CN101824363B - Method for extracting docosahexaenoic acid grease - Google Patents
Method for extracting docosahexaenoic acid grease Download PDFInfo
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- CN101824363B CN101824363B CN2010101822221A CN201010182222A CN101824363B CN 101824363 B CN101824363 B CN 101824363B CN 2010101822221 A CN2010101822221 A CN 2010101822221A CN 201010182222 A CN201010182222 A CN 201010182222A CN 101824363 B CN101824363 B CN 101824363B
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- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound 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 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 52
- 235000020669 docosahexaenoic acid Nutrition 0.000 title claims abstract description 45
- 239000004519 grease Substances 0.000 title claims abstract description 24
- 229940090949 docosahexaenoic acid Drugs 0.000 title claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 74
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 51
- 238000000605 extraction Methods 0.000 claims abstract description 40
- 239000003960 organic solvent Substances 0.000 claims abstract description 30
- 238000000855 fermentation Methods 0.000 claims abstract description 10
- 230000004151 fermentation Effects 0.000 claims abstract description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 34
- 239000007788 liquid Substances 0.000 claims description 33
- 238000003756 stirring Methods 0.000 claims description 27
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 16
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- 239000003921 oil Substances 0.000 claims description 14
- 239000008346 aqueous phase Substances 0.000 claims description 13
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 11
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 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 claims description 6
- 230000002255 enzymatic effect Effects 0.000 claims description 5
- 241000003595 Aurantiochytrium limacinum Species 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- 108010059892 Cellulase Proteins 0.000 claims description 3
- 108010022172 Chitinases Proteins 0.000 claims description 3
- 102000012286 Chitinases Human genes 0.000 claims description 3
- 241000199912 Crypthecodinium cohnii Species 0.000 claims description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 3
- 241000199914 Dinophyceae Species 0.000 claims description 3
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 claims description 3
- 102000035195 Peptidases Human genes 0.000 claims description 3
- 108091005804 Peptidases Proteins 0.000 claims description 3
- 101710173866 Seminase Proteins 0.000 claims description 3
- 241001467333 Thraustochytriaceae Species 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- 108010031616 deoxyribonuclease gamma Proteins 0.000 claims description 3
- 229940059442 hemicellulase Drugs 0.000 claims description 3
- 108010002430 hemicellulase Proteins 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 2
- 230000008020 evaporation Effects 0.000 claims description 2
- 238000005265 energy consumption Methods 0.000 abstract description 4
- 210000002421 cell wall Anatomy 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 241001052560 Thallis Species 0.000 abstract 1
- 235000014113 dietary fatty acids Nutrition 0.000 abstract 1
- 229930195729 fatty acid Natural products 0.000 abstract 1
- 239000000194 fatty acid Substances 0.000 abstract 1
- 150000004665 fatty acids Chemical class 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 17
- 239000011259 mixed solution Substances 0.000 description 15
- 239000000284 extract Substances 0.000 description 14
- 235000019198 oils Nutrition 0.000 description 13
- 239000000463 material Substances 0.000 description 11
- 239000010779 crude oil Substances 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 150000002632 lipids Chemical class 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 238000002390 rotary evaporation Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 235000008452 baby food Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 235000021323 fish oil Nutrition 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241000598397 Schizochytrium sp. Species 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- 235000020978 long-chain polyunsaturated fatty acids Nutrition 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Fats And Perfumes (AREA)
Abstract
The invention discloses a method for extracting docosahexaenoic acid grease. After the method enzymatically breaks the cell walls of fermentation broth containing docosahexaenoic acid, organic solvent is first adopted to carry out first-stage water separation, thalli are enriched, the organic solvent is used again for carrying out second-stage extraction, and thereby crude grease is obtained. The method has the advantages of simple operation, no equipment investment and low energy consumption, the usage of ethanol and the organic solvent is greatly reduced, moreover, the yield of the grease can reach 47g/L to 53g/L, the DHA accounts for 43 percent to 52 percent of the content of fatty acid, and thereby the application value is high.
Description
Technical field
The invention belongs to the grease technical field, relate to a kind of method of extracting docosahexaenoic acid grease.
