CN104313068B - A kind of phosphatide type DHA fermentation preparation - Google Patents
A kind of phosphatide type DHA fermentation preparation Download PDFInfo
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Abstract
The invention discloses a kind of phosphatide type DHA fermentation preparation, it comprises the following steps：(1) using produce DHA bacterial strain as starting strain, carry out high density fermentation in liquid medium within, obtain the somatic cells rich in glycerine epoxy-type DHA；(2) carbon source, nitrogen source, phosphorus source, amino acid are added in the zymotic fluid after terminating to step (1) high density fermentation, continuing to ferment obtains the somatic cells rich in phosphatide type DHA；(3) somatic cells rich in phosphatide type DHA that collection step (2) is obtained, the grease rich in phosphatide type DHA is obtained through broken, extraction.The present invention finally gives 15 40% polarity grease, and wherein main component is that DHA content reaches 48.16% in phosphatide, polarity grease.The grease that the present invention will be enriched in glycerine ester type DHA is converted into the grease rich in phosphatide type DHA, and effectively increases phospholipid in lipid type DHA content.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of phosphatide type DNA fermentation preparation.
Background technology
Docosahexaenoic acid (DHA) is a kind of important omega-3 long-chain polyunsaturated fatty acids, is brain and view
The constituent of cell membrane in membrane tissue, with enhancing eyesight, promotes brain cell development and preventing hypertension, artery sclerosis, the heart
The physiological functions such as vascular diseases.Due to human body it is difficult to meet demand of the body to DHA by itself synthesis, it is therefore necessary to rely on
Obtained from extraneous food.
At present, DHA main source is fish oil, and is mainly taken in the form of triglycerides by human body.DHA's is another
Existence form is phosphatide type DHA (DHA-containing PL).Phosphatide is the main component of biomembrane, and research shows that it has
Reducing blood lipid, the effect such as antiatherosclerosis.DHA existence form is different, absorption pattern and absorption efficiency in human body
It is different.Glycerine ester type DHA is mainly absorbed in human body in the way of Passive diffusion, and absorptivity is 50% or so.And phosphatide type
DHA is mainly absorbed in human body in the way of active absorption, and absorptivity is close to 100%.Kafrawy et al. research discoveries, with
Other phosphatide type aliphatic acid are compared, and phosphatide type DHA has bigger toxicity to mouse leukemia cell.Study and also found, phosphatide
Type DHA can effectively reduce T-CHOL and content of triglyceride in mice plasma and liver, improve the blood fat of normal mouse
Level.Gisbert et al. researchs find for phosphatide type DHA is compared with glycerine ester type DHA, there is more preferable promotion to the growth of prelarva
Effect.Schaefer et al. researchs find that the increase of phosphatide type DHA levels can effectively reduce the incidence of disease of dementia in blood plasma.
Phosphatide type DHA is existed only in yolk, and content is extremely low.At present, domestic phosphatide type DHA research is concentrated mainly on
The following aspects：(1) phosphatide type DHA application；(2) phosphatide type DHA extraction；(4) phosphatide type DHA physiological action is ground
Study carefully.Wherein, phosphatide type DHA relevant report is not found to prepare using biological fermentation process.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of phosphatide type DHA fermentation preparation to fill up domestic empty
In vain.
To solve above technical problem, the technical solution adopted by the present invention is as follows：
A kind of phosphatide type DHA fermentation preparation, it is characterised in that it comprises the following steps：
(1) using produce DHA bacterial strain as starting strain, carry out high density fermentation in liquid medium within, obtain and be rich in glycerine
Epoxy-type DHA somatic cells；
(2) nutriment is added in the zymotic fluid after terminating to step (1) high density fermentation, continuing to ferment to obtain is rich in phosphorus
Epoxy-type DHA somatic cells；
(3) somatic cells rich in phosphatide type DHA that collection step (2) is obtained, obtain through broken, extraction and are rich in phosphatide type
DHA grease；
Wherein, described nutriment is any one or a few the combination in carbon source, nitrogen source, phosphorus source, amino acid.
