CN104480152A - Method for increasing content of docosahexaenoic acid in schizochytrium limacinum grease - Google Patents
Method for increasing content of docosahexaenoic acid in schizochytrium limacinum grease Download PDFInfo
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- CN104480152A CN104480152A CN201410667988.7A CN201410667988A CN104480152A CN 104480152 A CN104480152 A CN 104480152A CN 201410667988 A CN201410667988 A CN 201410667988A CN 104480152 A CN104480152 A CN 104480152A
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- 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
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Abstract
The invention relates to a method for increasing content of docosahexaenoic acid (DHA) in schizochytrium limacinum grease. The method is characterized by comprising the following steps: lowering metabolic flux of a pentose phosphate pathway in thallus by selecting a fermentable carbon source, and lowering the metabolic flux of the pentose phosphate pathway in thallus by further adding an exogenous regulation factor; inhibiting the activity of malic enzyme in a tricarboxylic acid transfer system to greatly lower the content of nicotinamide adenine dinucleotide phosphate (NADPH) in the schizochytrium limacinum thaluus, promote the large-scale synthesis of DHA and enable the content of DHA in the grease of the obtained thallus to be as high as 55.3%, wherein the fermentation carbon source is 1,2-propylene glycol or any one or a mixture of 1,2-propylene glycol and acetate, the exogenous regulation factor is preferably sesamol which is added after filtering and degerming when a fermentation culture medium is sterilized, and the addition amount of the sesamol is 0.5-3.0mM/L. The method disclosed by the invention is simple and easy to perform, obvious in effect, capable of greatly increasing the content of DHA in the thallus grease and easy for industrial application.
Description
Technical field
The present invention relates to field of microbial fermentation, be specifically related to a kind of method improving docosahexenoic acid (DHA) content in schizochytrium limacinum grease.
Background technology
Docosahexenoic acid (DHA) belongs to polyunsaturated fatty acid, has very important physiological function, is that human body self can not synthesize but indispensable important nutrient in a large number.DHA is present in human body retina and corticocerebral nervous tissue in a large number, has and promotes brain and visual acuity, improves intelligence, the effect of Improving memory power and eyesight; In addition, it can also stop cholesterol to deposit on vessel wall, prevents or alleviate the generation of atherosclerosis and coronary heart disease.
Schizochytrium limacinum (Schizochytrium) is a kind of resource of excellent production polyunsaturated fatty acid grease.At present, utilize schizochytrium limacinum large scale fermentation production polyunsaturated fatty acid grease to obtain the concern of a lot of scientific research institutions and unit, and its production process is conducted in-depth research.Application number be 201010000055.4 patent document discloses a kind of thalassiomycetes schizochytrium limacinum, wherein docosahexenoic acid triglyceride level accounts for the 35-40% of grease total amount; Application number be 201310571413.0 patent discloses the method utilizing stalk hydrolyzed solution fermentative production docosahexenoic acid, the final DHA obtained can account for the 42.1-45.3% of grease gross weight; Application number is the method that patent discloses exterior addition factor promotion Microbe synthesis docosahexenoic acid of 201010504635.7, the method is pointed out, when adding any one or a few combination in acetic acid, citric acid and Simvastatin in the microbiological culture media that can produce DHA, DHA content in microbe can effectively be improved, and the highlyest brings up to 45.00% from initial 35.51%.
Aforesaid method has a certain upgrade to the Technology tool that schizochytrium limacinum fermentation produces DHA, but not yet there is DHA synthesis path specific in combine with technique schizochytrium limacinum body at present to regulate and control its metabolic pathway, to realizing the further lifting of DHA content in schizochytrium limacinum institute Lipid-producing; And, promote the increase of DHA content further, effectively can promote the quality of schizochytrium limacinum institute Lipid-producing, also indirectly reduce fermentation costs, add production efficiency.
Summary of the invention
For the existing deficiency utilizing schizochytrium limacinum fermentation to produce DHA grease technology, the invention provides a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease, according to the feature of DHA synthesis path specific in schizochytrium limacinum body, in schizochytrium limacinum fermentation process, endobacillary phosphopentose pathway metabolic flux is reduced by selecting fermenting carbon source, to improve the content of DHA in schizochytrium limacinum institute Lipid-producing, promote the quality of DHA grease, simultaneously, enhance productivity, and reduce production cost.