Background technology
DHA (Docosahexaenoic acid, 22:6 Δ 4.7.10.13.16.19, full name docosahexenoic acid) is that a kind of important n-3 is long chain polyunsaturated fatty acids (polyunsaturated fatty acid; Be called for short PUFA); Have and increase intelligence promotion brain cell development, effects such as treatment cardiovascular and cerebrovascular diseases; Be described as the functional health factor of new generation, be widely used in infant or baby food additive and pharmaceutical industries.
Traditional DHA source is mainly fish oil, but owing to the restriction of factors such as the kind that receives fish, season, geographical position makes that instabilities such as DHA lubricant component, quality that fish oil is produced are not high yet.Therefore, microbe fermentation method prepares DHA technology and receives extensive concern.The realization that domestic and international many enterprises are all successful microbial fermentation produce the greasy industrial production of DHA, but production technique tradition relatively mostly.
Owing to have a large amount of water in the fermented liquid, cause a large amount of ethanol and the organic solvents of consumption in the extraction process, therefore extract a very important step in the process that preceding branch water is the crude oil extraction.Dividing method for water commonly used in the industry is for adding the flocculation agent agent, like alum, Tai-Ace S 150, anhydrous CaCl
2, iron trichloride, polymer alumina, bodied ferric sulfate or SEPIGEL 305 etc., then through leaving standstill, filtration, centrifugal or oven dry supervisor.But these methods or time that need be long (generally need more than the placement 12h; Reduced production efficiency) or (whizzer etc. belong to consumptive material to expend bigger energy; The far away commentaries on classics consumed energy; The input of equipment and maintenance also need cost, and oven dry then needs bigger energy consumption), economical inadequately for industrial production.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of extracting docosahexaenoic acid grease.This method is based on enzymatic shell-broken and extracts the greasy method of DHA, can reduce the energy consumption of production process greatly and practice thrift cost.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
A kind of method of extracting docosahexaenoic acid grease, the fermented liquid that will contain docosahexenoic acid are carried out one-level earlier and are divided water behind enzymatic shell-broken, and thalline is carried out enrichment, carry out the secondary extraction again, obtain thick oil after extraction solvent is removed in evaporation;
Wherein,
Described one-level divides water to do, the fermented liquid behind the broken wall, ethanol and organic solvent 1: 0.5 by volume～1.5: 0.1～0.8 mixed, leaves standstill, and abandons lower floor's water; Through leaving standstill, the gained mixed solution is understood fast hierarchical, and lower floor is a water; The upper strata is organic phase and thalline; If the time of repose lengthening, solution will be divided into three layers, is respectively organic solvent layer, thalline layer and water layer from top to bottom; As long as remove lower floor's water, keep mixture or the upper strata organic solvent layer and the middle level thalline layer on upper strata;
Described secondary extraction is that the upper oil phase (being the part of aqueous phase discarded), ethanol and the organic solvent 1: 0.6 by volume～1.2: 0.6～1.2 that divide water to obtain one-level mix, leave standstill, and collect upper organic phase; Through leaving standstill, mixed solution is understood layering, and the upper strata is an organic phase, and the middle layer is a thalline, and lower floor is the ethanol phase that comprises the small amount of residual fermented liquid;
It is any one or a few the mixture of arbitrary proportion in hexanaphthene, normal hexane, ETHYLE ACETATE, acetone, sherwood oil, ether, toluene, chloroform, No. four solvents and No. six solvents that one-level is divided the organic solvent described in the extraction of water and secondary.Preferred normal hexane, acetone or sherwood oil.It is identical that one-level divides water and secondary to extract employed organic solvent.
Wherein, The fermented liquid that contain docosahexenoic acid of described fermented liquid for obtaining by fermentation by thraustochytriale, schizochytrium limacinum, Crypthecodinium cohnii or dino flagellate; The inventive method does not have any restriction to the bacterial classification of fermentation use and the fermentation process of employing; The bacterial strain of fermentative prodn DHA can use so long as have the ability, and can use so long as can fermentative prodn go out the method for DHA.Certainly, through optimal conditions, make and be rich in DHA in the fermented liquid that the fermented liquid of this DHA of being rich in is more suitable for carrying out with the inventive method the extraction of docosahexaenoic acid grease.
