CN102391955A - Mutagenic screening method of DHA-producing strain crypthecodinium. cohnii - Google Patents
Mutagenic screening method of DHA-producing strain crypthecodinium. cohnii Download PDFInfo
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- CN102391955A CN102391955A CN2011103893813A CN201110389381A CN102391955A CN 102391955 A CN102391955 A CN 102391955A CN 2011103893813 A CN2011103893813 A CN 2011103893813A CN 201110389381 A CN201110389381 A CN 201110389381A CN 102391955 A CN102391955 A CN 102391955A
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Abstract
The invention discloses a mutagenic screening method of a DHA-producing strain crypthecodinium. cohnii. According to the invention, a protoplast of a DHA-producing strain crypthecodinium. cohnii ATCC30556 is prepared; single or composite mutagenesis of ultraviolet + LiCl and diethyl sulfate is carried out upon the protoplast; a basic thought of metabolism regulation and fermentation cultivation is adopted, and oriented screening is carried out by using malonic acid and/or a high-glucose resistance plate; and preliminary screening is carried out by using a solid cultivation medium of red tetrazoline (TTC), such that screening efficiency can be effectively improved, screening links can be reduced, and work load can be reduced. The strain obtained from the preliminary screening step is processed through shake-flask secondary screening and hereditary stability verification. As a result, the growth activity of the strain after mutagenesis is improved. Compared to an initial strain, the DHA biosynthesis amount of an YVD-4 mutagenic strain is improved by 4.7 times, and reaches 45.3g/L. Therefore, the method has an excellent prospect of industrialized production.
Description
Technical field
The present invention relates to the mutagenesis screening method that a kind of DHA produces the bacterium dino flagellate.
Background technology
DHA (docosahexenoic acid) is a kind of polyene fatty acid with special construction (6 cis-configurations of non-conjugated system two keys), and it belongs to the typical long-chain highly unsaturated fatty acid of ω-3 series according to the position of last two key of its methyl end.The intravital DHA of people mainly is distributed in cerebral gray matter, retina cell's acromere, neural system, breast milk, cardiac muscle, eosinophil leucocyte, sperm etc. and locates, and its physiological action has: suppress platelet aggregation; Reduce neutral lipid in the blood; Reduce the SUV and the high density lipoprotein increasing SUV of vldl and low-density lipoprotein; Blood viscosity lowering; Mend the brain brain tonic, the prevention senile dementia; Improve eyesight, control myopia; Retarding effect through to archaeal dna polymerase and DNA topoisomerase reaches preventing cancer effect etc.
The DHA that tradition is extracted from fish oil, limited amount is difficult to satisfy the ever-increasing market requirement.Therefore, DHA produces research gradually to utilizing the development of biotechnology compound direction.Utilize the synthetic DHA of biotechnology to have the cycle weak point, lipid acid constitutes simple and bigger advantages such as production potential is arranged, and does not receive the restriction of raw material, and the fishy smell of the DHA that the DHA of Production by Microorganism Fermentation does not extract from fish oil more helps the application of DHA.
Mikrobe commonly used comprises thraustochytriale, schizochytrium limacinum and marine microalgae etc.Compare with other mikrobe, marine microalgae is ideal DHA source, and cultivation of algae is produced the productivity ratio of DHA and utilized fungi and microbial culture to exceed 1 ~ 2 one magnitude.The physiologically active substance of the uniqueness that marine microalgae contained makes them have multiple pharmaceutical use and nourishing function, utilizes marine microalgae to produce the especially research of DHA of pufas, has become the focus of various countries scientific research personnel research and development.Dino flagellate (
Crypthecodinium. cohniiATCC30556) DHA content reaches more than 30%, and its high-biomass, high growth rates, high DHA content, heterotrophism incubation growth are good, and do not contain EPA (timnodonic acid), are one of better generation bacterium of DHA.At present disclosed the utilization in the patent that marine microalgae produces DHA, the greasy output of DHA is all not high, and reason is relevant with the fermentating culturing process of used bacterial classification and employing.Therefore, how to select better dino flagellate strain and the culture process of optimizing this bacterium, become one of problem of researcher research to improve the market competitiveness.
