CN104313068A - Fermentation preparation method of phospholipid-type DHA - Google Patents
Fermentation preparation method of phospholipid-type DHA Download PDFInfo
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Abstract
The invention discloses a fermentation preparation method of phospholipid-type DHA. The preparation method comprises the following steps: (1) conducting high-density fermentation in a fluid nutrient medium by taking a DHA strain as an original strain to obtain bacterium cells rich in glyceride-type DHA; (2) adding a carbon source, a nitrogen source, a phosphor source and amino acid to fermentation liquor obtained from the high-density fermentation in the step (1), and further fermenting to obtain bacterium cells rich in phospholipid-type DHA; and (3) collecting the bacterium cells rich in phospholipid-type DHA obtained from the step (2), breaking and extracting the cells to obtain grease rich in phospholipid-type DHA. According to the preparation method disclosed by the invention, 15-40% of polar grease is finally obtained; the polar grease mainly contains phospholipid, and the content of DHA in the polar grease reaches 48.16%. The grease rich in glyceride-type DHA is converted into the grease rich in phospholipid-type DHA, and the content of phospholipid-type DHA in the grease is effectively improved.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the fermentation preparation of a kind of phosphatide type DNA.
Background technology
Docosahexenoic acid (DHA) is a kind of important omega-3 long chain polyunsaturated fatty acids, be the moiety of cytolemma in brain and retinal tissue, there is vision enhancing, promote the physiological function such as brain cell development and preventing hypertension, arteriosclerosis, cardiovascular disorder.Because human body is difficult to meet the demand of body to DHA by self synthesis, therefore must relies on and obtain from extraneous food.
At present, the main source of DHA is fish oil, and is mainly taken in by human body with the form of triglyceride level.The another kind of existence form of DHA is phosphatide type DHA (DHA-containing PL).Phosphatide is biomembranous main component, and research shows the effects such as it has reducing blood-fat, atherosclerosis.The existence form of DHA is different, and the absorption pattern in human body and assimilated efficiency are also different.Glycerine ester type DHA is mainly absorbed in the mode of Passive diffusion in human body, and specific absorption is about 50%.And phosphatide type DHA is mainly absorbed in the mode of active absorption in human body, specific absorption is close to 100%.The people such as Kafrawy study discovery, and compared with other phosphatide type lipid acid, phosphatide type DHA has larger toxicity to mouse leukemia cell.Research also finds, phosphatide type DHA effectively can reduce total cholesterol in mice plasma and liver and content of triglyceride, improves the blood lipid level of normal mouse.The people such as Gisbert study discovery, and phosphatide type DHA is glycerine ester type DHA comparatively, have better promoter action to the growth of prelarva.The people such as Schaefer study discovery, and in blood plasma, the increase of phosphatide type DHA level effectively can reduce dull-witted sickness rate.
Phosphatide type DHA is only present in yolk, and content is extremely low.At present, the research of domestic phosphatide type DHA mainly concentrates on the following aspects: the application of (1) phosphatide type DHA; (2) extraction of phosphatide type DHA; (4) the physiological action research of phosphatide type DHA.Wherein, do not find to utilize biological fermentation process to prepare the relevant report of phosphatide type DHA.
Summary of the invention
Technical problem to be solved by this invention is to provide the fermentation preparation of a kind of phosphatide type DHA to fill the domestic gaps.
For solving above technical problem, the technical solution used in the present invention is as follows:
A fermentation preparation of phosphatide type DHA, is characterized in that, it comprises the following steps:
(1) to produce the bacterial strain of DHA for starting strain, in liquid medium within, carry out high density fermentation, obtain the somatic cells being rich in glycerine epoxy-type DHA;
(2) add nutritive substance in the fermented liquid after terminating to step (1) high density fermentation, continue fermentation and obtain the somatic cells being rich in phosphatide type DHA;
(3) collect the somatic cells being rich in phosphatide type DHA that step (2) obtains, obtain through broken, extraction the grease being rich in phosphatide type DHA;
Wherein, described nutritive substance is any one or a few the combination in carbon source, nitrogenous source, phosphorus source, amino acid.
Wherein, the bacterial strain of described product DHA is schizochytrium limacinum genus, genus thraustochytrium, my Ken Shi kettle Trentepohlia, be preferably schizochytrium limacinum to belong to, most preferably be CCTCC No:M209059 bacterial strain (it is in the Chinese patent of 200910033869.5 that this bacterial strain has been disclosed in application number).