Wherein, described enzymatic shell-broken carries out broken wall for using proteolytic enzyme, N,O-Diacetylmuramidase, LSD, cellulase, chitinase, seminase or hemicellulase etc.The inventive method does not have any restriction to the enzyme of broken wall; Can use so long as can the strain cell wall be carried out broken wall breaking enzyme; The inventive method also has no restriction to the method for broken wall, can use so long as utilize enzyme that the strain cell wall is carried out broken method.
Wherein, described one-level is divided in the water, and the fermented liquid behind the broken wall, ethanol and organic solvent volume ratio be preferably 1: 0.6～and 1.3: 0.2～0.6.
Wherein, described one-level is divided in the water, follows stirring in the fermented liquid behind the broken wall, ethanol and the organic solvent mixing process, and churning time is 5min～3h.The purpose of stirring is in order to promote mixing of material, realize the part extraction function of organic solvent that the inventive method does not have any restriction to mixing the speed that stirs, and evenly is to get final product prerequisite stirring for some time with mixing of materials.
Wherein, described one-level is divided water, and time of repose is more than 5 minutes, and the present invention does not have any restriction to time of repose, and to realize that layering is as the criterion, the consideration working hour that can be suitable arranges.
Wherein, in the extraction of described secondary, upper oil phase (being the part of aqueous phase discarded), ethanol and the volume of organic solvent ratio that one-level divides water to obtain be preferably 1: 0.8～and 1.1: 0.8～1.1.
Wherein, described secondary extraction is followed stirring in upper oil phase, ethanol and the organic solvent mixing process that one-level divides water to obtain, and churning time is 15min～3h.The purpose that stirs is to extract purpose greatly in order to promote mixing of material, organic solvent fully to be contacted with DHA, reaching, and the inventive method does not have any restriction to mixing the speed that stirs, and evenly is to get final product prerequisite stirring for some time with mixing of materials.
Wherein, described secondary extraction, time of repose is more than 5 minutes, and the present invention does not have any restriction to time of repose, and to realize that layering is as the criterion, the consideration working hour that can be suitable arranges.
Wherein, described secondary extraction can improve yield like this for repeatedly extraction, and working method is: upper oil phase, ethanol and the organic solvent 1: 0.6 by volume～1.2: 0.6～1.2 that divides water to obtain one-level mixes, leaves standstill, and collects upper organic phase; Lower floor adds organic solvent and continues to mix, leave standstill, and repeats 1～10 time, merges upper organic phase; The secondary extraction is adopted when repeatedly extracting, and only adds ethanol in the first time in the extraction process, and extraction is afterwards only added organic solvent and need not to add ethanol; The type of organic solvent is with extraction phase is same for the first time, and the add-on of organic solvent has no restriction, can how can lack; Purpose is the DHA that extracts for maximum possible, improves yield.
Beneficial effect: the branch water that do not carry out of method of the present invention and routine directly adds ethanol and carries out method of extraction with organic solvent and compare, and the ethanol of this method and normal hexane consumption can reduce to 66% and 24%; And compare with the working method of industry water extraction in last original minute, this method adopts organic solvent to carry out branch water, has promptly realized dividing the purpose of water, can play the part effect of extracting again; Improved yield, this method is simple to operate, and is consuming time few; Need not any equipment input, energy consumption is low, and has improved extraction intensity and efficient; The grease productive rate can reach 47～53g/L, and the content that DHA accounts for lipid acid reaches 43～52%, has good using value.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
The fermented liquid of following examples utilizes schizochytrium limacinum (Schizochytrium sp.) (CCTCC No:M 209059), gets according to the method preparation of patent 200910033869.5.
Certainly the inventive method is not limited to above-mentioned bacterial strains and above-mentioned fermentation process, and the fermented liquid that contains docosahexenoic acid that thraustochytriale, schizochytrium limacinum, Crypthecodinium cohnii or dino flagellate obtain through any fermentation process may be used to the separation and Extraction of following examples.
Above-mentioned fermented liquid adopts following method broken wall:
The Sumizyme MP that in fermented liquid, adds 1～20g/L, 50～70 ℃ of following insulated and stirred 1～5h.