Summary of the invention
The object of the invention; Be to provide a kind of DHA to produce the mutagenesis screening method of bacterium dino flagellate, it abandons the defective of prior art, adopts the protoplastis induced-mutation technique; And the basic ideas of combination metabolic regulation fermentation breeding; Directive breeding goes out the higher dino flagellate bacterial strain of DHA content, and the culture process of this bacterial strain is further optimized, and obtains the positive mutant of high yield.
The present invention is achieved in that said a kind of DHA produces the mutagenesis screening method of bacterium dino flagellate, and its step is following:
(1) cultivation of seed
With dino flagellate (
Crypthecodinium. cohniiATCC30556) be inoculated in the seed culture medium, the inoculum size volume percent is 5 ~ 10%, and culture temperature is 26 ~ 30 ℃, and shaking speed 160 ~ 200r/min cultivates 24 ~ 48h to logarithmic phase;
(2) pre-treatment of seed liquor
Get the cell that step 1 is cultured to logarithmic phase, centrifugal 5 ~ 10min under the 4000 r/min rotating speeds, 0.2mol/L phosphate buffered saline buffer washing 2 ~ 3 times, dilute 40 ~ 80 times after, break up through sterile glass beads, 6 layers of sterile gauze are crossed and are filtered single-cell suspension liquid;
(3) preparation of protoplastis and ultraviolet mutagenesis are handled
With an amount of protoplast formation reaction solution, promptly step 2 single-cell suspension liquid, enzyme liquid and osmotic pressure regulator are handled 20 ~ 30 min in the mixed solution of 1:1 ~ 4:20 ~ 35 ratios composition under 25 ~ 30 ℃ of conditions; Enzymolysis solution filters, and spinning washs and be diluted to 10
6Individual/mL, get the protoplastis refined solution;
This protoplastis refined solution carries out pre-treatment with 0.8% LiCl earlier, places the sterile petri dish of magnetic stirring bar then, and bacterium liquid thickness is no more than 2mm, adds a cover; Open uv lamp preheating 20min, under 15W ~ 30W uv lamp, on the magnetic stirring apparatus apart from the 30cm place; Place petridish, slowly stir, open the ware lid and carry out uv irradiating 5 ~ 30 min; Take out petridish, gradient dilution, whole process is operated under ruddiness; Cultivate 4 ~ 7d for 26 ~ 30 ℃;
(4) ethyl sulfate mutagenic treatment
Getting protoplastis refined solution that step 3 obtains, to add percent by volume be 10% ethyl sulfate, in 160 ~ 200r/min vibration combination treatment, 5 ~ 10min, adds the Sulfothiorine termination reaction of 1mL 25%; With 500 ~ 1000 times of reaction solution dilutions and on the solid medium that has added redox agent TCC (TTC), cultivate 4 ~ 7d for 26 ~ 30 ℃;
(5) screening of anti-propanedioic acid and high sugared bacterial strain
It is the strongest to select the TTC activity; Be that the speed of growth is very fast; Dyeing more deeply, the bacterial strain that obtains as primary dcreening operation of bigger bacterium colony, the accumulation of these bacterial strains DHA is more relatively, after these positive mutants are reactivated; The preparation single cell suspension, and coat respectively and contain on the dull and stereotyped and/or high sugared plating medium of 0.05% malonic acid 26 ~ 30 ℃ and cultivate 4 ~ 7d;
(6) shake the multiple sieve of bottle
With growth on the above-mentioned resistant panel comparatively fast, cultivations of in seed culture medium, going down to posterity of bigger colony inoculation, carry out shake flask fermentation then respectively, in fermention medium, 26 ~ 30 ℃ of cultivation 3 ~ 5d; Measure fatty acid content and the DHA content of thalline in the fermented liquid, and with this as screening index, the positive mutant of acquisition high yield.