Wherein, described production DHA bacterial strain is Schizochytrium, genus thraustochytrium, my Ken Shi kettle Trentepohlias, preferably fragmentation
Chytridium, most preferably CCTCC No：(bacterial strain has been proposed in Application No. 200910033869.5 to M209059 bacterial strains
In Chinese patent).
High density fermentation in above-mentioned steps (1) comprises the following steps：
(1a) accesses the bacterial strain being stored in glycerol tube in the 500ml shaking flasks equipped with 100ml seed culture mediums, 20~
In 30 DEG C of shaking table, with 150~200rpm rotating speed, 24~48h is cultivated, primary seed solution is obtained；
The primary seed solution that (1b) obtains step (1a) is according to the access of 2~10%v/v inoculum concentrations equipped with the training of 100ml seeds
In the 500ml shaking flasks for supporting base, in 20~30 DEG C of shaking table, with 150~200rpm rotating speed, 24~48h is cultivated, two grades are obtained
Seed liquor；
The secondary seed solution that (1c) obtains step (1b) is according to the access of 2~10%v/v inoculum concentrations equipped with the training of 100ml seeds
In the 500ml shaking flasks for supporting base, in 20~30 DEG C of shaking table, with 150~200rpm rotating speed, 24~48h is cultivated, three-level is obtained
Seed liquor；
The three-level seed liquor obtained in step (1c) is equipped with fermented and cultured by (1d) according to the access of 2~10%v/v inoculum concentrations
In the fermentation tank of base, defoamer is then added into fermentation tank, is fermented, strain fermentation production is rich in glycerine ester type DHA's
Grease.
The throughput of fermentation condition is 0.2~1vvm, preferably 0.6vvm in above-mentioned steps (1d).
The mixing speed of fermentation condition is 100~200rpm, preferably 200rpm in above-mentioned steps (1d).
The tank temperature of fermentation condition is 20~30 DEG C, preferably 30 DEG C in above-mentioned steps (1d).
The incubation time of fermentation condition is 90~150h, preferably 120h in above-mentioned steps (1d).
The fermentation process used in above-mentioned steps (1d) is Intermittent fermentation or feed supplement formula fermentation process.
Above-specified high density fermentation in seed culture medium carbon content be 10~30g/L, preferably 15~25g/L, it is optimal
Elect 20g/L as.
Above-specified high density fermentation in fermentation medium carbon content be 20~60g/L, preferably 30~40g/L, most preferably
For 40g/L；Nitrogen content is 2.1~2.5g/L, most preferably preferably 1.8~2g/L, 2g/L；Sulfur content is 2~3g/L, preferably
For 2.5~3g/L, most preferably 3g/L；Phosphorus content is that 0.4~1.5g/L is preferably 1.2~1.5g/L, most preferably 1.5g/L.
In described seed culture medium and fermentation medium, carbon source is glucose, fructose or glycerine, preferably glucose；Nitrogen
Source is yeast extract, corn steep liquor, beef extract, peptone, ammonium nitrate or ammonium sulfate, preferred yeast cream；Phosphorus source be potassium dihydrogen phosphate or
Dipotassium hydrogen phosphate, preferably phosphoric acid potassium dihydrogen；Sulphur source is magnesium sulfate or ammonium sulfate, preferably sulfuric acid ammonium.
Defoamer described in above-mentioned steps (1d) is silicone SE-2, and addition is 0.3g/L.
The fermentation process used in above-mentioned steps (1d) is Intermittent fermentation or feed supplement formula fermentation process.
Wherein, in nutriment,
Described carbon source is any one or a few the mixture in ethanol, sodium acetate；The addition of carbon source be 2~
4g/L, preferably 4g/L.
Described nitrogen source is any one or a few the mixture in yeast extract, ammonium sulfate, sodium glutamate, and nitrogen source adds
Dosage is 5~20g/L, most preferably preferably 15~20g/L, 20g/L.