High density fermentation in above-mentioned steps (1) comprises the steps:
(1a) bacterial strain be kept in glycerine pipe access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in the shaking table of 20 ~ 30 DEG C, with the rotating speed of 150 ~ 200rpm, cultivates 24 ~ 48h, obtain primary seed solution;
(1b) primary seed solution step (1a) obtained is equipped with in the 500ml shaking flask of 100ml seed culture medium according to the access of 2 ~ 10%v/v inoculum size, in the shaking table of 20 ~ 30 DEG C, with the rotating speed of 150 ~ 200rpm, cultivate 24 ~ 48h, obtain secondary seed solution;
(1c) secondary seed solution step (1b) obtained is equipped with in the 500ml shaking flask of 100ml seed culture medium according to the access of 2 ~ 10%v/v inoculum size, in the shaking table of 20 ~ 30 DEG C, with the rotating speed of 150 ~ 200rpm, cultivate 24 ~ 48h, obtain three grades of seed liquor;
(1d) the three grades of seed liquor obtained in step (1c) are equipped with in the fermentor tank of fermention medium according to the access of 2 ~ 10%v/v inoculum size, then in fermentor tank, defoamer is added, ferment, make strain fermentation produce the grease being rich in glycerine ester type DHA.
In above-mentioned steps (1d), the air flow of fermentation condition is 0.2 ~ 1vvm, is preferably 0.6vvm.
In above-mentioned steps (1d), the stirring velocity of fermentation condition is 100 ~ 200rpm, is preferably 200rpm.
In above-mentioned steps (1d), the tank temperature of fermentation condition is 20 ~ 30 DEG C, is preferably 30 DEG C.
In above-mentioned steps (1d), the incubation time of fermentation condition is 90 ~ 150h, is preferably 120h.
The fermentation process adopted in above-mentioned steps (1d) is Intermittent fermentation or feed supplement formula fermentation process.
The carbon content of the seed culture medium in above-specified high density fermentation is 10 ~ 30g/L, is preferably 15 ~ 25g/L, most preferably is 20g/L.
Fermention medium carbon content in above-specified high density fermentation is 20 ~ 60g/L, is preferably 30 ~ 40g/L, most preferably is 40g/L; Nitrogen content is 2.1 ~ 2.5g/L, is preferably 1.8 ~ 2g/L, most preferably is 2g/L; Sulphur content is 2 ~ 3g/L, is preferably 2.5 ~ 3g/L, most preferably is 3g/L; Phosphorus content is that 0.4 ~ 1.5g/L is preferably 1.2 ~ 1.5g/L, most preferably is 1.5g/L.
In described seed culture medium and fermention medium, carbon source is glucose, fructose or glycerine, preferred glucose; Nitrogenous source is yeast extract paste, corn steep liquor, extractum carnis, peptone, ammonium nitrate or ammonium sulfate, preferred yeast cream; Phosphorus source is potassium primary 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 adopted in above-mentioned steps (1d) is Intermittent fermentation or feed supplement formula fermentation process.
Wherein, in nutritive substance,
Described carbon source is any one or a few the mixture in ethanol, sodium acetate; The addition of carbon source is 2 ~ 4g/L, is preferably 4g/L.
Described nitrogenous source is any one or a few the mixture in yeast extract paste, ammonium sulfate, Sodium Glutamate, and the addition of nitrogenous source is 5 ~ 20g/L, is preferably 15 ~ 20g/L, most preferably is 20g/L.
Described phosphorus source is in potassium primary phosphate, and the addition in phosphorus source is 2 ~ 4g/L, is preferably 3 ~ 4g/L, most preferably is 4g/L.
Described amino acid is proline(Pro), Methionin, and addition is 0.1 ~ 0.5g/L, is preferably 0.2 ~ 0.3g/L, most preferably is 0.3g/L.
The preferred Sodium Glutamate of described nutritive substance, ammonium sulfate, ethanol add separately, and ammonium sulfate and ethanol two-cocoon feeding add, and ammonium sulfate and proline(Pro) two-cocoon feeding add, and most preferably ammonium sulfate adds separately, and ammonium sulfate and proline(Pro) two-cocoon feeding add.
Wherein, in step (2), the air flow of described continuation fermentation condition is 0.5 ~ 1vvm, is preferably 0.6vvm.
Wherein, in step (2), the stirring velocity 100 ~ 200rpm of described continuation fermentation condition, is preferably 170rpm.