Seed culture medium described in the step 1 of the present invention is formed: glucose 40 ~ 80 g/L, NaCl 15 ~ 25g/L, MgSO
4.7H
2O 4 ~ 8 g/L, KH
2PO
40.5 ~ 1.0 g/L, KNO
31 ~ 5 g/L, yeast powder 2 ~ 8 g/L, (NH
4)
2SO
40.2 ~ 0.5 g/L, NaHCO
30.08 ~ 0.12 g/L, MnSO
40.5 ~ 1.0 μ g/L, vitamins B
10.01 ~ 0.04mg/L, vitamins B
60.001 ~ 0.006mg/L, initial pH6.0 ~ 7.0.
The said solid medium of step 4 of the present invention is formed: glucose 40 ~ 100 g/L, NaCl 15 ~ 25g/L, MgSO
4.7H
2O 4 ~ 8 g/L, KH
2PO
40.5 ~ 1.0 g/L, KNO
31 ~ 5 g/L, yeast powder 2 ~ 8 g/L, (NH
4)
2SO
40.2 ~ 0.5 g/L, NaHCO
30.08 ~ 0.12 g/L, MnSO
40.5 ~ 1.0 μ g/L, vitamins B
10.01 ~ 0.04mg/L, vitamins B
60.001 ~ 0.01mg/L, agar powder 20 g/L, initial pH6.0 ~ 7.0.
The sugared plate culture medium of the said height of step 5 of the present invention is formed: NaCl 10 ~ 30g/L, MgSO
4.7H
2O 5 ~ 10 g/L, KH
2PO
40.2 ~ 0.8 g/L, (NH
4)
2SO
40.2 ~ 1.0 g/L, NaHCO
30.1 ~ 0.5 g/L, vitamins B
10.01 ~ 0.1mg/L, vitamins B
60.001 ~ 0.01mg/L, KNO
30.5 ~ 1.5 g/L, glucose 200 ~ 350 g/L, yeast powder 1 ~ 5 g/L, agar powder 20 g/L.
The said fermention medium of step 6 of the present invention is formed: glucose 40 ~ 100 g/L, NaCl 15 ~ 25g/L, MgSO
4.7H
2O 4 ~ 8 g/L, yeast powder 2 ~ 8 g/L, KNO
31 ~ 5 g/L, KH
2PO
40.5 ~ 1.0 g/L, (NH
4)
2SO
40.2 ~ 0.5 g/L, CaCO
38 ~ 10 g/L, NaHCO
30.08 ~ 0.12 g/L, MnSO
40.5 ~ 1.0 μ g/L, vitamins B
10.01 ~ 0.04mg/L, vitamins B
60.001 ~ 0.01mg/L, initial pH6.0 ~ 7.0.
The invention has the beneficial effects as follows, through preparation DHA produce the bacterium dino flagellate (
Crypthecodinium. cohniiATCC30556) protoplastis; And it is carried out the single or complex mutation of ultraviolet+LiCl, ethyl sulfate; The basic ideas that combine metabolic regulation fermentation breeding simultaneously adopt the resistant panel that contains propanedioic acid and/or high sugar to carry out directed screening, and carry out primary dcreening operation with the solid medium of TCC (TTC); Effectively improve screening efficiency, reduce screening link and workload.The bacterial strain that primary dcreening operation obtains is through shaking multiple sieve of bottle and genetic stability checking; The strain growth activity improves after finding mutagenesis, compares with starting strain, and the DHA biosynthesizing amount of YVD-4 mutagenic fungi has improved 4.7 times; Reach 45.3 g/L, possess excellent suitability for industrialized production prospect.
Embodiment
A kind of DHA according to the invention produces the mutagenesis screening method of bacterium dino flagellate, and its step is following:
1, the pre-treatment of the cultivation of seed and seed liquor
With dino flagellate (
Crypthecodinium. cohniiATCC30556) be inoculated in the seed culture medium, the inoculum size volume percent is 10%, and culture temperature is 26 ℃, and shaking speed 200r/min cultivates 36h to logarithmic phase.
