.
6. produce the method for arachidonic acid oil according to the Mortierella alpina mutant strain that utilizes described in claim 4 or 5, it is characterized in that: described carbon source is selected from glucose, sucrose or starch; Nitrogenous source is selected from dusty yeast, 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 post processing of described step (4) is that the wet thallus after separation of fermentative broth is dried and obtained dry mycelium, then adds extractant to extract.
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