CN104480152B - A kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease - Google Patents

A kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease Download PDF

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CN104480152B
CN104480152B CN201410667988.7A CN201410667988A CN104480152B CN 104480152 B CN104480152 B CN 104480152B CN 201410667988 A CN201410667988 A CN 201410667988A CN 104480152 B CN104480152 B CN 104480152B
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schizochytrium limacinum
grease
docosahexaenoic acid
acid content
ethanol
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CN104480152A (en
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余龙江
陈伟
朱圆敏
周蓬蓬
金文闻
余金龙
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WUHAN HUASHITE INDUSTRIAL BIOTECHNOLOGY DEVELOPMENT Co Ltd
Huazhong University of Science and Technology
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WUHAN HUASHITE INDUSTRIAL BIOTECHNOLOGY DEVELOPMENT Co Ltd
Huazhong University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone

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Abstract

The present invention relates to a kind of method for improving docosahexaenoic acid (DHA) content in schizochytrium limacinum grease, it is characterized in that, endobacillary pentose phosphate pathway metabolic flux is reduced by selecting fermenting carbon source, external source regulatory factor can also be added, reduce endobacillary pentose phosphate pathway metabolic flux, the activity to the malate dehydrogenase in tricarboxylic acids delivery system suppresses simultaneously, nicotinamide-adenine dinucleotide phosphate (NADPH) content in schizochytrium limacinum body is set to decline to a great extent, promote DHA a large amount of synthesis, DHA content in the grease of final acquired thalline is set to reach as high as 55.3%.Fermenting carbon source is 1,2 propane diols, or 1,2 propane diols and any in ethanol and acetate or two kinds mixtures, and external source regulatory factor is preferably sesamol, after fermentation medium sterilizing, added after filtration sterilization；The addition of sesamol can be 0.5 3.0mM/L.The inventive method is simple and easy to apply, and effect substantially, can greatly improve DHA content in thalline grease, and be easy to industrial applications.

Description

A kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease
Technical field
The present invention relates to field of microbial fermentation, and in particular to one kind improves docosahexaenoic acid in schizochytrium limacinum grease (DHA) method of content.
Background technology
Docosahexaenoic acid (DHA) belongs to polyunsaturated fatty acid, with highly important physiological function, be human body from Body can not be synthesized largely but indispensable important nutrient.DHA is largely present in human body retina and corticocerebral god Promote brain and visual acuity through in tissue, having, improve intelligence, improve the effect of memory and eyesight；In addition, it can also be prevented Cholesterol is deposited on vascular wall, prevention or the generation for mitigating atherosclerosis and coronary heart disease.
Schizochytrium limacinum (Schizochytrium) is a kind of resource of excellent production polyunsaturated fatty acid grease.Mesh Before, the concern that polyunsaturated fatty acid grease obtains many scientific research institutions and unit is produced using schizochytrium limacinum large scale fermentation, And its production process is conducted in-depth research.The patent document of Application No. 201010000055.4 discloses a kind of ocean Fungi schizochytrium limacinum, wherein docosahexaenoic acid triglycerides account for the 35-40% of grease total amount；Application No. 201310571413.0 patent discloses the method using stalk hydrolyzate fermenting and producing docosahexaenoic acid, final to obtain DHA can account for the 42.1-45.3% of grease gross weight；The patent of Application No. 201010504635.7 discloses exterior addition factor Promote the method for Microbe synthesis docosahexaenoic acid, this method is pointed out, second is added into the microbiological culture media that can produce DHA During any one or a few combination in acid, citric acid and Simvastatin, the DHA content in microbial body can be carried effectively Height, 45.00% can be brought up to from 35.51% initial highest.
The technology tool that the above method produces DHA to schizochytrium limacinum fermentation has a certain upgrade, but not yet has technology at present The DHA synthesis paths with reference to specific in schizochytrium limacinum body regulate and control its metabolic pathway, to realizing schizochytrium limacinum institute Lipid-producing The further lifting of middle DHA content；Moreover, further promoting the increase of DHA content, schizochytrium limacinum institute Lipid-producing can be effectively lifted Quality, fermentation costs are also reduced indirectly, production efficiency is added.