Get the above-mentioned cell that is cultured to logarithmic phase of 2.5 mL, centrifugal 10min under the 4000 r/min rotating speeds, 0.2mol/L phosphate buffered saline buffer washing 3 times, and after diluting 50 times, break up through sterile glass beads, 6 layers of sterile gauze are crossed and are filtered single-cell suspension liquid.
2, the preparation of protoplastis and ultraviolet mutagenesis are handled
The protoplast formation reaction solution is 5 mL, comprising the osmotic pressure regulator of single-cell suspension liquid, 0.2 mL Snailase enzyme liquid and 4.6 mL of 0.2 mL.Under 25 ℃ of conditions, handle 30 min.Enzymolysis solution filters, and spinning washs and be diluted to 10
6Individual/mL, get the protoplastis refined solution.
This refined solution carries out pre-treatment with 0.8% LiCl earlier, places the sterile petri dish of magnetic stirring bar then, and bacterium liquid thickness is no more than 2mm, adds a cover; Open uv lamp preheating 20min, under the 15W uv lamp, on the magnetic stirring apparatus apart from the 30cm place; Place petridish, slowly stir, open the ware lid and carry out uv irradiating 5,10,15,20,25 min; Take out petridish, gradient dilution, whole process is operated under ruddiness.Cultivate 6d for 26 ℃.Obtain 15 strains bacterial strain to be screened.
3, ethyl sulfate mutagenic treatment
Getting protoplastis refined solution that 5mL obtains, to add percent by volume be 10% ethyl sulfate, in 180r/min vibration combination treatment 8min, adds the Sulfothiorine termination reaction of 1mL 25%.With 1000 times of reaction solution dilutions, stop fully to guarantee mutagenesis reaction, and then gradient dilution successively, and on the solid medium that has added redox agent TCC (TTC) 26 ℃ cultivate 4d.Obtain 15 strains bacterial strain to be screened.
4, the ultraviolet of protoplastis, ethyl sulfate complex mutation are handled
Under ruddiness, getting protoplastis refined solution that the 5mL ultraviolet handled, to add volume(tric)fraction be 10% ethyl sulfate, in 160r/min vibration combination treatment 5min, adds the Sulfothiorine termination reaction of 1mL 25%.With 1000 times of reaction solution dilutions, stop fully to guarantee mutagenesis reaction, and then gradient dilution and coat the solid medium that has added TCC (TTC) successively, cultivate 4d for 26 ℃.Obtain 63 strains bacterial strain to be screened.
5, dull and stereotyped primary dcreening operation and shake the multiple sieve of bottle
(1) bacterial strain that step 2 is obtained, ruddiness condition descending stair degree dilution is coated on the solid medium that has added 1.5% TTC, cultivate 3d after, picking 35 strain growing ways better, TTC dyeing more deeply, bigger bacterial strain to be to treat multiple sieve.
(2) 35 strain bacterial strains through primary dcreening operation are gone to the high sugar that has added 0.05% propanedioic acid and/or 20% ~ 35% glucose and cultivate 4d for dull and stereotyped last 26 ℃.Observe flat board, select the bacterial strain that the 20 strain speeds of growth are very fast, bacterium colony is bigger, be seeded to respectively in the seed culture fluid, subsequent use.
(3) bacterial strain that the resistant panel screening is obtained is seeded in the fermention medium respectively; It is 0 that 26 ℃ of fermentation culture 3d consume to sugar; Measure dried cell weight, fatty acid content and DHA content respectively; Through the comparative analysis of each index, obtain the higher bacterial strain of 4 strain DHA output, respectively called after YV-1, YV-2, YV-3, YV-4.
According to the treatment step treatment step 3 of (1) ~ (3), the bacterial strain that step 4 obtains; Respectively obtain the higher bacterial strain of 5 strain DHA output respectively, distinguish called after YD-1, YD-2, YD-3, YD-4, YD-5 and YVD-1, YVD-2, YVD-3, YVD-4, YVD-5 separately.