Certainly the inventive method is not limited to above-mentioned wall breaking enzyme and wall-breaking method; Any enzyme (like proteolytic enzyme, N,O-Diacetylmuramidase, LSD, cellulase, chitinase, seminase, hemicellulase etc.) is as long as can realize the broken wall of fermentation strain; Realize the release of DHA, may be used to the separation and Extraction of following examples.
Embodiment 1:
Get the fermented liquid 200mL behind the broken wall, the fermented liquid behind the broken wall, ethanol and normal hexane are mixed according to volume ratio at 1: 0.6: 0.2, leave standstill 10min after stirring 10min, carry out branch water, thalline is carried out enrichment, gained mixed solution fast hierarchical is abandoned lower floor's water.
Divide upper strata mixed solution (being the part of aqueous phase discarded), ethanol and normal hexane behind the water to mix in 1: 1: 1 by volume one-level, leave standstill 10min after stirring 15min, extract; Collect upper strata normal hexane phase; Continuation is added and the normal hexane mixing that extracts equal volume for the first time in the material of middle level and lower floor, leaves standstill 10min behind the stirring 15min, repeats lixiviate three times; Merge upper strata normal hexane phase; Rotary evaporation gets DHA crude oil 10.3g after removing normal hexane, and the grease productive rate is 51.5g/L, and the content that DHA accounts for lipid acid reaches 49.7%.The ethanol of this method and normal hexane consumption are respectively 155mL and 145mL.
Comparative Examples 1:
Do not carry out branch water, get the fermented liquid 200mL behind the broken wall, directly the fermented liquid behind the broken wall, ethanol and normal hexane were mixed in 1: 1: 1 by volume; Leave standstill 10min after stirring 15min, extract, get supernatant; The normal hexane that adds same amount repeats lixiviate three times, merges upper strata normal hexane phase, and rotary evaporation gets DHA crude oil 9.7g after removing normal hexane; The grease productive rate is 48.5g/L, and the content that DHA accounts for lipid acid reaches 47.2%, and the ethanol of this method and normal hexane consumption are respectively 200mL and 600mL.
Embodiment 2:
Get the fermented liquid 200mL behind the broken wall, the fermented liquid behind the broken wall, ethanol and chloroform are mixed according to volume ratio at 1: 0.7: 0.4, leave standstill 10min after stirring 10min, carry out branch water, thalline is carried out enrichment, gained mixed solution fast hierarchical is abandoned lower floor's water.
Divide upper strata mixed solution (being the part of aqueous phase discarded), ethanol and chloroform behind the water to mix in 1: 0.8: 1 by volume one-level, leave standstill 10min after stirring 15min, extract; Collect upper strata chloroform phase; Continuation is added and the chloroform mixing that extracts equal volume for the first time in the material of middle level and lower floor, leaves standstill 10min behind the stirring 15min, repeats lixiviate three times; Merge upper strata chloroform phase; Rotary evaporation gets DHA crude oil 9.9g after removing and desolvating, and the grease productive rate is 49.5g/L, and the content that DHA accounts for lipid acid is 50.3%.
Embodiment 3:
Get the fermented liquid 200mL behind the broken wall, the fermented liquid behind the broken wall, ethanol and ether are mixed according to volume ratio at 1: 1.3: 0.6, leave standstill 10min after stirring 10min, carry out branch water, thalline is carried out enrichment, gained mixed solution fast hierarchical is abandoned lower floor's water.
Divide upper strata mixed solution (being the part of aqueous phase discarded), ethanol and ether behind the water to mix in 1: 0.8: 1.1 by volume one-level, leave standstill 10min after stirring 15min, extract; Collect upper strata ether phase; Continuation is added and the ether mixing that extracts equal volume for the first time in the material of middle level and lower floor, leaves standstill 10min behind the stirring 15min, repeats lixiviate once; Merge upper strata ether phase; Rotary evaporation gets DHA crude oil 9.8g after removing ether, and the grease productive rate is 49g/L, and the content that DHA accounts for lipid acid reaches 44.8%.