To achieve these goals, the invention provides a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease, the method selects the fermenting carbon source in fermention medium to reduce endobacillary phosphopentose pathway metabolic flux, Triphosphopyridine nucleotide, reduced (NADPH) content in schizochytrium limacinum body is declined to a great extent, promotes a large amount of synthesis of DHA; Described 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-PD, ethanol and acetate.
As the improvement of technique scheme, the method adds external source regulatory factor in the fermentation medium, endobacillary phosphopentose pathway metabolic flux is reduced, the activity of the malic enzyme in tricarboxylic acid delivery system is suppressed simultaneously, NADPH content in schizochytrium limacinum body is declined to a great extent, promotes a large amount of synthesis of DHA.Wherein, external source regulatory factor is preferably sesamol, after fermention medium sterilizing, adds after carrying out filtration sterilization; The addition of sesamol can be 0.5-3.0mM/L.
The invention has the advantages that by selecting fermenting carbon source, phosphopentose pathway metabolic flux in thalline is declined, and select fermenting carbon source further simultaneously and add external source regulatory factor, endobacillary phosphopentose pathway metabolic flux is reduced, the activity of the malic enzyme (ME) in tricarboxylic acid delivery system is suppressed simultaneously, reducing power NADPH content in schizochytrium limacinum body is declined to a great extent, carbon metabolism flow is caused to turn to polyketide synthase path to synthesize DHA, thus promote a large amount of synthesis of DHA, significantly improve the content of DHA in schizochytrium limacinum grease, significantly improve the quality of DHA grease.
Embodiment
The present invention is by selecting fermenting carbon source, and add the modes such as external source regulatory factor further, endobacillary phosphopentose pathway metabolic flux is reduced, the activity of the malic enzyme in tricarboxylic acid delivery system is suppressed simultaneously, NADPH content in schizochytrium limacinum body is declined to a great extent, thus impels fatty acid precursor material-acetyl-CoA to transform to polyketide synthase approach.
The specific implementation step of the inventive method is:
(1) the schizochytrium limacinum kind activation culture of cryopreservation is become schizochytrium limacinum seed liquor;
The various components that the seed culture medium that activation culture adopts can adopt prior art to provide, its preferred component (g/L) is: glucose 15.0-30.0, yeast powder 4.0-8.0, extractum carnis 0-5.0 peptone 0-5.0, corn steep liquor 2.0-6.0, MgSO
47H
2o 0.2-1.0, KH
2pO
40.5-2.0, sea crystal 10.0-20.0.
(2) schizochytrium limacinum seed liquor is accessed fermention medium according to volume percent 4%-10% (preferably 8%) to cultivate;
The various components that fermention medium can adopt prior art to provide, just the glucose wherein as fermenting carbon source is replaced with 1,2-propylene glycol, or be 1, the mixing solutions of 2-propylene glycol and ethanol and/or acetate, ethanol need degerming after filtration after add in the fermention medium after sterilizing, wherein, the preferred sodium acetate of acetate.
The preferred component of fermention medium (g/L) is: fermenting carbon source 40.0-60.0, yeast extract 6.0-15.0, Sodium Glutamate 0-10.0, MgSO
47H
2o 0.2-1.0, KH
2pO
40.5-2.0, (NH
4)
2sO
40-5.0, NaNO
31.0-3.0, Na
2sO
45.0-12.0.
When fermenting carbon source is many kinds of substance mixing, its component (g/L) is:
1,2-PD and acetate: 1,2-PD 40-60; Sodium acetate 0-10;
1,2-PD and ethanol: 1,2-PD 40-60; Ethanol 0-10;
1,2-PD, ethanol and acetate: 1,2-PD 40-60; Ethanol 0-10; Acetate 0-10.
For improving technique effect of the present invention further, after fermention medium sterilizing, external source regulatory factor after carrying out filtration sterilization, can be added.
External source regulatory factor can preferred sesamol, and its addition is 0.5-3.0mM/L.
(3) after terminating fermentation, collect wet thallus and vacuum-drying, until thalline keeps constant weight, and weigh.
(4) broken wall is carried out to the thalline of drying, add organic solvent (as sherwood oil and normal hexane etc.) and extract, thus obtain the grease being rich in docosahexenoic acid, carry out the vapor detection analysis after esterification, the content of DHA can be obtained.
Step (3), (4) all can adopt prior art, do not repeat them here.