The content of the invention
The deficiency of DHA grease technology is produced for existing utilization schizochytrium limacinum fermentation, fragmentation is improved the invention provides one kind The method of docosahexaenoic acid content in chytrid grease, according to specific in schizochytrium limacinum body the characteristics of DHA synthesis paths, During schizochytrium limacinum fermentation, endobacillary pentose phosphate pathway metabolic flux is reduced by selecting fermenting carbon source, to carry DHA content in high schizochytrium limacinum institute Lipid-producing, lifts the quality of DHA grease, meanwhile, production efficiency is improved, and reduce production Cost.
To achieve these goals, docosahexaenoic acid content in schizochytrium limacinum grease is improved the invention provides one kind Method, the fermenting carbon source in this method selection fermentation medium reduces endobacillary pentose phosphate pathway metabolic flux, makes Nicotinamide-adenine dinucleotide phosphate (NADPH) content declines to a great extent in schizochytrium limacinum body, promotes DHA a large amount of synthesis；Institute Fermenting carbon source is stated for 1,2-PD, 1,2-PD and ethanol, 1,2-PD and acetate, or 1,2-PD, ethanol With any one in the combination of acetate.
As the improvement of above-mentioned technical proposal, this method adds external source regulatory factor in the fermentation medium, makes in thalline Pentose phosphate pathway metabolic flux reduction, while to the malate dehydrogenase in tricarboxylic acids delivery system activity suppress, make NADPH contents decline to a great extent in schizochytrium limacinum body, promote DHA a large amount of synthesis.Wherein, external source regulatory factor is preferably sesame Phenol, after fermentation medium sterilizing, add after filtration sterilization；The addition of sesamol can be 0.5-3.0mM/L.
The advantage of the invention is that by selecting fermenting carbon source, decline the pentose phosphate pathway metabolic flux in thalline, And further simultaneous selection fermenting carbon source and addition external source regulatory factor, endobacillary pentose phosphate pathway metabolic flux drops It is low, while the activity to the malate dehydrogenase (ME) in tricarboxylic acids delivery system suppresses, make the reducing power in schizochytrium limacinum body NADPH contents decline to a great extent, and cause carbon metabolism flow to turn to polyketide synthase path to synthesize DHA, so as to promote DHA a large amount of conjunctions Into greatly improving the content of DHA in schizochytrium limacinum grease, be obviously improved the quality of DHA grease.
Embodiment
The present invention makes endobacillary phosphorus by selecting fermenting carbon source, and the further mode such as addition external source regulatory factor Sour pentose pathway metabolic flux reduction, while the activity to the malate dehydrogenase in tricarboxylic acids delivery system suppresses, makes fragmentation NADPH contents decline to a great extent in chytrid body, so as to promote fatty acid precursor material-acetyl coenzyme A to turn to polyketide synthase approach Change.
The step that implements of the inventive method is：
(1) by the schizochytrium limacinum kind activation culture of cryopreservation into schizochytrium limacinum seed liquor；
The various components that the seed culture medium that activation culture is used can be provided using prior art, its preferred component (g/L) it is：Glucose 15.0-30.0, dusty yeast 4.0-8.0, beef extract 0-5.0 peptone 0-5.0, corn steep liquor 2.0-6.0, MgSO4·7H2O 0.2-1.0, KH2PO40.5-2.0, sea crystal 10.0-20.0.
(2) schizochytrium limacinum seed liquor is carried out according to percent by volume 4%-10% (preferably 8%) access fermentation mediums Culture；
The various components that fermentation medium can be provided using prior art, simply will wherein be used as the grape of fermenting carbon source Sugar replaces with 1,2-PD, or is 1,2-PD and ethanol and/or the mixed solution of acetate, and ethanol need to be by filtering out Added after bacterium in the fermentation medium after having sterilized, wherein, the preferred sodium acetate of acetate.
The preferred component of fermentation medium (g/L) is：Fermenting carbon source 40.0-60.0, yeast extract 6.0-15.0, paddy ammonia Sour sodium 0-10.0, MgSO4·7H2O 0.2-1.0, KH2PO40.5-2.0, (NH4)2SO40-5.0, NaNO31.0-3.0, Na2SO4 5.0-12.0。
When fermenting carbon source is that many kinds of substance is mixed, its component (g/L) is：
1,2- propane diols and acetate：1,2- propane diols 40-60；Sodium acetate 0-10；
1,2- propane diols and ethanol：1,2- propane diols 40-60；Ethanol 0-10；
1,2- propane diols, ethanol and acetate：1,2- propane diols 40-60；Ethanol 0-10；Acetate 0-10.
Further to improve the technique effect of the present invention, it can add after fermentation medium sterilizing after carrying out filtration sterilization Plus external source regulatory factor.