Embodiment 4:
Get the fermented liquid 5.3m behind the broken wall
3, the fermented liquid behind the broken wall, ethanol and normal hexane are mixed according to volume ratio at 1: 0.6: 0.3, leave standstill 1h behind the stirring 1h, carry out branch water, thalline is carried out enrichment, gained mixed solution fast hierarchical,, abandon lower floor's water.
Divide upper strata mixed solution (being the part of aqueous phase discarded), ethanol and normal hexane behind the water to mix in 1: 1: 0.8 by volume one-level, leave standstill 1h after stirring 2h, extract; Collect upper strata normal hexane phase, the continuation interpolation mixes with the normal hexane that extracts for the first time equal volume in the material of middle level and lower floor, leaves standstill 1h behind the stirring 2h; Repeat lixiviate three times, merge the upper strata normal hexane, boil off behind the normal hexane DHA crude oil 280kg; The grease productive rate is 52.8g/L, and the content that DHA accounts for lipid acid reaches 48.6%.
Embodiment 5:
Get the fermented liquid 4.9m behind the broken wall
3, the fermented liquid behind the broken wall, ethanol and sherwood oil were mixed in 1: 0.7: 0.2 by volume, leave standstill 1h behind the stirring 30min, carry out branch water, thalline is carried out enrichment, gained mixed solution fast hierarchical is abandoned lower floor's water.
Divide upper strata mixed solution (being the part of aqueous phase discarded), ethanol and sherwood oil behind the water to mix in 1: 1.1: 0.9 by volume one-level, leave standstill 1h after stirring 1.5h, extract; Collect upper strata sherwood oil phase; Continuation is added and the sherwood oil mixing that extracts equal volume for the first time in the material of middle level and lower floor, leaves standstill 1h behind the stirring 1.5h, repeats lixiviate three times; Merge upper strata sherwood oil phase; Get DHA crude oil 256kg after boiling off solvent, extraction yield is 52.2g/L, and the content that DHA accounts for lipid acid reaches 50.5%.
Embodiment 6:
Method with embodiment 1 is identical, and different is that the fermented liquid behind the broken wall, ethanol and normal hexane are mixed according to volume ratio at 1: 0.5: 0.1.
Embodiment 7
Method with embodiment 1 is identical, and different is that the fermented liquid behind the broken wall, ethanol and normal hexane are mixed according to volume ratio at 1: 1.5: 0.8.
Embodiment 8:
Method with embodiment 1 is identical, and different is divides upper strata mixed solution (being the part of aqueous phase discarded), ethanol and normal hexane behind the water to mix in 1: 1.2: 0.6 by volume one-level.
Embodiment 9:
Method with embodiment 1 is identical, and different is divides upper strata mixed solution (being the part of aqueous phase discarded), ethanol and normal hexane behind the water to mix in 1: 0.6: 1.2 by volume one-level.
Embodiment 10:
Method with embodiment 1 is identical, and different is that normal hexane is replaced with hexanaphthene.
Embodiment 11:
Method with embodiment 1 is identical, and different is with mixed solvent (volume ratio 1: the 2) replacement of normal hexane with sherwood oil and acetone.
Embodiment 12:
Method with embodiment 1 is identical, and different is with No. four solvent replacing with normal hexane.
Embodiment 13:
Method with embodiment 1 is identical, and different is with No. six solvent replacing with normal hexane.
Embodiment 14:
Method with embodiment 1 is identical, and different is with mixed solvent (volume ratio 1: the 1) replacement of normal hexane with ETHYLE ACETATE and toluene.
Embodiment 15:
One-level divides water identical with the method for embodiment 1; Different is secondary extraction only carrying out single extraction; The upper strata mixed solution (being the part of aqueous phase discarded), ethanol and the normal hexane that are about to after one-level is divided water mixed in 1: 1: 1 by volume, left standstill 10min behind the stirring 15min, only this time extracted; Collect upper strata normal hexane phase, rotary evaporation gets the DHA crude oil after removing normal hexane.