Described phosphorus source is in potassium dihydrogen phosphate, the addition of phosphorus source is 2~4g/L, preferably 3~4g/L, is most preferably
4g/L。
Described amino acid be proline, lysine, addition be 0.1~0.5g/L, preferably 0.2~0.3g/L, most
Preferably 0.3g/L.
The preferred sodium glutamate of described nutriment, ammonium sulfate, ethanol are individually added, and ammonium sulfate and ethanol two-cocoon feeding add, sulphur
Sour ammonium and proline two-cocoon feeding adds, and most preferably ammonium sulfate is individually added, and ammonium sulfate and proline two-cocoon feeding add.
Wherein, in step (2), the throughput of described continuation fermentation condition is 0.5~1vvm, preferably 0.6vvm.
Wherein, in step (2), the described 100~200rpm of mixing speed, preferably 170rpm that continue fermentation condition.
Wherein, in step (2), described 20~30 DEG C of tank temperature for continuing fermentation condition, preferably 30 DEG C.
Wherein, in step (2), the incubation time of described continuation fermentation condition is 48~72h, preferably 72h.
Step (3) detailed process is：Somatic cells are collected, using wall breaking enzyme smudge cells, then extract with organic solvent 4~
8h, obtains the grease rich in phosphatide type DHA.
Wherein, described organic solvent is any one or a few the mixing in n-hexane, petroleum ether, ethanol and ether
Thing, according to organic solvent and smudge cells liquid volume ratio 1:0.5~2 amount addition.
Wherein, described wall breaking enzyme is protease, and addition is 2~8g/L.
It is obtained by extraction after grease, oil component is separated using column chromatography method, polar lipid and neutral lipid is obtained respectively,
Described column chromatography method is as follows：
Take 100~200 mesh to chromatograph silicon special rubber powder in drying 24h in 120 DEG C of baking ovens, make its water content 5% or so, treat
With.First 30ml petroleum ethers are added in chromatographic column, the bubble removed in core is patted；A certain amount of silica white is taken into beaker, plus
Enter a certain amount of petroleum ether, stirring is poured into chromatographic column to bubble-free, then slowly, makes its uniform settlement, with neutral eluant, eluent (oil
Ether/ether, 9:1, v/v) pillar 2h is rinsed, makes pillar consolidation.2.0g or so greases are weighed, are dissolved with neutral eluant, eluent.By sample
Product are slowly added into chromatographic column along post jamb, first obtain neutral fats with the neutral eluents of 500ml, then with polar elution agent (first
Alcohol) it is eluted to silicagel column and turns white, rotary evaporation obtains each separation component respectively.
Detect the fatty acid profile situation in total grease, neutral lipid and polar lipid respectively using gas-chromatography, it is described
Gas chromatography analysis method adopt with the following method：
Esterification method：0.5g or so greases are taken, 10ml is settled to n-hexane；1ml to 10ml appearances are pipetted with pipette
Measuring bottle, adds 3ml 0.5mol/L KOH- methanol solutions, in 65 DEG C of 15~20min of water-bath, cooling；Add 2ml BF3- second
Ether-methanol solution (BF3- ether:Methanol=3:7, v/v), 65 DEG C of water-bath, 5~10min, cooling；Again plus saturation NaCl solution,
Each 2ml of n-hexane, stands after vibration, is layered, take upper strata into another volumetric flask, with being settled to after n-hexane extraction 2-3 times
10ml, takes 100 μ l extracts and adds the mixing of 100 μ l Nonadecanoic acid methylesters (known to concentration) solution, can be used as DHA analyses.
Gas phase analysis condition：Chromatographic column：DB-23(60m*0.25mm*0.25μm)；Detector：FID；Carrier gas：Nitrogen；Point
Stream ratio：30/1；Injector temperature：250℃；Detector temperature：280℃；Sample size：1μl；Heating schedule：Initial column temperature is 100
DEG C, 196 DEG C first are risen to 25 DEG C/min speed, then 220 DEG C are risen to 2 DEG C/min speed, keep 12min.Column flow rate：
3.0ml/min；Tail wind drift speed：30ml/min；Hydrogen flow rate：40ml/min；Air velocity：400ml/min.