Wherein, in step (2), the tank temperature of described continuation fermentation condition 20 ~ 30 DEG C, is preferably 30 DEG C.
Wherein, in step (2), the incubation time of described continuation fermentation condition is 48 ~ 72h, is preferably 72h.
Step (3) detailed process is: collect somatic cells, adopts wall breaking enzyme smudge cells, then carries 4 ~ 8h with organic solvent extracting, obtain the grease being rich in phosphatide type DHA.
Wherein, described organic solvent is any one or a few the mixture in normal hexane, sherwood oil, ethanol and ether, adds according to the amount of organic solvent and smudge cells liquid volume ratio 1:0.5 ~ 2.
Wherein, described wall breaking enzyme is proteolytic enzyme, and addition is 2 ~ 8g/L.
After extraction obtains grease, utilize column chromatography method to be separated oil component, obtain polar lipid and neutral lipid respectively, described column chromatography method is as follows:
Get 100 ~ 200 order chromatography silicon special rubber powders and dry 24h in 120 DEG C of baking ovens, make its water content about 5%, stand-by.First 30ml sherwood oil is added in chromatography column, pat the bubble in removing core; Get a certain amount of silica-gel powder in beaker, add a certain amount of sherwood oil, be stirred to bubble-free, more slowly pour in chromatography column, make its uniform settling, rinse pillar 2h with neutral eluent (petroleum ether/ethyl ether, 9:1, v/v), make pillar consolidation.Take about 2.0g grease, dissolve with neutral eluent.Slowly added in chromatography column along post jamb by sample, first obtain neutral fat with the neutral eluent of 500ml, then use polar elution agent (methyl alcohol) to be eluted to silicagel column to turn white, rotary evaporation obtains each separated portion respectively.
Utilize gas-chromatography to detect fatty acid distribution situation in total grease, neutral lipid and polar lipid respectively, described gas chromatography analysis method is adopted with the following method:
Esterification method: get about 0.5g grease, be settled to 10ml with normal hexane; Pipette 1ml to 10ml volumetric flask with transfer pipet, add the KOH-methanol solution of 3ml 0.5mol/L, in 65 DEG C of water-bath 15 ~ 20min, cooling; Add 2ml BF3-ether-methanol solution (BF3-ether: methyl alcohol=3:7, v/v) again, water-bath 65 DEG C, 5 ~ 10min, cooling; Add saturated NaCl solution, each 2ml of normal hexane again, standing, layering after vibration, get upper strata in another volumetric flask, 10ml is settled to after n-hexane extraction 2-3 time, get 100 μ l extraction liquids and add 100 μ l Nonadecanoic acid methylester (concentration is known) solution mixings, can be used as DHA and analyze.
Gas phase analysis condition: chromatographic column: DB-23 (60m*0.25mm*0.25 μm); Detector: FID; Carrier gas: nitrogen; Splitting ratio: 30/1; Injector temperature: 250 DEG C; Detector temperature: 280 DEG C; Sample size: 1 μ l; Heating schedule: initial column temperature is 100 DEG C, first rises to 196 DEG C with the speed of 25 DEG C/min, then rises to 220 DEG C with the speed of 2 DEG C/min, keeps 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 preparation method that the invention provides a kind of phosphatide type DHA, produces on the basis of DHA in thalassiomycetes fermentation, and adopt the method for two stage fermentation, the grease of phosphatide type DHA is rich in preparation.In the grease obtained, it is 15%-40% that polar lipid accounts for total grease percentage composition, and in polar lipid, DHA content can reach 48.16%, and wherein major part is phosphatide type DHA.And the glyceryl ester of the research utilizing fermentable to prepare DHA grease at present mainly in enrichment born of the same parents, little to the research of phosphatide, the phosphatide preparing process technique that this patent provides is simple, and production cost is lower, and thalassiomycetes growth is very fast, is easier to expand the scale of production.Therefore, the proposition of the method, is conducive to the physiological action that we study phosphatide type DHA further, effectively improves the application efficiency of DHA product.
Accompanying drawing explanation
Fig. 1 schizochytrium limacinum DHA Unicell Oils and Fats TLC analyzes: Fig. 1-A shows that in schizochytrium limacinum Unicell Oils and Fats, neutral fat is formed primarily of triglyceride level (TAG), triglyceride (DAG) and monoglyceride (MAG); Schizochytrium limacinum Unicell Oils and Fats is carried out tlc analysis in polarity developing agent, and Fig. 1-B shows in polar lipid based on Yelkin TTS.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
The schizochytrium limacinum (Schizochytrium sp.) that the bacterial strain of the product DHA used in following examples is CCTCC No:M209059 for preserving number.