Be clearly and completely described to the technical scheme in the embodiment of the present invention below.Obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
(1) in 250mL triangular flask, 50mL seed culture medium is added.The schizochytrium limacinum kind of access cryopreservation, culture temperature 25 DEG C, cultivates 2 days under shaking speed 200rpm condition, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed liquor;
Seed culture medium component (g/L): glucose 15.0, yeast powder 6.0, extractum carnis 3.0, peptone 2.0, corn steep liquor 4.0, MgSO
47H
2o 0.2, KH
2pO
42.0, sea crystal 15.0.
(2) in 250mL triangular flask, add 50mL fermention medium, by schizochytrium limacinum with 10% inoculum size access fermention medium, 26 DEG C of constant temperature culture 3 days, rotating speed 200rpm;
Fermention medium component (g/L): 1,2-PD 40.0, yeast extract 6.0, MgSO
47H
2o0.6, KH
2pO
40.5, NaNO
31.0, Na
2sO
45.0;
CK (g/L): glucose 40.0, yeast extract 6.0, MgSO
47H
2o 0.6, KH
2pO
40.5, NaNO
31.0, Na
2sO
45.0;
Often kind of different substratum prepares 3 Duplicate Samples, 121 DEG C, sterilizing 25min, cools for subsequent use;
(3) cultured continuously terminates fermentation after 3 days, fermented liquid carries out 8000g centrifugal 15 minutes, and with after single steaming washing 2-3 time, recentrifuge, collecting wet thallus and be placed in vacuum drying oven, is 0.9Mpa in vacuum tightness, temperature is under 80 DEG C of conditions, about baking 10 hours, until thalline keeps constant weight, and weighs.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, take 1g broken wall bacterium powder in test tube, the sherwood oil adding 30-60 boiling range carries out equal-volume and extracts three times, adds sherwood oil 5mL at every turn, collects petroleum ether extraction liquid, and be placed in vacuum drying oven, in 80 DEG C, vacuum tightness is under 0.9Mpa condition, removing sherwood oil, thus obtain the grease being rich in docosahexenoic acid, and carry out the vapor detection analysis after esterification.
Fermentation results is as follows:
Endobacillary glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) activity change contrast in table 1 fermenting process
Endobacillary NADPH content contrast in table 2 fermenting process
Table 3 1,2-PD group and glucose group fermentation contrast
The application of fermenting carbon source 1,2-PD, causes the schizochytrium limacinum active obviously decline of G6PDH during the fermentation, phosphopentose pathway metabolic flux declines obviously, in thalline, NADPH content declines obviously, causes DHA content to promote obviously, thus the final DHA output obtained also significantly raises accordingly.
Embodiment 2
(1) in 250mL triangular flask, 50mL seed culture medium is added.The schizochytrium limacinum kind of access cryopreservation, culture temperature 25 DEG C, cultivates 2 days under shaking speed 200rpm condition, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed liquor;
Seed culture medium component (g/L): glucose 25.0, yeast powder 8.0, peptone 5.0, corn steep liquor 2.0, MgSO
47H
2o 0.6, KH
2pO
41.5, sea crystal 10.0.
(2) in 250mL triangular flask, add 50mL fermention medium, by schizochytrium limacinum with 8% inoculum size access fermention medium, 26 DEG C of constant temperature culture 3 days, rotating speed 200rpm;
Fermention medium component (g/L): 1,2-PD 50.0, yeast extract 10.0, Sodium Glutamate 5.0, MgSO
47H
2o 0.2, KH
2pO
42.0, (NH
4)
2sO
43.0, NaNO
32.0, Na
2sO
48.0;
CK (g/L): glucose 50.0, yeast extract 10.0, Sodium Glutamate 5.0, MgSO
47H
2o0.2, KH
2pO
42.0, (NH
4)
2sO
43.0, NaNO
32.0, Na
2sO
48.0;
Often kind of different substratum prepares 3 Duplicate Samples, 121 DEG C, sterilizing 25min, cools for subsequent use;
(3) cultured continuously terminates fermentation after 3 days, fermented liquid carries out 8000g centrifugal 15 minutes, and with after single steaming washing 2-3 time, recentrifuge, collecting wet thallus and be placed in vacuum drying oven, is 0.9Mpa in vacuum tightness, temperature is under 80 DEG C of conditions, about baking 10 hours, until thalline keeps constant weight, and weighs.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, take 1g broken wall bacterium powder in test tube, the sherwood oil adding 30-60 boiling range carries out equal-volume and extracts three times, adds sherwood oil 5mL at every turn, collects petroleum ether extraction liquid, and be placed in vacuum drying oven, in 80 DEG C, vacuum tightness is under 0.9Mpa condition, removing sherwood oil, thus obtain the grease being rich in docosahexenoic acid, and carry out the vapor detection analysis after esterification.