Beneficial effect：The invention provides a kind of phosphatide type DHA preparation method, the base for the production DHA that fermented in marine fungi
On plinth, the method fermented using dual-stage prepares the grease rich in phosphatide type DHA.In obtained grease, polar lipid accounts for total grease
Percentage composition is that DHA content can reach 48.16% in 15%-40%, polar lipid, and wherein most is phosphatide type DHA.And
The research for preparing DHA grease currently with microbial fermentation is mainly enriched with the glyceride of intracellular, and the research to phosphatide is seldom, this
The phosphatide preparing process technique that patent is provided is simple, and production cost is relatively low, and marine fungi growth is very fast, it is easier to expanding production
Scale.Therefore, the proposition of this method, is conducive to us further to study phosphatide type DHA physiological action, effectively increases DHA
The application efficiency of product.
Brief description of the drawings
Fig. 1 schizochytrium limacinum DHA Unicell Oils and Fats TLC is analyzed：Fig. 1-A show neutral fats master in schizochytrium limacinum Unicell Oils and Fats
To be made up of triglycerides (TAG), diglyceride (DAG) and monoglyceride (MAG)；By schizochytrium limacinum Unicell Oils and Fats in polarity
Tlc analysis is carried out in developing agent, Fig. 1-B show in polar lipid based on lecithin.
Embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should be also without limitation on sheet described in detail in claims
Invention.
The production DHA used in following examples bacterial strain is that preserving number is CCTCC No：M209059 schizochytrium limacinum
(Schizochytrium sp.)。
Embodiment 1：Prepared by first stage fermentation is rich in glycerine ester type DHA grease.
The DHA strains being stored in glycerol tube are accessed in the 500ml shaking flasks equipped with 100ml seed culture mediums, in shaking table
The upper activation for carrying out seed, 30 DEG C of temperature, rotating speed 170rpm cultivates 24h, obtains the higher seed liquor of vigor.According to same
After method activation three generations, seed liquor is accessed in the fermentation tank equipped with fermentation medium, inoculum concentration 10% (v/v), throughput
0.6vvm, speed of agitator 200rpm, 30 DEG C of tank temperature add defoamer silicone SE-2, addition is 0.3g/L, fermentation training
120h is supported, obtains being rich in glyceride DHA grease.Fermentation results are shown in Table 1.
1 first stage of table fermentation results
Embodiment 2：Metabolite analysis in fermentation process.
Component separation is carried out to the grease of different fermentations time in first stage fermentation process using column chromatography method, investigated
Different oil components change with time situation.Analysis result is shown in Table 2.From Table 2, it can be seen that prolonging with fermentation time
Long, neutral fats content is constantly raised in somatic cells, and polar lipid content is gradually reduced.Therefore can be by analyzing cell growth
The situation of change of stage, earlier fermentation and the various metabolite contents of later stage intracellular that ferment, so that it is determined that needed for the accumulation of polar lipid
The nutrient wanted.
Oil component changes in 2 first stage of table fermentation process
Analysis detection and analysis is carried out to the metabolin in each stage in fermentation process using GC-MS, metabolites kinds are found
Mainly include machine acid, amino acid, carbohydrate and phosphate compound.Each stage metabolite content change of fermenting is shown in Table 3.
Each stage intermediate metabolites changes of contents situation of the fermentation of table 3
As shown in Table 3, the content of most of metabolin changes with the change of fermentation time during the fermentation.By table 2
Understand, 0~48h polar lipid contents are higher, and polar lipid content declines after 48h, until fermentation ends.0~48h cells are in
Growth phase and earlier fermentation, combining with fermentation each stage intracellular metabolite content mutation analysis discovery, phosphoric acid, lysine, dried meat
Propylhomoserin is higher in this stage content, and the fermentation later stage constantly declined in polar lipid content, and phosphoric acid, lysine, proline exist
The content of intracellular has all declined.Therefore, the material such as phosphoric acid, lysine, proline probably has close with the accumulation of early stage phosphatide
Cut relation.Therefore, it is the synthesis of promotion phosphatide, such material is used as to second stage fermentation addition nutriment.