The preferable sesamol of external source regulatory factor, its addition is 0.5-3.0mM/L.
(3) terminate after fermentation, collect wet thallus and be dried in vacuo, until thalline keeps constant weight, and weigh.
(4) broken wall is carried out to dry thalline, adds organic solvent (such as petroleum ether and n-hexane) and extracted, so that The grease rich in docosahexaenoic acid is obtained, the vapor detection analysis after esterification is carried out, DHA content can be obtained.
Step (3), (4) can use prior art, will not be repeated here.
The technical scheme in the embodiment of the present invention will be clearly and completely described below.Obviously, described implementation Example only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, this area is common The every other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model that the present invention is protected Enclose.
Embodiment 1
(1) 50mL seed culture mediums are added in 250mL triangular flasks.The schizochytrium limacinum kind of cryopreservation is accessed, in culture Cultivated 2 days under the conditions of 25 DEG C of temperature, shaking speed 200rpm, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed Liquid；
Seed culture medium component (g/L)：Glucose 15.0, dusty yeast 6.0, beef extract 3.0, peptone 2.0, corn steep liquor 4.0, MgSO4·7H2O 0.2, KH2PO42.0, sea crystal 15.0.
(2) 50mL fermentation mediums are added in 250mL triangular flasks, schizochytrium limacinum is accessed with 10% inoculum concentration and fermented Culture medium, 26 DEG C incubated 3 days, rotating speed 200rpm；
Fermentation medium component (g/L)：1,2-PD 40.0, yeast extract 6.0, MgSO4·7H2O 0.6, KH2PO40.5, NaNO31.0, Na2SO45.0；
CK(g/L)：Glucose 40.0, yeast extract 6.0, MgSO4·7H2O 0.6, KH2PO40.5, NaNO31.0, Na2SO45.0；
Every kind of different culture medium prepares 3 Duplicate Samples, and 121 DEG C, sterilize 25min, cools down standby；
(3) continuous culture terminates fermentation after 3 days, and zymotic fluid carries out 8000g and centrifuged 15 minutes, and is washed 2-3 times with single steam Afterwards, centrifuge again, collect wet thallus be placed in vacuum drying chamber, vacuum be 0.9Mpa, temperature be 80 DEG C under the conditions of, greatly About dry 10 hours, until thalline keeps constant weight, and weigh.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, weighs 1g broken wall bacterium powders in test tube, add 30-60 boiling ranges Petroleum ether carry out equal-volume extraction three times, petroleum ether 5mL is added every time, petroleum ether extraction liquid is collected, vacuum drying is placed in In case, in 80 DEG C, vacuum is under the conditions of 0.9Mpa, removes petroleum ether, so that the grease rich in docosahexaenoic acid is obtained, And carry out the analysis of the vapor detection after esterification.
Fermentation results are as follows：
Endobacillary glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) activity change contrast in the fermentation process of table 1
Endobacillary NADPH changes of contents contrast in the fermentation process of table 2
The 1,2- propane diols group of table 3 and glucose group fermentation contrast
The application of fermenting carbon source 1,2-PD, causing schizochytrium limacinum, G6PDH activity is decreased obviously during the fermentation, phosphorus Sour pentose pathway metabolic flux declines substantially, and NADPH contents decline substantially in thalline, causes DHA content lifting obvious, so that most The DHA yield obtained eventually is also significantly raised accordingly.
Embodiment 2
(1) 50mL seed culture mediums are added in 250mL triangular flasks.The schizochytrium limacinum kind of cryopreservation is accessed, in culture Cultivated 2 days under the conditions of 25 DEG C of temperature, shaking speed 200rpm, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed Liquid；
Seed culture medium component (g/L)：Glucose 25.0, dusty yeast 8.0, peptone 5.0, corn steep liquor 2.0, MgSO4· 7H2O 0.6, KH2PO41.5, sea crystal 10.0.