6, shake flask fermentation
The higher bacterial strain of 14 strain DHA output that screening is obtained carries out the genetic stability investigation respectively, warp 10 generation cultured continuously, and obvious variation does not take place in the living weight of bacterial strain, fat content and three indexs of DHA content, all shows more stable leavening property.Therefrom choose higher YD-1 of two strain output and YVD-4 and be inoculated in the seed culture fluid, 26 ℃, 200r/min cultivates 36h.Cultivate overdue seed liquor and be forwarded to fermention medium with 10% inoculum size, 26 ℃, 200r/min continues fermentation culture 72h.Do the controlled trial of original strain simultaneously.Fermentation result such as table 1:
The contrast of table 1 DHA high productive mutant and starting strain
| Bacterial strain | Living weight (g/L) | Fat content (%) | DHA content (%) | DHA output (g/L) |
| Original strain | 58.2 | 30.6 | 35.4 | 9.6 |
| YD-1 | 76.8 | 48.1 | 49.7 | 39.4 |
| YVD-4 | 79.6 | 50.2 | 53.6 | 45.3 |
Fermentation the contrast experiment show: strain growth is active after the mutagenesis improves; The DHA biosynthesizing amount of comparing the YD-1 mutagenic fungi with starting strain has improved 4.1 times approximately; Reach 39.4 g/L, the DHA biosynthesizing amount of YVD-4 mutagenic fungi has then improved about 4.7 times, reaches 45.3 g/L.
The substratum of using is following:
Seed culture medium is formed: glucose 50g/L, NaCl 25g/L, MgSO
4.7H
2O 6 g/L, KH
2PO
40.5g/L, KNO
32 g/L, yeast powder 5 g/L, (NH
4)
2SO
40.2 g/L, NaHCO
30.08 g/L, MnSO
40.5 μ g/L, vitamins B
10.01mg/L, vitamins B
60.003mg/L, initial pH6.0 ~ 7.0.
Solid medium is formed: glucose 40 ~ 100 g/L, NaCl 15 ~ 25g/L, MgSO
4.7H
2O 4 ~ 8 g/L, KH
2PO
40.5 ~ 1.0 g/L, KNO
31 ~ 5 g/L, yeast powder 2 ~ 8 g/L, (NH
4)
2SO
40.2 ~ 0.5 g/L, NaHCO
30.08 ~ 0.12 g/L, MnSO
40.5 ~ 1.0 μ g/L, vitamins B
10.01 ~ 0.04mg/L, vitamins B
60.001 ~ 0.01mg/L, agar powder 20 g/L, initial pH6.0 ~ 7.0.
High sugared plate culture medium is formed: NaCl 25g/L, MgSO
4.7H
2O 5 g/L, KH
2PO
40.5 g/L, (NH
4)
2SO
40.2 g/L, NaHCO
30.1 g/L, vitamins B
10.01mg/L, vitamins B
60.001mg/L, KNO
31 g/L, glucose 200 ~ 350 g/L, yeast powder 2 g/L, agar powder 20 g/L.
Fermention medium is formed: glucose 60g/L, NaCl 15g/L, MgSO
4.7H
2O 4g/L, yeast powder 5g/L, KNO
32 g/L, KH
2PO
40.5 g/L, (NH
4)
2SO
40.2g/L, CaCO
38 g/L, NaHCO
30.08 g/L, MnSO
41.0 μ g/L, vitamins B
10.01mg/L, vitamins B
60.01mg/L, initial pH6.0 ~ 7.0.