Embodiment 3：The addition fermentation of second stage list nutriment is investigated.
After first stage fermentation ends, the metabolite profile according to obtained by metabolism group adds Different Nutrition thing into zymotic fluid
Matter, addition nitrogen source provides amino acid, and potassium dihydrogen phosphate provides phosphoric acid.Nutriment and addition be respectively 2g/L sodium acetates,
40ml/L ethanol, 4g/L yeast extracts, 20g/L sodium glutamates, 3.6g/L ammonium sulfate, 4g/L potassium dihydrogen phosphates.Second stage is fermented
Condition it is as follows：Throughput is 0.6vvm, and mixing speed is 170rpm, and tank temperature is 30 DEG C, and incubation time is 72h.Fermentation ends
Afterwards, oil component separation is carried out to the grease of acquisition using column chromatography method, in the zymotic fluid for investigating addition different nutriments
The component of grease is investigated, and investigation the results are shown in Table 4.
Table 4 adds different single nutrient zymotic fluid grease analysis results
From fat content angle, when first stage fermentation ends compared with, after second stage culture, zymotic fluid
In fat content have different degrees of reduction.Wherein, be not added with the control group (33.624g/L) of any nutrient with
(52.847g/L) is compared during first stage fermentation ends, and fat content reduces 36.37%.Sodium glutamate, ethanol, ammonium sulfate
The addition of three kinds of nutrients can preferably alleviate the consumption of grease.
Total grease percentage composition angle analysis is accounted for from different oil components, compared with first stage fermentation ends, by
After two-stage fermentation, polar lipid accounts for the percentage composition of total grease and all changed in zymotic fluid grease.Fermented with the first stage
Terminate (6.88%) to compare, control group (9.21%) grease polar lipid/total grease adds 33.87%.Compared with control group, add
Plus in the grease of different nutrients, polar lipid/total grease also increased.Wherein, 3.6g/L ammonium sulfate additive effect is most
For obvious (14.59%), compared with control group, polar lipid accounts for total fat content and adds 58.41%.
From oil component content angle analysis, polar lipid content is 3.636g/L during with first stage fermentation ends, is passed through
Second stage is fermented, and control group and be with the addition of polar lipid content in the grease of yeast extract and has been declined, and with the addition of sodium glutamate,
Polar lipid content has different degrees of increase in ethanol, sodium acetate, the grease of ammonium sulfate and potassium dihydrogen phosphate.Wherein, sulfuric acid
Ammonium, ethanol additive effect are the most obvious, compared with first stage fermentation ends, and 53.19%, 68.87% is added respectively.Implement
Example 4：The addition fermentation of second stage two nutrient material is investigated.
On the basis of embodiment 3, two nutrient material is added after fermentation ends into zymotic fluid in the first stage.Nutrition
Material and addition are respectively 40ml/L ethanol, 3.6g/L ammonium sulfate, 4g/L potassium dihydrogen phosphates, 0.3g/L proline, 0.2g/L
Lysine.And fat content and oil component are investigated, investigation the results are shown in Table 5.
Table 5 adds two nutrient fermentation of materials liquid grease analysis result
As can be seen from the above table, nutriment combination addition can effectively improve the percentage that polar lipid in grease accounts for total grease
Content and polar lipid content.Add after ammonium sulfate and proline, the percentage composition that polar lipid accounts for total grease can reach 38.54%,
Compared with the control (9.21%), 3.18 times are added, polar lipid content can reach 13.87g/L, compared with the control
(3.10g/L), adds 3.47 times.
Embodiment 5：Second stage fermentation DHA content is investigated.
The grease finally given using column chromatography for separation embodiment 1, embodiment 3 and each experimental group of embodiment 4, utilizes meteorology
Chromatogram is analyzed.The DHA content of different oil components is investigated in zymotic fluid to adding Different Nutrient Elements, investigates knot
Fruit is shown in Table 6.