(2) 50mL fermentation mediums are added in 250mL triangular flasks, schizochytrium limacinum is accessed with 8% inoculum concentration and fermented Culture medium, 26 DEG C incubated 3 days, rotating speed 200rpm；
Fermentation medium component (g/L)：1,2-PD 50.0, yeast extract 10.0, sodium glutamate 5.0, MgSO4· 7H2O 0.2, KH2PO42.0, (NH4)2SO43.0, NaNO32.0, Na2SO48.0；
CK(g/L)：Glucose 50.0, yeast extract 10.0, sodium glutamate 5.0, MgSO4·7H2O 0.2, KH2PO4 2.0, (NH4)2SO43.0, NaNO32.0, Na2SO48.0；
Every kind of different culture medium prepares 3 Duplicate Samples, and 121 DEG C, sterilize 25min, cools down standby；
(3) continuous culture terminates fermentation after 3 days, and zymotic fluid carries out 8000g and centrifuged 15 minutes, and is washed 2-3 times with single steam Afterwards, centrifuge again, collect wet thallus be placed in vacuum drying chamber, vacuum be 0.9Mpa, temperature be 80 DEG C under the conditions of, greatly About dry 10 hours, until thalline keeps constant weight, and weigh.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, weighs 1g broken wall bacterium powders in test tube, add 30-60 boiling ranges Petroleum ether carry out equal-volume extraction three times, petroleum ether 5mL is added every time, petroleum ether extraction liquid is collected, vacuum drying is placed in In case, in 80 DEG C, vacuum is under the conditions of 0.9Mpa, removes petroleum ether, so that the grease rich in docosahexaenoic acid is obtained, And carry out the analysis of the vapor detection after esterification.
Fermentation results are as follows.
The 1,2- propane diols group of table 4 and glucose group fermentation contrast
From result above, the application of fermenting carbon source 1,2-PD can effectively improve the content of DHA in grease, from And the DHA yield increase finally obtained is obvious.
Embodiment 3
(1) 50mL seed culture mediums are added in 250mL triangular flasks.The schizochytrium limacinum kind of cryopreservation is accessed, in culture Cultivated 2 days under the conditions of 25 DEG C of temperature, shaking speed 200rpm, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed Liquid；
Seed culture medium component (g/L)：Glucose 30.0, dusty yeast 4.0, beef extract 5.0, corn steep liquor 6.0, MgSO4· 7H2O 1.0, KH2PO40.5, sea crystal 20.0.
(2) 50mL fermentation mediums are added in 250mL triangular flasks, schizochytrium limacinum is accessed with 4% inoculum concentration and fermented Culture medium, 26 DEG C incubated 3 days, rotating speed 200rpm；
Fermentation medium component (g/L)：1,2-PD 60.0, yeast extract 15.0, sodium glutamate 10.0, MgSO4·7H2O 1.0, KH2PO41.5, (NH4)2SO45.0, NaNO33.0, Na2SO412.0；
After fermentation medium sterilizing, carry out adding external source regulatory factor sesamol after filtration sterilization, its addition is 0.5mM/L。
CK(g/L)：Glucose 60.0, yeast extract 15.0, sodium glutamate 10.0, MgSO4·7H2O 1.0, KH2PO4 1.5, (NH4)2SO45.0, NaNO33.0, Na2SO412.0；
Every kind of different culture medium prepares 3 Duplicate Samples, and 121 DEG C, sterilize 25min, cools down standby；
(3) continuous culture terminates fermentation after 3 days, and zymotic fluid carries out 8000g and centrifuged 15 minutes, and is washed 2-3 times with single steam Afterwards, centrifuge again, collect wet thallus be placed in vacuum drying chamber, vacuum be 0.9Mpa, temperature be 80 DEG C under the conditions of, greatly About dry 10 hours, until thalline keeps constant weight, and weigh.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, weighs 1g broken wall bacterium powders in test tube, add 30-60 boiling ranges Petroleum ether carry out equal-volume extraction three times, petroleum ether 5mL is added every time, petroleum ether extraction liquid is collected, vacuum drying is placed in In case, in 80 DEG C, vacuum is under the conditions of 0.9Mpa, removes petroleum ether, so that the grease rich in docosahexaenoic acid is obtained, And carry out the analysis of the vapor detection after esterification.
Fermentation results are as follows.
Endobacillary G6PDH activity changes contrast in the fermentation process of table 5
Endobacillary malate dehydrogenase (ME) activity change contrast in the fermentation process of table 6
Endobacillary NADPH changes of contents contrast in the fermentation process of table 7
1,2- propane diols+0.5mM/L sesamols the group of table 8 and glucose group fermentation contrast
The application of fermenting carbon source 1,2-PD and external source regulatory factor sesamol, effectively inhibits the phosphorus in schizochytrium limacinum Sour pentose pathway and tricarboxylic acids delivery system, causes NADPH contents to decline obvious, is not influenceing schizochytrium limacinum biomass and grease On the basis of content, the raising of DHA content can be obviously promoted, so that DHA content lifting is obvious.