Fermentation results is as follows.
Table 4 1,2-PD group and glucose group fermentation contrast
From above result, the application of fermenting carbon source 1,2-PD, effectively can improve the content of DHA in grease, thus the final DHA output obtained increases obviously.
Embodiment 3
(1) in 250mL triangular flask, 50mL seed culture medium is added.The schizochytrium limacinum kind of access cryopreservation, culture temperature 25 DEG C, cultivates 2 days under shaking speed 200rpm condition, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed liquor;
Seed culture medium component (g/L): glucose 30.0, yeast powder 4.0, extractum carnis 5.0, corn steep liquor 6.0, MgSO
47H
2o 1.0, KH
2pO
40.5, sea crystal 20.0.
(2) in 250mL triangular flask, add 50mL fermention medium, by schizochytrium limacinum with 4% inoculum size access fermention medium, 26 DEG C of constant temperature culture 3 days, rotating speed 200rpm;
Fermention medium component (g/L): 1,2-PD 60.0, yeast extract 15.0, Sodium Glutamate 10.0, MgSO
47H
2o 1.0, KH
2pO
41.5, (NH
4)
2sO
45.0, NaNO
33.0, Na
2sO
412.0;
After fermention medium sterilizing, add external source regulatory factor sesamol after carrying out filtration sterilization, its addition is 0.5mM/L.
CK (g/L): glucose 60.0, yeast extract 15.0, Sodium Glutamate 10.0, MgSO
47H
2o1.0, KH
2pO
41.5, (NH
4)
2sO
45.0, NaNO
33.0, Na
2sO
412.0;
Often kind of different substratum prepares 3 Duplicate Samples, 121 DEG C, sterilizing 25min, cools for subsequent use;
(3) cultured continuously terminates fermentation after 3 days, fermented liquid carries out 8000g centrifugal 15 minutes, and with after single steaming washing 2-3 time, recentrifuge, collecting wet thallus and be placed in vacuum drying oven, is 0.9Mpa in vacuum tightness, temperature is under 80 DEG C of conditions, about baking 10 hours, until thalline keeps constant weight, and weighs.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, take 1g broken wall bacterium powder in test tube, the sherwood oil adding 30-60 boiling range carries out equal-volume and extracts three times, adds sherwood oil 5mL at every turn, collects petroleum ether extraction liquid, and be placed in vacuum drying oven, in 80 DEG C, vacuum tightness is under 0.9Mpa condition, removing sherwood oil, thus obtain the grease being rich in docosahexenoic acid, and carry out the vapor detection analysis after esterification.
Fermentation results is as follows.
Endobacillary G6PDH activity change contrast in table 5 fermenting process
Endobacillary malic enzyme (ME) activity change contrast in table 6 fermenting process
Endobacillary NADPH content contrast in table 7 fermenting process
Table 8 1,2-PD+0.5mM/L sesamol group and glucose group fermentation contrast
Fermenting carbon source 1, the application of 2-propylene glycol and external source regulatory factor sesamol, effectively inhibit the phosphopentose pathway in schizochytrium limacinum and tricarboxylic acid delivery system, NADPH content is caused to decline obviously, on the basis not affecting schizochytrium limacinum biomass and fat content, obviously can promote the raising of DHA content, thus DHA content promotes obviously.
Embodiment 4
(1) in 250mL triangular flask, 50mL seed culture medium is added.The schizochytrium limacinum kind of access cryopreservation, culture temperature 25 DEG C, cultivates 2 days under shaking speed 200rpm condition, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed liquor;
Seed culture medium component (g/L): glucose 15.0, yeast powder 6.0, extractum carnis 3.0, peptone 2.0, corn steep liquor 4.0, MgSO
47H
2o 0.2, KH
2pO
42.0, sea crystal 15.0.