The DHA content analysis result of different oil components in the addition Different Nutrient Elements after fermentation liquid of table 6
After examples detailed above, second stage fermentation ends, different component in the grease of different nutriments is added
DHA content can all change.Wherein, compared with first stage fermentation ends, the total grease DHA content of control group is added
27.31%, it with the addition of total grease DHA content in the fermentation of yeast extract and add 31.78%, the DHA content of polar lipid reaches
48.42%.The DHA content for adding polar lipid in the zymotic fluid of ammonium sulfate reaches 48.16%, is added compared with control group
8.59%.
Embodiment 6：Unicell Oils and Fats TLC is analyzed.
Smudge cells embodiment 4 and 5 with the addition of the cell obtained after nutriment fermentation as follows, and extract
To grease：Somatic cells are collected, 4h is extracted using wall breaking enzyme smudge cells, then with organic solvent, are obtained rich in phosphatide type DHA
Grease.Organic solvent is n-hexane and ethanol by volume 1:1 mixture, according to organic solvent and smudge cells liquid volume ratio
1:1 amount addition.Wall breaking enzyme is protease, and addition is 3g/L.Thin-layer chromatographic analysis are carried out to schizochytrium limacinum Unicell Oils and Fats,
Analysis result is as shown in Figure 1.
Schizochytrium limacinum Unicell Oils and Fats are subjected to tlc analysis in neutral developing agent, show schizochytrium limacinum Unicell Oils and Fats
Middle neutral fats is mainly made up of (see Fig. 1-A) triglycerides (TAG), diglyceride (DAG) and monoglyceride (MAG)；By fragmentation
Chytrid Unicell Oils and Fats carry out tlc analysis in polarity developing agent, show in polar lipid based on lecithin (see Fig. 1-B).
Claims (7)
1. a kind of phosphatide type DHA fermentation preparation, it is characterised in that it comprises the following steps：
(1) using produce DHA bacterial strain as starting strain, carry out high density fermentation in liquid medium within, obtain and be rich in glycerine epoxy-type
DHA somatic cells；
(2) nutriment is added in the zymotic fluid after terminating to step (1) high density fermentation, continuing to ferment to obtain is rich in phosphatide type
DHA somatic cells；
(3) somatic cells rich in phosphatide type DHA that collection step (2) is obtained, obtain through broken, extraction and are rich in phosphatide type DHA
Grease；
Wherein, described nutriment is carbon source, nitrogen source, phosphorus source, any one or a few combination of amino acid；
Described carbon source is any one or a few the mixture in ethanol, sodium acetate, and the addition of carbon source is 2~4g/L；
Described nitrogen source is any one or a few the mixture in ammonium sulfate, sodium glutamate, the addition of nitrogen source for 5~
20g/L；
Described phosphorus source is potassium dihydrogen phosphate, and the addition of phosphorus source is 2~4g/L；
Described amino acid is proline or lysine, and addition is 0.1~0.5g/L；
Described production DHA bacterial strain is CCTCC No：M209059；
In step (1), described high density fermentation comprises the following steps：
(1a) accesses the bacterial strain being stored in glycerol tube in the 500ml shaking flasks equipped with 100ml seed culture mediums, at 20~30 DEG C
Shaking table in, with 150~200rpm rotating speed, cultivate 24~48h, obtain primary seed solution；
The primary seed solution that (1b) obtains step (1a) is equipped with 100ml seed culture mediums according to the access of 2~10%v/v inoculum concentrations
500ml shaking flasks in, in 20~30 DEG C of shaking table, with 150~200rpm rotating speed, cultivate 24~48h, obtain secondary seed
Liquid；
The secondary seed solution that (1c) obtains step (1b) is equipped with 100ml seed culture mediums according to the access of 2~10%v/v inoculum concentrations
500ml shaking flasks in, in 20~30 DEG C of shaking table, with 150~200rpm rotating speed, cultivate 24~48h, obtain three-level seed
Liquid；
(1d) accesses the three-level seed liquor obtained in step (1c) equipped with fermentation medium according to 2~10%v/v inoculum concentrations
In fermentation tank, defoamer is then added into fermentation tank, throughput be 0.2~1vvm, mixing speed be 100~200rpm,
Tank temperature is 90~150h of culture under conditions of 20~30 DEG C, makes grease of the strain fermentation production rich in glycerine ester type DHA.