Embodiment 1: first stage fermentation is standby is rich in glycerine ester type DHA grease.
The DHA bacterial classification be kept in glycerine pipe access is equipped with in the 500ml shaking flask of 100ml seed culture medium, shaking table carries out the activation of seed, temperature 30 DEG C, rotating speed 170rpm, cultivate 24h, obtain the seed liquor that vigor is higher.After activating three generations after the same method, seed liquor access is equipped with in the fermentor tank of fermention medium, inoculum size 10% (v/v), air flow 0.6vvm, mixing speed 200rpm, tank temperature 30 DEG C, add defoamer silicone SE-2, addition is 0.3g/L, fermentation culture 120h, obtains being rich in glyceryl ester DHA grease.Fermentation results is in table 1.
Table 1 first stage fermentation results
Embodiment 2: metabolite analysis in fermenting process.
Utilize column chromatography method to carry out Component seperation to the grease of different fermentations time in first stage fermenting process, investigate different oil component situation over time.Analytical results is in table 2.As can be seen from Table 2, along with the prolongation of fermentation time, in somatic cells, neutral fat content constantly raises, and polar lipid content reduces gradually.Therefore can pass through the changing conditions of various metabolite content in analysis of cells growth phase, earlier fermentation and fermentation later stage born of the same parents, thus determine the nutritive element required for accumulation of polar lipid.
Oil component change in table 2 first stage fermenting process
Utilize GC-MS to carry out analyzing and testing analysis to the metabolite in each stage in fermenting process, find that metabolites kinds mainly includes machine acid, amino acid, carbohydrate and phosphate compound.Ferment each stage metabolite content change in table 3.
Table 3 ferments each stage intermediate metabolites content situation
As shown in Table 3, the content of most of metabolite changes with the change of fermentation time during the fermentation.As shown in Table 2,0 ~ 48h polar lipid content is higher, and after 48h, polar lipid content declines, until fermentation ends.0 ~ 48h cell is in growth phase and earlier fermentation, the born of the same parents' intracellular metabolite thing content analysis of each stage of combining with fermentation finds, phosphoric acid, Methionin, proline(Pro) are higher at this stage content, and in the fermentation later stage that polar lipid content constantly declines, phosphoric acid, Methionin, the proline(Pro) content in born of the same parents declines all to some extent.Therefore, the material such as phosphoric acid, Methionin, proline(Pro) is very possible accumulates substantial connection with phosphatide in early stage.Therefore, for promoting the synthesis of phosphatide, this type of material is added nutritive substance as subordinate phase fermentation.
Embodiment 3: subordinate phase list nutritive substance adds fermentation and investigates.
After first stage fermentation ends, the metabolite profile according to metabolism group gained adds different nutriments in fermented liquid, and adding nitrogenous source provides amino acid, and potassium primary phosphate provides phosphoric acid.Nutritive substance and addition are respectively 2g/L sodium acetate, 40ml/L ethanol, 4g/L yeast extract paste, 20g/L Sodium Glutamate, 3.6g/L ammonium sulfate, 4g/L potassium primary phosphate.The condition of subordinate phase fermentation is as follows: air flow is 0.6vvm, and stirring velocity is 170rpm, and tank temperature is 30 DEG C, and incubation time is 72h.After fermentation ends, utilize column chromatography method to carry out oil component separation to the grease obtained, the component investigating grease in the fermented liquid adding different nutriments is investigated, and investigates and the results are shown in Table 4.
Table 4 adds different single nutritive element fermented liquid oil analyses result
From fat content angle, compared with during first stage fermentation ends, after subordinate phase is cultivated, the fat content in fermented liquid has had minimizing in various degree.Wherein, do not add the control group (33.624g/L) of any nutritive element compared with (52.847g/L) during first stage fermentation ends, fat content reduces 36.37%.The interpolation of Sodium Glutamate, ethanol, ammonium sulfate three kinds of nutritive elements can alleviate the consumption of grease preferably.