(2) in 250mL triangular flask, add 50mL fermention medium, by schizochytrium limacinum with 10% inoculum size access fermention medium, 26 DEG C of constant temperature culture 3 days, rotating speed 200rpm;
Fermention medium component (g/L): 1,2-PD 50.0, sodium acetate 10.0, yeast extract 6.0, MgSO
47H
2o 0.6, KH
2pO
40.5, NaNO
31.0, Na
2sO
45.0;
CK (g/L): glucose 60.0, yeast extract 6.0, MgSO
47H
2o 0.6, KH
2pO
40.5, NaNO
31.0, Na
2sO
45.0;
Often kind of different substratum prepares 3 Duplicate Samples, 121 DEG C, sterilizing 25min, cools for subsequent use;
(3) cultured continuously terminates fermentation after 3 days, fermented liquid carries out 8000g centrifugal 15 minutes, and with after single steaming washing 2-3 time, recentrifuge, collecting wet thallus and be placed in vacuum drying oven, is 0.9Mpa in vacuum tightness, temperature is under 80 DEG C of conditions, about baking 10 hours, until thalline keeps constant weight, and weighs.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, take 1g broken wall bacterium powder in test tube, the sherwood oil adding 30-60 boiling range carries out equal-volume and extracts three times, adds sherwood oil 5mL at every turn, collects petroleum ether extraction liquid, and be placed in vacuum drying oven, in 80 DEG C, vacuum tightness is under 0.9Mpa condition, removing sherwood oil, thus obtain the grease being rich in docosahexenoic acid, and carry out the vapor detection analysis after esterification.
Fermentation results is as follows.
Table 9 1,2-PD+sodium acetate group and glucose group fermentation contrast
The application of fermenting carbon source 1,2-PD and sodium acetate, effectively facilitates the accumulation of DHA in grease, and cause DHA content to promote obviously, DHA output also significantly raises accordingly.
Embodiment 5
(1) in 250mL triangular flask, 50mL seed culture medium is added.The schizochytrium limacinum kind of access cryopreservation, culture temperature 25 DEG C, cultivates 2 days under shaking speed 200rpm condition, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed liquor;
Seed culture medium component (g/L): glucose 15.0, yeast powder 6.0, extractum carnis 3.0, peptone 2.0, corn steep liquor 4.0, MgSO
47H
2o 0.2, KH
2pO
42.0, sea crystal 15.0.
(2) in 250mL triangular flask, add 50mL fermention medium, by schizochytrium limacinum with 10% inoculum size access fermention medium, 26 DEG C of constant temperature culture 3 days, rotating speed 200rpm;
Fermention medium component (g/L): 1,2-PD 55.0, sodium acetate 5.0, yeast extract 6.0, MgSO
47H
2o 0.6, KH
2pO
40.5, NaNO
31.0, Na
2sO
45.0;
After fermention medium sterilizing, add external source regulatory factor sesamol after carrying out filtration sterilization, its addition is 1.0mM/L.
CK (g/L): glucose 60.0, yeast extract 6.0, MgSO
47H
2o 0.6, KH
2pO
40.5, NaNO
31.0, Na
2sO
45.0;
Often kind of different substratum prepares 3 Duplicate Samples, 121 DEG C, sterilizing 25min, cools for subsequent use;
(3) cultured continuously terminates fermentation after 3 days, fermented liquid carries out 8000g centrifugal 15 minutes, and with after single steaming washing 2-3 time, recentrifuge, collecting wet thallus and be placed in vacuum drying oven, is 0.9Mpa in vacuum tightness, temperature is under 80 DEG C of conditions, about baking 10 hours, until thalline keeps constant weight, and weighs.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, take 1g broken wall bacterium powder in test tube, the sherwood oil adding 30-60 boiling range carries out equal-volume and extracts three times, adds sherwood oil 5mL at every turn, collects petroleum ether extraction liquid, and be placed in vacuum drying oven, in 80 DEG C, vacuum tightness is under 0.9Mpa condition, removing sherwood oil, thus obtain the grease being rich in docosahexenoic acid, and carry out the vapor detection analysis after esterification.
Fermentation results is as follows.
Table 10 1,2-PD+sodium acetate+1.0mM/L sesamol interpolation group and glucose group fermentation contrast
Result shows, fermenting carbon source 1,2-PD and sodium acetate, and the application of sesamol, significantly improves the content of DHA, and the final DHA output obtained also increases obviously.
Embodiment 6
(1) in 250mL triangular flask, 50mL seed culture medium is added.The schizochytrium limacinum kind of access cryopreservation, culture temperature 25 DEG C, cultivates 2 days under shaking speed 200rpm condition, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed liquor;
Seed culture medium component (g/L): glucose 25.0, yeast powder 8.0, peptone 5.0, corn steep liquor 2.0, MgSO
47H
2o 0.6, KH
2pO
41.5, sea crystal 10.0.