2. phosphatide type DHA according to claim 1 fermentation preparation, it is characterised in that described seed culture medium
Carbon content be 10~30g/L, described fermentation medium carbon content is 20~60g/L, and nitrogen content is 2.1~2.5g/L, sulphur
Content is 2~3g/L, and phosphorus content is 0.4~1.5g/L；In above-mentioned culture medium, carbon source is glucose, fructose or glycerine；Nitrogen source is
Yeast extract, corn steep liquor, beef extract, peptone, ammonium nitrate or ammonium sulfate；Phosphorus source is potassium dihydrogen phosphate or dipotassium hydrogen phosphate, sulphur source
For magnesium sulfate or ammonium sulfate.
3. phosphatide type DHA according to claim 1 fermentation preparation, it is characterised in that described in step (1d)
Defoamer is silicone SE-2, and addition is 0.3g/L.
4. phosphatide type DHA according to claim 1 fermentation preparation, it is characterised in that described in step (2)
Continuing fermentation condition is：0.5~1vvm of throughput, 100~200rpm of mixing speed, 20~30 DEG C of tank temperature, continuation culture 48~
72h。
5. phosphatide type DHA according to claim 1 fermentation preparation, it is characterised in that step (3) detailed process
For：Somatic cells are collected, 4~8h is extracted using wall breaking enzyme smudge cells, then with organic solvent, are obtained rich in phosphatide type DHA
Grease.
6. phosphatide type DHA according to claim 5 fermentation preparation, it is characterised in that described organic solvent is
Any one or a few mixture in n-hexane, petroleum ether, ethanol and ether, according to organic solvent and smudge cells liquid
Product compares 1:0.5~2 amount addition.
7. phosphatide type DHA according to claim 5 fermentation preparation, it is characterised in that described wall breaking enzyme is egg
White enzyme, addition is 2~8g/L.
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| CN104962589B (en) * | 2015-08-05 | 2019-02-26 | 嘉必优生物技术（武汉）股份有限公司 | A kind of microbial oil and preparation method rich in phosphatide type polyunsaturated fatty acid |
| CN105420122A (en) * | 2015-12-23 | 2016-03-23 | 通威股份有限公司 | Schizochytrium limacinum suitable for high-density culture and method for producing grease rich in DHA |
| US10851395B2 (en) | 2016-06-10 | 2020-12-01 | MARA Renewables Corporation | Method of making lipids with improved cold flow properties |
| CN106834370A (en) * | 2017-01-20 | 2017-06-13 | 武汉净宇微藻科技有限公司 | A kind of method of raising schizochytrium limacinum DHA and carotenoids yield |
| CN108486177A (en) * | 2018-02-12 | 2018-09-04 | 广州富诺健康科技股份有限公司 | A method of it is prepared using algae oil and is rich in omega-fatty acid phosphatide |
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| EP2163641A1 (en) * | 2007-06-04 | 2010-03-17 | National University Corporation Hokkaido University | Method for production of dha-containing phospholipid through microbial fermentation |
| CN101519676A (en) * | 2009-04-03 | 2009-09-02 | 湖北福星生物科技有限公司 | Method for producing docosahexenoic acid by fermenting schizochytrium |
| CN101575584A (en) * | 2009-06-18 | 2009-11-11 | 南京工业大学 | Schizochytrium sp. and method for producing DHA lipa by using same |
| CN102181320A (en) * | 2011-03-29 | 2011-09-14 | 青岛佰福得科技有限公司 | Extraction method of DHA (docosahexaenoic acid) algae oil by biological fermentation |
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| 裂殖壶菌Schizochytrium sp. FJU-512 DHA单细胞油脂的研究;张娟梅;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20070630;第36页 * |
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