Claims (5)
1. a DHA produces the mutagenesis screening method of bacterium dino flagellate, and its step is following:
(1) cultivation of seed
With dino flagellate (
Crypthecodinium. cohniiATCC30556) be inoculated in the seed culture medium, the inoculum size volume percent is 5 ~ 10%, and culture temperature is 26 ~ 30 ℃, and shaking speed 160 ~ 200r/min cultivates 24 ~ 48h to logarithmic phase;
(2) pre-treatment of seed liquor
Get the cell that step 1 is cultured to logarithmic phase, centrifugal 5 ~ 10min under the 4000 r/min rotating speeds, 0.2mol/L phosphate buffered saline buffer washing 2 ~ 3 times, dilute 40 ~ 80 times after, break up through sterile glass beads, 6 layers of sterile gauze are crossed and are filtered single-cell suspension liquid;
(3) preparation of protoplastis and ultraviolet mutagenesis are handled
With an amount of protoplast formation reaction solution, promptly step 2 single-cell suspension liquid, enzyme liquid and osmotic pressure regulator are handled 20 ~ 30 min in the mixed solution of 1:1 ~ 4:20 ~ 35 ratios composition under 25 ~ 30 ℃ of conditions; Enzymolysis solution filters, and spinning washs and be diluted to 10
6Individual/mL, get the protoplastis refined solution;
This protoplastis refined solution carries out pre-treatment with 0.8% LiCl earlier, places the sterile petri dish of magnetic stirring bar then, and bacterium liquid thickness is no more than 2mm, adds a cover; Open uv lamp preheating 20min, under 15W ~ 30W uv lamp, on the magnetic stirring apparatus apart from the 30cm place; Place petridish, slowly stir, open the ware lid and carry out uv irradiating 5 ~ 30 min; Take out petridish, gradient dilution, whole process is operated under ruddiness; Cultivate 4 ~ 7d for 26 ~ 30 ℃;
(4) ethyl sulfate mutagenic treatment
Getting protoplastis refined solution that step 3 obtains, to add percent by volume be 10% ethyl sulfate, in 160 ~ 200r/min vibration combination treatment, 5 ~ 10min, adds the Sulfothiorine termination reaction of 1mL 25%; With 500 ~ 1000 times of reaction solution dilutions and on the solid medium that has added redox agent TCC (TTC), cultivate 4 ~ 7d for 26 ~ 30 ℃;
(5) screening of anti-propanedioic acid and high sugared bacterial strain
It is the strongest to select the TTC activity; Be that the speed of growth is very fast; Dyeing more deeply, the bacterial strain that obtains as primary dcreening operation of bigger bacterium colony, the accumulation of these bacterial strains DHA is more relatively, after these positive mutants are reactivated; The preparation single cell suspension, and coat respectively and contain on the dull and stereotyped and/or high sugared plating medium of 0.05% malonic acid 26 ~ 30 ℃ and cultivate 4 ~ 7d;
(6) shake the multiple sieve of bottle
With growth on the above-mentioned resistant panel comparatively fast, cultivations of in seed culture medium, going down to posterity of bigger colony inoculation, carry out shake flask fermentation then respectively, in fermention medium, 26 ~ 30 ℃ of cultivation 3 ~ 5d; Measure fatty acid content and the DHA content of thalline in the fermented liquid, and with this as screening index, the positive mutant of acquisition high yield.
2. produce the mutagenesis screening method of bacterium dino flagellate according to the said a kind of DHA of claim 1, it is characterized in that: seed culture medium described in the step 1 is formed: glucose 40 ~ 80 g/L, NaCl 15 ~ 25g/L, MgSO
4.7H
2O 4 ~ 8 g/L, KH
2PO
40.5 ~ 1.0 g/L, KNO
31 ~ 5 g/L, yeast powder 2 ~ 8 g/L, (NH
4)
2SO
40.2 ~ 0.5 g/L, NaHCO
30.08 ~ 0.12 g/L, MnSO
40.5 ~ 1.0 μ g/L, vitamins B
10.01 ~ 0.04mg/L, vitamins B
60.001 ~ 0.006mg/L, initial pH6.0 ~ 7.0.
3. produce the mutagenesis screening method of bacterium dino flagellate according to the said a kind of DHA of claim 1, it is characterized in that: the said solid medium of step 4 is formed: glucose 40 ~ 100 g/L, NaCl 15 ~ 25g/L, MgSO
4.7H
2O 4 ~ 8 g/L, KH
2PO
40.5 ~ 1.0 g/L, KNO
31 ~ 5 g/L, yeast powder 2 ~ 8 g/L, (NH
4)
2SO
40.2 ~ 0.5 g/L, NaHCO
30.08 ~ 0.12 g/L, MnSO
40.5 ~ 1.0 μ g/L, vitamins B
10.01 ~ 0.04mg/L, vitamins B
60.001 ~ 0.01mg/L, agar powder 20 g/L, initial pH6.0 ~ 7.0.