Embodiment 4
(1) 50mL seed culture mediums are added in 250mL triangular flasks.The schizochytrium limacinum kind of cryopreservation is accessed, in culture Cultivated 2 days under the conditions of 25 DEG C of temperature, shaking speed 200rpm, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed Liquid；
Seed culture medium component (g/L)：Glucose 15.0, dusty yeast 6.0, beef extract 3.0, peptone 2.0, corn steep liquor 4.0, MgSO4·7H2O 0.2, KH2PO42.0, sea crystal 15.0.
(2) 50mL fermentation mediums are added in 250mL triangular flasks, schizochytrium limacinum is accessed with 10% inoculum concentration and fermented Culture medium, 26 DEG C incubated 3 days, rotating speed 200rpm；
Fermentation medium component (g/L)：1,2-PD 50.0, sodium acetate 10.0, yeast extract 6.0, MgSO4· 7H2O 0.6, KH2PO40.5, NaNO31.0, Na2SO45.0；
CK(g/L)：Glucose 60.0, yeast extract 6.0, MgSO4·7H2O 0.6, KH2PO40.5, NaNO31.0, Na2SO45.0；
Every kind of different culture medium prepares 3 Duplicate Samples, and 121 DEG C, sterilize 25min, cools down standby；
(3) continuous culture terminates fermentation after 3 days, and zymotic fluid carries out 8000g and centrifuged 15 minutes, and is washed 2-3 times with single steam Afterwards, centrifuge again, collect wet thallus be placed in vacuum drying chamber, vacuum be 0.9Mpa, temperature be 80 DEG C under the conditions of, greatly About dry 10 hours, until thalline keeps constant weight, and weigh.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, weighs 1g broken wall bacterium powders in test tube, add 30-60 boiling ranges Petroleum ether carry out equal-volume extraction three times, petroleum ether 5mL is added every time, petroleum ether extraction liquid is collected, vacuum drying is placed in In case, in 80 DEG C, vacuum is under the conditions of 0.9Mpa, removes petroleum ether, so that the grease rich in docosahexaenoic acid is obtained, And carry out the analysis of the vapor detection after esterification.
Fermentation results are as follows.
The 1,2- propane diols of table 9+sodium acetate group and glucose group fermentation contrast
The application of fermenting carbon source 1,2-PD and sodium acetate, has effectively facilitated the accumulation of DHA in grease, has caused DHA to contain Amount lifting is obvious, and DHA yield is also significantly raised accordingly.
Embodiment 5
(1) 50mL seed culture mediums are added in 250mL triangular flasks.The schizochytrium limacinum kind of cryopreservation is accessed, in culture Cultivated 2 days under the conditions of 25 DEG C of temperature, shaking speed 200rpm, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed Liquid；
Seed culture medium component (g/L)：Glucose 15.0, dusty yeast 6.0, beef extract 3.0, peptone 2.0, corn steep liquor 4.0, MgSO4·7H2O 0.2, KH2PO42.0, sea crystal 15.0.
(2) 50mL fermentation mediums are added in 250mL triangular flasks, schizochytrium limacinum is accessed with 10% inoculum concentration and fermented Culture medium, 26 DEG C incubated 3 days, rotating speed 200rpm；
Fermentation medium component (g/L)：1,2-PD 55.0, sodium acetate 5.0, yeast extract 6.0, MgSO4·7H2O 0.6, KH2PO40.5, NaNO31.0, Na2SO45.0；
After fermentation medium sterilizing, carry out adding external source regulatory factor sesamol after filtration sterilization, its addition is 1.0mM/L。
CK(g/L)：Glucose 60.0, yeast extract 6.0, MgSO4·7H2O 0.6, KH2PO40.5, NaNO31.0, Na2SO45.0；
Every kind of different culture medium prepares 3 Duplicate Samples, and 121 DEG C, sterilize 25min, cools down standby；
(3) continuous culture terminates fermentation after 3 days, and zymotic fluid carries out 8000g and centrifuged 15 minutes, and is washed 2-3 times with single steam Afterwards, centrifuge again, collect wet thallus be placed in vacuum drying chamber, vacuum be 0.9Mpa, temperature be 80 DEG C under the conditions of, greatly About dry 10 hours, until thalline keeps constant weight, and weigh.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, weighs 1g broken wall bacterium powders in test tube, add 30-60 boiling ranges Petroleum ether carry out equal-volume extraction three times, petroleum ether 5mL is added every time, petroleum ether extraction liquid is collected, vacuum drying is placed in In case, in 80 DEG C, vacuum is under the conditions of 0.9Mpa, removes petroleum ether, so that the grease rich in docosahexaenoic acid is obtained, And carry out the analysis of the vapor detection after esterification.