(2) in 250mL triangular flask, add 50mL fermention medium, by schizochytrium limacinum with 8% inoculum size access fermention medium, 26 DEG C of constant temperature culture 3 days, rotating speed 200rpm;
Fermention medium component (g/L): 1,2-PD 50.0, ethanol 10.0, yeast extract 10.0, Sodium Glutamate 5.0, MgSO
47H
2o 0.2, KH
2pO
42.0, (NH
4)
2sO
43.0, NaNO
32.0, Na
2sO
48.0;
CK (g/L): glucose 60.0, yeast extract 10.0, Sodium Glutamate 5.0, MgSO
47H
2o0.2, KH
2pO
42.0, (NH
4)
2sO
43.0, NaNO
32.0, Na
2sO
48.0;
Often kind of different substratum prepares 3 Duplicate Samples, 121 DEG C, sterilizing 25min, cools for subsequent use;
(3) cultured continuously terminates fermentation after 3 days, fermented liquid carries out 8000g centrifugal 15 minutes, and with after single steaming washing 2-3 time, recentrifuge, collecting wet thallus and be placed in vacuum drying oven, is 0.9Mpa in vacuum tightness, temperature is under 80 DEG C of conditions, about baking 10 hours, until thalline keeps constant weight, and weighs.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, take 1g broken wall bacterium powder in test tube, the sherwood oil adding 30-60 boiling range carries out equal-volume and extracts three times, adds sherwood oil 5mL at every turn, collects petroleum ether extraction liquid, and be placed in vacuum drying oven, in 80 DEG C, vacuum tightness is under 0.9Mpa condition, removing sherwood oil, thus obtain the grease being rich in docosahexenoic acid, and carry out the vapor detection analysis after esterification.
Fermentation results is as follows.
Table 11 1,2-PD+ethanol group and glucose group fermentation contrast
From above result, the application of fermenting carbon source 1,2-PD and ethanol, effectively can improve the content of DHA in grease, and the DHA output that final fermentation obtains increases obviously.
Embodiment 7
(1) in 250mL triangular flask, 50mL seed culture medium is added.The schizochytrium limacinum kind of access cryopreservation, culture temperature 25 DEG C, cultivates 2 days under shaking speed 200rpm condition, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed liquor;
Seed culture medium component (g/L): glucose 25.0, yeast powder 8.0, peptone 5.0, corn steep liquor 2.0, MgSO
47H
2o 0.6, KH
2pO
41.5, sea crystal 10.0.
(2) in 250mL triangular flask, add 50mL fermention medium, by schizochytrium limacinum with 8% inoculum size access fermention medium, 26 DEG C of constant temperature culture 3 days, rotating speed 200rpm;
Fermention medium component (g/L): 1,2-PD 55.0, ethanol 5.0, yeast extract 10.0, Sodium Glutamate 5.0, MgSO
47H
2o 0.2, KH
2pO
42.0, (NH
4)
2sO
43.0, NaNO
32.0, Na
2sO
48.0;
After fermention medium sterilizing, add external source regulatory factor sesamol after carrying out filtration sterilization, its addition is 2.0mM/L.
CK (g/L): glucose 60.0, yeast extract 10.0, Sodium Glutamate 5.0, MgSO
47H
2o0.2, KH
2pO
42.0, (NH
4)
2sO
43.0, NaNO
32.0, Na
2sO
48.0;
Often kind of different substratum prepares 3 Duplicate Samples, 121 DEG C, sterilizing 25min, cools for subsequent use;
(3) cultured continuously terminates fermentation after 3 days, fermented liquid carries out 8000g centrifugal 15 minutes, and with after single steaming washing 2-3 time, recentrifuge, collecting wet thallus and be placed in vacuum drying oven, is 0.9Mpa in vacuum tightness, temperature is under 80 DEG C of conditions, about baking 10 hours, until thalline keeps constant weight, and weighs.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, take 1g broken wall bacterium powder in test tube, the sherwood oil adding 30-60 boiling range carries out equal-volume and extracts three times, adds sherwood oil 5mL at every turn, collects petroleum ether extraction liquid, and be placed in vacuum drying oven, in 80 DEG C, vacuum tightness is under 0.9Mpa condition, removing sherwood oil, thus obtain the grease being rich in docosahexenoic acid, and carry out the vapor detection analysis after esterification.
Fermentation results is as follows.