4. produce the mutagenesis screening method of bacterium dino flagellate according to the said a kind of DHA of claim 1, it is characterized in that:
The sugared plate culture medium of the said height of step 5 is formed: NaCl 10 ~ 30g/L, MgSO
4.7H
2O 5 ~ 10 g/L, KH
2PO
40.2 ~ 0.8 g/L, (NH
4)
2SO
40.2 ~ 1.0 g/L, NaHCO
30.1 ~ 0.5 g/L, vitamins B
10.01 ~ 0.1mg/L, vitamins B
60.001 ~ 0.01mg/L, KNO
30.5 ~ 1.5 g/L, glucose 200 ~ 350 g/L, yeast powder 1 ~ 5 g/L, agar powder 20 g/L.
5. produce the mutagenesis screening method of bacterium dino flagellate according to the said a kind of DHA of claim 1, it is characterized in that: the said fermention medium of step 6 is formed: glucose 40 ~ 100 g/L, NaCl 15 ~ 25g/L, MgSO
4.7H
2O 4 ~ 8 g/L, yeast powder 2 ~ 8 g/L, KNO
31 ~ 5 g/L, KH
2PO
40.5 ~ 1.0 g/L, (NH
4)
2SO
40.2 ~ 0.5 g/L, CaCO
38 ~ 10 g/L, NaHCO
30.08 ~ 0.12 g/L, MnSO
40.5 ~ 1.0 μ g/L, vitamins B
10.01 ~ 0.04mg/L, vitamins B
60.001 ~ 0.01mg/L, initial pH6.0 ~ 7.0.
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|---|---|---|---|
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966273A (en) * | 2014-04-29 | 2014-08-06 | 内蒙古金达威药业有限公司 | Method for producing DHA through Crypthecodinium cohnii fermentation |
| CN105647956A (en) * | 2015-04-20 | 2016-06-08 | 昆明藻能生物科技有限公司 | Method for transforming gene of crypthecodinium cohnii producing docosahexaenoic acid (DHA) |
| CN105647820A (en) * | 2016-03-22 | 2016-06-08 | 广西生众生物科技开发有限公司 | Efficient preparation method of morchella protoplast |
| CN105713894A (en) * | 2016-01-26 | 2016-06-29 | 李晓白 | Culture method of trichoderma viride mutant strain |
| CN106591186A (en) * | 2016-12-13 | 2017-04-26 | 北京林业大学 | Preparation method of spirulina platensis protoplasts |
| CN111057653A (en) * | 2018-10-17 | 2020-04-24 | 天津大学 | Breeding method of high-yield docosahexaenoic acid thraustochytrid strains |
-
2011
- 2011-11-30 CN CN2011103893813A patent/CN102391955A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966273A (en) * | 2014-04-29 | 2014-08-06 | 内蒙古金达威药业有限公司 | Method for producing DHA through Crypthecodinium cohnii fermentation |
| CN105647956A (en) * | 2015-04-20 | 2016-06-08 | 昆明藻能生物科技有限公司 | Method for transforming gene of crypthecodinium cohnii producing docosahexaenoic acid (DHA) |
| CN105647956B (en) * | 2015-04-20 | 2019-08-23 | 昆明藻能生物科技有限公司 | The method that a kind of pair of hidden dinoflagellate of production docosahexaenoic acid carries out genetic transformation |
| CN105713894A (en) * | 2016-01-26 | 2016-06-29 | 李晓白 | Culture method of trichoderma viride mutant strain |
| CN105647820A (en) * | 2016-03-22 | 2016-06-08 | 广西生众生物科技开发有限公司 | Efficient preparation method of morchella protoplast |
| CN106591186A (en) * | 2016-12-13 | 2017-04-26 | 北京林业大学 | Preparation method of spirulina platensis protoplasts |
| CN111057653A (en) * | 2018-10-17 | 2020-04-24 | 天津大学 | Breeding method of high-yield docosahexaenoic acid thraustochytrid strains |
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