Claims (8)
1. a method of extracting docosahexaenoic acid grease is characterized in that the fermented liquid that contains docosahexenoic acid behind enzymatic shell-broken, carries out one-level earlier and divides water, and thalline is carried out enrichment, carries out the secondary extraction again, obtains thick oil after extraction solvent is removed in evaporation;
Wherein,
Described one-level divides water to do, with 1: 0.5 by volume～1.5: 0.1～0.8 mixing of the fermented liquid behind the broken wall, ethanol and organic solvent, standing demix, abandons lower floor's water;
The extraction of described secondary does, one-level divided part, ethanol and the organic solvent 1: 0.6 by volume～1.2: 0.6～1.2 of the aqueous phase discarded that water obtains mixes, standing demix, collects upper organic phase;
It is any one or a few the mixture of arbitrary proportion in hexanaphthene, normal hexane, ETHYLE ACETATE, acetone, sherwood oil, ether, toluene, chloroform, No. four solvents and No. six solvents that one-level is divided the organic solvent described in the extraction of water and secondary.
2. the method for extraction docosahexaenoic acid grease according to claim 1 is characterized in that the fermented liquid that contain docosahexenoic acid of described fermented liquid for being obtained by fermentation by thraustochytriale, schizochytrium limacinum, Crypthecodinium cohnii or dino flagellate.
3. the method for extraction docosahexaenoic acid grease according to claim 1 is characterized in that described enzymatic shell-broken carries out broken wall for using proteolytic enzyme, N,O-Diacetylmuramidase, LSD, cellulase, chitinase, seminase or hemicellulase.
4. the method for extraction docosahexaenoic acid grease according to claim 1 is characterized in that described one-level divides water, follows stirring in the fermented liquid behind the broken wall, ethanol and the organic solvent mixing process, and churning time is 5min～3h.
5. the method for extraction docosahexaenoic acid grease according to claim 1 is characterized in that described one-level divides water, and time of repose is more than 5 minutes.
6. the method for extraction docosahexaenoic acid grease according to claim 1 is characterized in that the extraction of described secondary, and one-level is divided in part, ethanol and the organic solvent mixing process of the aqueous phase discarded that water obtains and followed stirring, and churning time is 15min～3h.
7. the method for extraction docosahexaenoic acid grease according to claim 1 is characterized in that described secondary extraction, and time of repose is more than 5 minutes.
8. the method for extraction docosahexaenoic acid grease according to claim 1; It is characterized in that described secondary extraction or be repeatedly extraction; One-level divided part, ethanol and the organic solvent 1: 0.6 by volume～1.2: 0.6～1.2 of the aqueous phase discarded that water obtains mixes, standing demix, collect upper organic phase; Remainder adds organic solvent and continues mixing, standing demix, collects upper organic phase, repeats 1～10 time, merges upper organic phase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101822221A CN101824363B (en) | 2010-05-25 | 2010-05-25 | Method for extracting docosahexaenoic acid grease |
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| CN102276444B (en) * | 2011-05-04 | 2013-10-09 | 厦门大学 | Fatty acid compounds and preparation method and application thereof |
| FR2975705B1 (en) * | 2011-05-27 | 2014-12-26 | Roquette Freres | PROCESS FOR EXTRACTING SQUALENE FROM MICROALGUES |
| CN102492544B (en) * | 2011-12-07 | 2013-09-25 | 湖北福星生物科技有限公司 | Method for preparing microalgal docosahexaenoic acid (DHA) oil by dry method |
| CN103173273B (en) * | 2011-12-21 | 2015-11-25 | 新奥科技发展有限公司 | Microalgae grease extraction method |
| FR2991337B1 (en) * | 2012-05-29 | 2016-09-02 | Roquette Freres | CONTINUOUS PROCESS FOR ENRICHING DHA WITH ETHYL ESTERS OF AN OIL PRODUCED BY MICROALGUES |
| JP6135144B2 (en) * | 2013-01-22 | 2017-05-31 | 富士電機株式会社 | Oil production method |
| CN104560402A (en) * | 2014-12-31 | 2015-04-29 | 广东东阳光药业有限公司 | Tricholoma matsutake extract oil and preparation method thereof |
| CN109181842B (en) * | 2018-08-20 | 2021-03-26 | 梁云 | Microbial oil and extraction method thereof |
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| CN101168501A (en) * | 2007-07-11 | 2008-04-30 | 南京工业大学 | Technique for extracting and refining DHA enriched fatty acid from Crypthecodinium cohnii |
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