Fermentation results are as follows.
The 1,2- propane diols of table 10+sodium acetate+1.0mM/L sesamol addition groups and glucose group fermentation contrast
As a result show, fermenting carbon source 1,2-PD and sodium acetate, and sesamol application, hence it is evident that improve containing for DHA Amount, the DHA yield finally obtained also increases substantially.
Embodiment 6
(1) 50mL seed culture mediums are added in 250mL triangular flasks.The schizochytrium limacinum kind of cryopreservation is accessed, in culture Cultivated 2 days under the conditions of 25 DEG C of temperature, shaking speed 200rpm, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed Liquid；
Seed culture medium component (g/L)：Glucose 25.0, dusty yeast 8.0, peptone 5.0, corn steep liquor 2.0, MgSO4· 7H2O 0.6, KH2PO41.5, sea crystal 10.0.
(2) 50mL fermentation mediums are added in 250mL triangular flasks, schizochytrium limacinum is accessed with 8% inoculum concentration and fermented Culture medium, 26 DEG C incubated 3 days, rotating speed 200rpm；
Fermentation medium component (g/L)：1,2-PD 50.0, ethanol 10.0, yeast extract 10.0, sodium glutamate 5.0, MgSO4·7H2O 0.2, KH2PO42.0, (NH4)2SO43.0, NaNO32.0, Na2SO48.0；
CK(g/L)：Glucose 60.0, yeast extract 10.0, sodium glutamate 5.0, MgSO4·7H2O 0.2, KH2PO4 2.0, (NH4)2SO43.0, NaNO32.0, Na2SO48.0；
Every kind of different culture medium prepares 3 Duplicate Samples, and 121 DEG C, sterilize 25min, cools down standby；
(3) continuous culture terminates fermentation after 3 days, and zymotic fluid carries out 8000g and centrifuged 15 minutes, and is washed 2-3 times with single steam Afterwards, centrifuge again, collect wet thallus be placed in vacuum drying chamber, vacuum be 0.9Mpa, temperature be 80 DEG C under the conditions of, greatly About dry 10 hours, until thalline keeps constant weight, and weigh.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, weighs 1g broken wall bacterium powders in test tube, add 30-60 boiling ranges Petroleum ether carry out equal-volume extraction three times, petroleum ether 5mL is added every time, petroleum ether extraction liquid is collected, vacuum drying is placed in In case, in 80 DEG C, vacuum is under the conditions of 0.9Mpa, removes petroleum ether, so that the grease rich in docosahexaenoic acid is obtained, And carry out the analysis of the vapor detection after esterification.
Fermentation results are as follows.
The 1,2- propane diols of table 11+ethanol group and glucose group fermentation contrast
From result above, the application of fermenting carbon source 1,2-PD and ethanol can effectively improve containing for DHA in grease Amount, the DHA yield increase that final fermentation is obtained is obvious.
Embodiment 7
(1) 50mL seed culture mediums are added in 250mL triangular flasks.The schizochytrium limacinum kind of cryopreservation is accessed, in culture Cultivated 2 days under the conditions of 25 DEG C of temperature, shaking speed 200rpm, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed Liquid；
Seed culture medium component (g/L)：Glucose 25.0, dusty yeast 8.0, peptone 5.0, corn steep liquor 2.0, MgSO4· 7H2O 0.6, KH2PO41.5, sea crystal 10.0.