Account for total grease percentage composition angle analysis from different oil component, compared with first stage fermentation ends, after subordinate phase fermentation, the percentage composition that fermented liquid grease Semi-polarity fat accounts for total grease all there occurs change.Compared with first stage fermentation ends (6.88%), control group (9.21%) grease polar lipid/total grease adds 33.87%.Compared with control group, add in the grease of different nutritive elements, polar lipid/total grease also increases to some extent.Wherein, the most obviously (14.59%), compared with control group, polar lipid accounts for total fat content and adds 58.41% 3.6g/L ammonium sulfate additive effect.
From oil component content angle analysis, be 3.636g/L with polar lipid content during first stage fermentation ends, ferment through subordinate phase, control group declines to some extent with the grease Semi-polarity fat content that with the addition of yeast extract paste, and the grease Semi-polarity fat content that with the addition of Sodium Glutamate, ethanol, sodium acetate, ammonium sulfate and potassium primary phosphate has increase in various degree.Wherein, ammonium sulfate, ethanol additive effect are the most obvious, compared with first stage fermentation ends, add 53.19%, 68.87% respectively.Embodiment 4: subordinate phase two nutrient material adds fermentation and investigates.
On the basis of embodiment 3, after first stage fermentation ends, in fermented liquid, add two nutrient material.Nutritive substance and addition are respectively 40ml/L ethanol, 3.6g/L ammonium sulfate, 4g/L potassium primary phosphate, 0.3g/L proline(Pro), 0.2g/L Methionin.And fat content and oil component are investigated, investigate and the results are shown in Table 5.
Table 5 adds two nutrient fermentation of materials liquid oil analyses result
As can be seen from the above table, nutritive substance combination is added and effectively can be improved percentage composition and the polar lipid content that grease Semi-polarity fat accounts for total grease.After adding ammonium sulfate and proline(Pro), the percentage composition that polar lipid accounts for total grease can reach 38.54%, compared with the control (9.21%), add 3.18 times, polar lipid content can reach 13.87g/L, compared with the control (3.10g/L), adds 3.47 times.
Embodiment 5: subordinate phase fermentation DHA content is investigated.
The grease utilizing column chromatography for separation embodiment 1, embodiment 3 and each experimental group of embodiment 4 finally to obtain, utilizes chromatography of gases analysis.The DHA content of different oil component in the fermented liquid of interpolation Different Nutrient Elements is investigated, investigates and the results are shown in Table 6.
Table 6 adds the DHA content analytical results of different oil component in Different Nutrient Elements secondary fermentation liquid
From above-mentioned example, after subordinate phase fermentation ends, the DHA content of adding different components in the grease of different nutriments all can change.Wherein, compared with first stage fermentation ends, the total grease DHA content of control group adds 27.31%, and with the addition of total grease DHA content in the fermentation of yeast extract paste and add 31.78%, the DHA content of polar lipid reaches 48.42%.The DHA content of adding the fermented liquid Semi-polarity fat of ammonium sulfate reaches 48.16%, adds 8.59% compared with control group.
Embodiment 6: Unicell Oils and Fats TLC analyzes.
Smudge cells embodiment 4 and 5 with the addition of the cell obtained after nutritive substance ferments as follows, and extraction obtains grease: collect somatic cells, adopt wall breaking enzyme smudge cells, then carry 4h with organic solvent extracting, obtain the grease being rich in phosphatide type DHA.Organic solvent is the mixture of normal hexane and ethanol 1:1 by volume, adds according to the amount of organic solvent and smudge cells liquid volume ratio 1:1.Wall breaking enzyme is proteolytic enzyme, and addition is 3g/L.Carry out thin-layer chromatographic analysis to schizochytrium limacinum Unicell Oils and Fats, analytical results as shown in Figure 1.
Schizochytrium limacinum Unicell Oils and Fats is carried out tlc analysis in neutral developing agent, shows that in schizochytrium limacinum Unicell Oils and Fats, neutral fat forms (see Fig. 1-A) primarily of triglyceride level (TAG), triglyceride (DAG) and monoglyceride (MAG); Schizochytrium limacinum Unicell Oils and Fats is carried out tlc analysis in polarity developing agent, shows in polar lipid based on Yelkin TTS (see Fig. 1-B).
Claims (10)
1. a fermentation preparation of phosphatide type DHA, is characterized in that, it comprises the following steps:
(1) to produce the bacterial strain of DHA for starting strain, in liquid medium within, carry out high density fermentation, obtain the somatic cells being rich in glycerine epoxy-type DHA;
(2) add nutritive substance in the fermented liquid after terminating to step (1) high density fermentation, continue fermentation and obtain the somatic cells being rich in phosphatide type DHA;
(3) collect the somatic cells being rich in phosphatide type DHA that step (2) obtains, obtain through broken, extraction the grease being rich in phosphatide type DHA;
Wherein, described nutritive substance is carbon source, nitrogenous source, phosphorus source, any one or a few combination amino acid whose.