Table 12 1,2-PD+ethanol+2.0mM/L sesamol group and glucose group fermentation contrast
From above result, fermenting carbon source 1,2-PD and ethanol, and the application of regulatory factor sesamol, effectively can improve the content of DHA in grease, and the final DHA output obtained is increased obviously.
Embodiment 8
(1) in 250mL triangular flask, 50mL seed culture medium is added.The schizochytrium limacinum kind of access cryopreservation, culture temperature 25 DEG C, cultivates 2 days under shaking speed 200rpm condition, the schizochytrium limacinum kind of cryopreservation is activated into schizochytrium limacinum seed liquor;
Seed culture medium component (g/L): glucose 30.0, yeast powder 4.0, extractum carnis 5.0, corn steep liquor 6.0, MgSO
47H
2o 1.0, KH
2pO
40.5, sea crystal 20.0.
(2) in 250mL triangular flask, add 50mL fermention medium, by schizochytrium limacinum with 4% inoculum size access fermention medium, 26 DEG C of constant temperature culture 3 days, rotating speed 200rpm;
Fermention medium component (g/L): 1,2-PD 40.0, sodium acetate 10.0, ethanol 10.0, yeast extract 15.0, Sodium Glutamate 10.0, MgSO
47H
2o 1.0, KH
2pO
41.5, (NH
4)
2sO
45.0, NaNO
33.0, Na
2sO
412.0;
After fermention medium sterilizing, add external source regulatory factor sesamol after carrying out filtration sterilization, its addition is 3.0mM/L.
CK (g/L): glucose 60.0, yeast extract 15.0, Sodium Glutamate 10.0, MgSO
47H
2o1.0, KH
2pO
41.5, (NH
4)
2sO
45.0, NaNO
33.0, Na
2sO
412.0;
Often kind of different substratum prepares 3 Duplicate Samples, 121 DEG C, sterilizing 25min, cools for subsequent use;
(3) cultured continuously terminates fermentation after 3 days, fermented liquid carries out 8000g centrifugal 15 minutes, and with after single steaming washing 2-3 time, recentrifuge, collecting wet thallus and be placed in vacuum drying oven, is 0.9Mpa in vacuum tightness, temperature is under 80 DEG C of conditions, about baking 10 hours, until thalline keeps constant weight, and weighs.
(4) mechanical breaking-wall method is carried out to vacuum drying thalline, take 1g broken wall bacterium powder in test tube, the sherwood oil adding 30-60 boiling range carries out equal-volume and extracts three times, adds sherwood oil 5mL at every turn, collects petroleum ether extraction liquid, and be placed in vacuum drying oven, in 80 DEG C, vacuum tightness is under 0.9Mpa condition, removing sherwood oil, thus obtain the grease being rich in docosahexenoic acid, and carry out the vapor detection analysis after esterification.
Fermentation results is as follows.
Table 13 1,2-PD+sodium acetate+ethanol+3.0mM/L sesamol group and glucose group fermentation contrast
Fermenting carbon source 1,2-propylene glycol, the application of sodium acetate and ethanol, and the interpolation of external source regulatory factor sesamol, effectively inhibit the phosphopentose pathway in schizochytrium limacinum and tricarboxylic acid delivery system, cause DHA content in schizochytrium limacinum grease to increase obviously, thus the DHA output that fermentation obtains promote obviously.
The inventive method is applicable to various schizochytrium limacinum, and as schizochytrium limacinum (Schizochytrium sp) S056, be preserved in China typical culture collection center CCTCC on September 29th, 2013, deposit number is CCTCC M 2013459, etc.
The above is preferred embodiment of the present invention, but the present invention should not be confined to the content disclosed in this embodiment.The equivalence completed under not departing from spirit disclosed in this invention so every or amendment, all fall into the scope of protection of the invention.
Claims (10)
1. one kind is improved the method for docosahexenoic acid content in schizochytrium limacinum grease, the method selects the fermenting carbon source in fermention medium to reduce endobacillary phosphopentose pathway metabolic flux, NADPH content in schizochytrium limacinum body is declined to a great extent, promotes a large amount of synthesis of DHA; Described 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-PD, ethanol and acetate.
2. the method improving docosahexenoic acid content in schizochytrium limacinum grease as claimed in claim 1, it is characterized in that, the method adds external source regulatory factor in the fermentation medium, endobacillary phosphopentose pathway metabolic flux is reduced, the activity of the malic enzyme in tricarboxylic acid delivery system is suppressed simultaneously, NADPH content in schizochytrium limacinum body is declined to a great extent, promotes a large amount of synthesis of DHA.