(2) 50mL fermentation mediums are added in 250mL triangular flasks, schizochytrium limacinum is accessed with 8% inoculum concentration and fermented Culture medium, 26 DEG C incubated 3 days, rotating speed 200rpm；
Fermentation medium component (g/L)：1,2-PD 55.0, ethanol 5.0, yeast extract 10.0, sodium glutamate 5.0, MgSO4·7H2O 0.2, KH2PO42.0, (NH4)2SO43.0, NaNO32.0, Na2SO48.0；
After fermentation medium sterilizing, carry out adding external source regulatory factor sesamol after filtration sterilization, its addition is 2.0mM/L。
CK(g/L)：Glucose 60.0, yeast extract 10.0, sodium glutamate 5.0, MgSO4·7H2O 0.2, KH2PO4 2.0, (NH4)2SO43.0, NaNO32.0, Na2SO48.0；
Every kind of different culture medium prepares 3 Duplicate Samples, and 121 DEG C, sterilize 25min, cools down standby；
(3) continuous culture terminates fermentation after 3 days, and zymotic fluid carries out 8000g and centrifuged 15 minutes, and is washed 2-3 times with single steam Afterwards, centrifuge again, collect wet thallus be placed in vacuum drying chamber, vacuum be 0.9Mpa, temperature be 80 DEG C under the conditions of, greatly About dry 10 hours, until thalline keeps constant weight, and weigh.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, weighs 1g broken wall bacterium powders in test tube, add 30-60 boiling ranges Petroleum ether carry out equal-volume extraction three times, petroleum ether 5mL is added every time, petroleum ether extraction liquid is collected, vacuum drying is placed in In case, in 80 DEG C, vacuum is under the conditions of 0.9Mpa, removes petroleum ether, so that the grease rich in docosahexaenoic acid is obtained, And carry out the analysis of the vapor detection after esterification.
Fermentation results are as follows.
The 1,2- propane diols of table 12+ethanol+2.0mM/L sesamols group and glucose group fermentation contrast
From result above, fermenting carbon source 1,2-PD and ethanol, and regulatory factor sesamol application, can be effective The content of DHA in grease is improved, the DHA yield finally obtained is increased obvious.
Embodiment 8
(1) 50mL seed culture mediums are added in 250mL triangular flasks.The schizochytrium limacinum kind of cryopreservation is accessed, in culture Cultivated 2 days under the conditions of 25 DEG C of temperature, shaking speed 200rpm, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed Liquid；
Seed culture medium component (g/L)：Glucose 30.0, dusty yeast 4.0, beef extract 5.0, corn steep liquor 6.0, MgSO4· 7H2O 1.0, KH2PO40.5, sea crystal 20.0.
(2) 50mL fermentation mediums are added in 250mL triangular flasks, schizochytrium limacinum is accessed with 4% inoculum concentration and fermented Culture medium, 26 DEG C incubated 3 days, rotating speed 200rpm；
Fermentation medium component (g/L)：1,2-PD 40.0, sodium acetate 10.0, ethanol 10.0, yeast extract 15.0, sodium glutamate 10.0, MgSO4·7H2O 1.0, KH2PO41.5, (NH4)2SO45.0, NaNO33.0, Na2SO4 12.0；
After fermentation medium sterilizing, carry out adding external source regulatory factor sesamol after filtration sterilization, its addition is 3.0mM/L。
CK(g/L)：Glucose 60.0, yeast extract 15.0, sodium glutamate 10.0, MgSO4·7H2O 1.0, KH2PO4 1.5, (NH4)2SO45.0, NaNO33.0, Na2SO412.0；
Every kind of different culture medium prepares 3 Duplicate Samples, and 121 DEG C, sterilize 25min, cools down standby；
(3) continuous culture terminates fermentation after 3 days, and zymotic fluid carries out 8000g and centrifuged 15 minutes, and is washed 2-3 times with single steam Afterwards, centrifuge again, collect wet thallus be placed in vacuum drying chamber, vacuum be 0.9Mpa, temperature be 80 DEG C under the conditions of, greatly About dry 10 hours, until thalline keeps constant weight, and weigh.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, weighs 1g broken wall bacterium powders in test tube, add 30-60 boiling ranges Petroleum ether carry out equal-volume extraction three times, petroleum ether 5mL is added every time, petroleum ether extraction liquid is collected, vacuum drying is placed in In case, in 80 DEG C, vacuum is under the conditions of 0.9Mpa, removes petroleum ether, so that the grease rich in docosahexaenoic acid is obtained, And carry out the analysis of the vapor detection after esterification.
Fermentation results are as follows.
The 1,2- propane diols of table 13+sodium acetate+ethanol+3.0mM/L sesamols group and glucose group fermentation contrast
The application of fermenting carbon source 1,2-PD, sodium acetate and ethanol, and external source regulatory factor sesamol addition, effectively The pentose phosphate pathway and tricarboxylic acids delivery system in schizochytrium limacinum are inhibited, DHA content increase in schizochytrium limacinum grease is caused Substantially, so that the DHA yield lifting for acquisition of fermenting is obvious.