2. the fermentation preparation of phosphatide type DHA according to claim 1, is characterized in that, the bacterial strain of described product DHA is genus thraustochytrium, schizochytrium limacinum belongs to, my Ken Shi kettle Trentepohlia.
3. the fermentation preparation of phosphatide type DHA according to claim 1, is characterized in that, in step (1), described high density fermentation comprises the steps:
(1a) bacterial strain be kept in glycerine pipe access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in the shaking table of 20 ~ 30 DEG C, with the rotating speed of 150 ~ 200rpm, cultivates 24 ~ 48h, obtain primary seed solution;
(1b) primary seed solution step (1a) obtained is equipped with in the 500ml shaking flask of 100ml seed culture medium according to the access of 2 ~ 10%v/v inoculum size, in the shaking table of 20 ~ 30 DEG C, with the rotating speed of 150 ~ 200rpm, cultivate 24 ~ 48h, obtain secondary seed solution;
(1c) secondary seed solution step (1b) obtained is equipped with in the 500ml shaking flask of 100ml seed culture medium according to the access of 2 ~ 10%v/v inoculum size, in the shaking table of 20 ~ 30 DEG C, with the rotating speed of 150 ~ 200rpm, cultivate 24 ~ 48h, obtain three grades of seed liquor;
(1d) the three grades of seed liquor obtained in step (1c) are equipped with in the fermentor tank of fermention medium according to the access of 2 ~ 10%v/v inoculum size, then in fermentor tank, defoamer is added, air flow be 0.2 ~ 1vvm, stirring velocity is 100 ~ 200rpm, tank temperature cultivates 90 ~ 150h under being the condition of 20 ~ 30 DEG C, makes strain fermentation produce the grease being rich in glycerine ester type DHA.
4. the fermentation preparation of phosphatide type DHA according to claim 3, it is characterized in that, the carbon content of described seed culture medium is 10 ~ 30g/L, described fermention medium carbon content is 20 ~ 60g/L, 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 substratum, carbon source is glucose, fructose or glycerine; Nitrogenous source is yeast extract paste, corn steep liquor, extractum carnis, peptone, ammonium nitrate or ammonium sulfate; Phosphorus source is potassium primary phosphate or dipotassium hydrogen phosphate, and sulphur source is magnesium sulfate or ammonium sulfate.
5. the fermentation preparation of phosphatide type DHA according to claim 3, is characterized in that, in step (1d), described defoamer is silicone SE-2, and addition is 0.3g/L.
6. the fermentation preparation of phosphatide type DHA according to claim 1, is characterized in that, 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 nitrogenous source is any one or a few the mixture in yeast extract paste, ammonium sulfate, Sodium Glutamate, and the addition of nitrogenous source is 5 ~ 20g/L;
Described phosphorus source is potassium primary phosphate, and the addition in phosphorus source is 2 ~ 4g/L;
Described amino acid is proline(Pro) or Methionin, and addition is 0.1 ~ 0.5g/L.
7. the fermentation preparation of phosphatide type DHA according to claim 1, is characterized in that, in step (2), described continuation fermentation condition is: air flow 0.5 ~ 1vvm, stirring velocity 100 ~ 200rpm, tank temperature 20 ~ 30 DEG C, continues cultivation 48 ~ 72h.
8. the fermentation preparation of phosphatide type DHA according to claim 1, it is characterized in that, step (3) detailed process is: collect somatic cells, adopts wall breaking enzyme smudge cells, carry 4 ~ 8h with organic solvent extracting again, obtain the grease being rich in phosphatide type DHA.
9. the fermentation preparation of phosphatide type DHA according to claim 8, it is characterized in that, described organic solvent is any one or a few the mixture in normal hexane, sherwood oil, ethanol and ether, adds according to the amount of organic solvent and smudge cells liquid volume ratio 1:0.5 ~ 2.
10. the fermentation preparation of phosphatide type DHA according to claim 8, is characterized in that, described wall breaking enzyme is proteolytic enzyme, and addition is 2 ~ 8g/L.
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| CN109196108B (en) * | 2016-06-10 | 2022-04-05 | 玛拉可再生能源公司 | Method for preparing lipid with improved cold flow characteristics |
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| 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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