3. a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease as claimed in claim 2, is characterized in that, external source regulatory factor is sesamol, after fermention medium sterilizing, adds after carrying out filtration sterilization.
4. a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease as claimed in claim 3, is characterized in that, the addition of sesamol is 0.5-3.0mM/L.
5., as a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease as described in arbitrary in Claims 1-4, it is characterized in that, when fermenting carbon source be 1,2-PD and acetate time, its proportioning is: 1,2-PD 40g/L-60g/L; Sodium acetate 0-10g/L; When fermenting carbon source be 1,2-PD and ethanol time, its proportioning is: 1,2-PD 40-60g/L; Ethanol 0-10g/L; When fermenting carbon source is 1,2-PD, ethanol and acetate, its proportioning is: 1,2-PD 40-60g/L; Ethanol 0-10g/L; Acetate 0-10g/L.
6. a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease as claimed in claim 1, is characterized in that, the specific implementation step of the method is:
(1) the schizochytrium limacinum kind activation culture of cryopreservation is become schizochytrium limacinum seed liquor;
(2) schizochytrium limacinum seed liquor is accessed fermention medium according to volume percent 4%-10% to cultivate;
(3) after terminating fermentation, collect wet thallus and vacuum-drying, until thalline keeps constant weight, and weigh;
(4) carry out broken wall to the thalline of drying, then extraction obtains the grease being rich in docosahexenoic acid.
7. a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease as claimed in claim 6, is characterized in that, in step (2), after fermention medium sterilizing, adds external source regulatory factor after carrying out filtration sterilization.
8. a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease as claimed in claims 6 or 7, is characterized in that, time in described fermenting carbon source containing ethanol, adds in the fermention medium after sterilizing after ethanol is degerming after filtration; Described acetate selects sodium acetate.
9. a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease as claimed in claims 6 or 7, it is characterized in that, seed culture medium component (g/L) in described step (1) is: glucose 15.0-30.0, yeast powder 4.0-8.0, extractum carnis 0-5.0 peptone 0-5.0, corn steep liquor 2.0-6.0, MgSO
47H
2o 0.2-1.0, KH
2pO
40.5-2.0, sea crystal 10.0-20.0.
10. a kind of method improving docosahexenoic acid content in schizochytrium limacinum grease as claimed in claims 6 or 7, it is characterized in that, fermention medium component (g/L) in described step (3) is: fermenting carbon source 40.0-60.0, yeast extract 6.0-15.0, Sodium Glutamate 0-10.0, MgSO
47H
2o0.2-1.0, KH
2pO
40.5-2.0, (NH
4)
2sO
40-5.0, NaNO
31.0-3.0, Na
2sO
45.0-12.0.
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| CN112481318A (en) * | 2020-12-25 | 2021-03-12 | 南京师范大学 | Application of catechin in improving yield of DHA (docosahexaenoic acid) grease of schizochytrium limacinum |
| CN116555054A (en) * | 2023-06-09 | 2023-08-08 | 陕西海斯夫生物工程有限公司 | Recombinant schizochytrium limacinum with high DHA yield, construction method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136162A (en) * | 2018-06-07 | 2019-01-04 | 昆明藻能生物科技有限公司 | A kind of method that multifactor coevolution improves hidden dinoflagellate DHA yield |
| CN109136162B (en) * | 2018-06-07 | 2021-09-28 | 昆明藻能生物科技有限公司 | Method for improving yield of DHA (docosahexaenoic acid) of Crypthecodinium cohnii through multi-factor coevolution |
| CN112481318A (en) * | 2020-12-25 | 2021-03-12 | 南京师范大学 | Application of catechin in improving yield of DHA (docosahexaenoic acid) grease of schizochytrium limacinum |
| CN112481318B (en) * | 2020-12-25 | 2022-06-03 | 南京师范大学 | Application of catechin in improving yield of DHA (docosahexaenoic acid) grease of schizochytrium limacinum |
| CN116555054A (en) * | 2023-06-09 | 2023-08-08 | 陕西海斯夫生物工程有限公司 | Recombinant schizochytrium limacinum with high DHA yield, construction method and application thereof |
| CN116555054B (en) * | 2023-06-09 | 2024-05-14 | 陕西海斯夫生物工程有限公司 | Recombinant schizochytrium limacinum with high DHA yield, construction method and application thereof |
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