The inventive method is applied to various schizochytrium limacinums, such as schizochytrium limacinum (Schizochytrium sp) S056, in 2013 , on September be preserved in China typical culture collection center CCTCC for 29, and deposit number is CCTCC M 2013459, etc..
It is described above be presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment Content.So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention is protected is both fallen within Enclose.

Claims (12)

1. in a kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease, this method selection fermentation medium Fermenting carbon source reduces endobacillary pentose phosphate pathway metabolic flux, the NADPH contents in schizochytrium limacinum body is declined to a great extent, and promotees Enter DHA a large amount of synthesis；The fermenting carbon source is 1,2-PD, 1,2-PD and ethanol, 1,2-PD and acetate, Or any one in the combination of 1,2- propane diols, ethanol and acetate；
The schizochytrium limacinum is Schizochytrium sp S056.
2. the method for docosahexaenoic acid content in schizochytrium limacinum grease is improved as claimed in claim 1, it is characterised in that This method adds external source regulatory factor in the fermentation medium, reduces endobacillary pentose phosphate pathway metabolic flux, simultaneously Activity to the malate dehydrogenase in tricarboxylic acids delivery system suppresses, and NADPH contents in schizochytrium limacinum body is declined to a great extent, and promotees Enter DHA a large amount of synthesis.
3. a kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease as claimed in claim 2, its feature It is, external source regulatory factor is sesamol, after fermentation medium sterilizing, add after filtration sterilization.
4. a kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease as claimed in claim 3, its feature It is, the addition of sesamol is 0.5-3.0mM/L.
5. the side of docosahexaenoic acid content in a kind of raising schizochytrium limacinum grease as described in any in Claims 1-4 Method, it is characterised in that when fermenting carbon source is 1,2-PD and acetate, its proportioning is：1,2- propane diols 40g/L-60g/ L；Sodium acetate 0-10g/L.
6. the side of docosahexaenoic acid content in a kind of raising schizochytrium limacinum grease as described in any in Claims 1-4 Method, it is characterised in that when fermenting carbon source is 1,2-PD and ethanol, its proportioning is：1,2- propane diols 40-60g/L；Ethanol 0-10g/L。
7. the side of docosahexaenoic acid content in a kind of raising schizochytrium limacinum grease as described in any in Claims 1-4 Method, it is characterised in that when fermenting carbon source is 1,2-PD, ethanol and acetate, its proportioning is：1,2- propane diols 40- 60g/L；Ethanol 0-10g/L；Acetate 0-10g/L.
8. a kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease as claimed in claim 1, its feature It is, the step that implements of this method is：
(1) by the schizochytrium limacinum kind activation culture of cryopreservation into schizochytrium limacinum seed liquor；
(2) schizochytrium limacinum seed liquor is cultivated according to percent by volume 4%-10% access fermentation mediums；
(3) terminate after fermentation, collect wet thallus and be dried in vacuo, until thalline keeps constant weight, and weigh；
(4) broken wall is carried out to dry thalline, the grease rich in docosahexaenoic acid is then obtained by extraction.
9. a kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease as claimed in claim 8, its feature It is, external source regulatory factor is also added with the fermentation medium in step (2), is after being sterilized to the fermentation medium, to enter External source regulatory factor is added after row filtration sterilization again.
10. a kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease as claimed in claim 8 or 9, its It is characterised by, when containing ethanol in the fermenting carbon source, ethanol is added after filtration sterilization in the fermentation medium after having sterilized；Institute State acetate and select sodium acetate.
11. a kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease as claimed in claim 8 or 9, its It is characterised by, the schizochytrium limacinum seed liquor component in the step (1) is calculated as by g/L：Glucose 15.0-30.0, dusty yeast 4.0-8.0, beef extract 0-5.0 peptone 0-5.0, corn steep liquor 2.0-6.0, MgSO4·7H2O 0.2-1.0, KH2PO4 0.5- 2.0, sea crystal 10.0-20.0.
12. a kind of method for improving docosahexaenoic acid content in schizochytrium limacinum grease as claimed in claim 8 or 9, its It is characterised by, the fermentation medium component in the step (2) is calculated as by g/L：Fermenting carbon source 40.0-60.0, yeast extract 6.0-15.0, sodium glutamate 0-10.0, MgSO4·7H2O 0.2-1.0, KH2PO40.5-2.0, (NH4)2SO40-5.0, NaNO3 1.0-3.0, Na2SO4 5.0-12.0。
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