CN102304479A - Culture medium added with plant hormone, and application thereof in schizochytrium limacinum fermentation - Google Patents
Culture medium added with plant hormone, and application thereof in schizochytrium limacinum fermentation Download PDFInfo
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- CN102304479A CN102304479A CN201110255188A CN201110255188A CN102304479A CN 102304479 A CN102304479 A CN 102304479A CN 201110255188 A CN201110255188 A CN 201110255188A CN 201110255188 A CN201110255188 A CN 201110255188A CN 102304479 A CN102304479 A CN 102304479A
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Abstract
The invention provides a culture medium added with a plant hormone, and an application thereof in schizochytrium limacinum fermentation. The culture medium comprises components of: a carbon source of glucose with a concentration of 10 to 100g/L; a nitrogen source of an yeast extract with a concentration of 10 to 80g/L; seawater which takes 20% to 80% of the total volume of the culture medium; and one component selected from 6-benzyladenine, furfuryl aminopurine, gibberellin, indole butyric acid, naphthalene acetic acid, and heteroauxin. According to the invention, a plant hormone with an appropriate concentration is added to the schizochytrium limacinum fermentation medium. The cytokinins of 6-benzyladenine, furfuryl aminopurine, gibberellin, indole butyric acid, naphthalene acetic acid, and heteroauxin all assist in substantially improving the growing speed and the DHA content of schizochytrium limacinum. Also, with the technical scheme provided by the invention, fermentation cost of schizochytrium limacinum can be substantially reduced, and fermentation efficiency can be improved, such that the development of the industry is promoted.
Description
Technical field
The invention belongs to technical field of microbe application, be specifically related to a kind of substratum and application in the schizochytrium limacinum fermentation thereof that is added with plant hormone.
Background technology
Research shows; With docosahexenoic acid (Docosahexaenoic acid; DHA, C22:6 n-3) is the n-3 pufas of representative (Polyunsaturated fatty acids; PUFAs) be necessary lipid acid, receive attention widely because have the healthy different physiological roles that is beneficial to man.DHA is pallium and amphiblestroid key component, to infant's vision and neural normal development, keeps vision and neural function is most important.In addition, also have physiological functions such as preventing cardiovascular disease, anticancer, anti-inflammatory.
Schizochytrium limacinum (Schizochytrium limacinum) is a kind of thalassiomycetes, and the outstanding feature of this fungi is a large amount of grease of accumulation in the cell, and the grease 90% or more exists with the form that human body is prone to the neutral grease-triglyceride of absorption.Unsaturated fatty acid content is very high in the total fatty acids, is mainly DHA.The schizochytrium limacinum fast growth is easy to carry out fermentative prodn.These species are safe in utilization, have no toxic side effect, are the ideal Biological resources of a kind of DHA of production.Since this century, DHA has obtained considerable progress with the schizochytrium limacinum fermentative prodn, becomes the main source of China DHA.
Plant hormone (plant hormone) is synthetic in plant materials, and is transported to other places from producing part, though concentration is very low, to the physiological process of plant and the significantly biologically active substance of effect of generation that grows.The plant hormone of having found at present has sour 5 types of phytokinin, growth hormone, Plant hormones regulators,gibberellins, ethene and fallen leaves, the hormone of former three for promoting to grow, and then both are negative sense hormones, can suppress to grow.Research shows that the fungi that has (like root nodule bacterium) can directly be synthesized or the synthetic plant hormone of inducing plant.Plant hormone has fungi growth and metabolism certain influence is also arranged, and Yang Zhongbao etc. (2005) have studied the influence that hormone infects and produce spore to arbuscular mycorrhiza (Arbuscular mycorrhizae); Yu Jianxin etc. (2009) studied mycorrhizal fungi growth and with the plant symbiosis process in can directly synthesize or inducing plant produces multiple hormonal substance, like growth hormone etc., mycorrhizal fungi is to the influence of plant hormone kind, form and content etc.But also do not see about the report of plant hormone to the influence of schizochytrium limacinum.
Summary of the invention
To the report that also schizochytrium limacinum is not influenced in the prior art relevant for plant hormone; The invention provides substratum and the application in the schizochytrium limacinum fermentation thereof of adding plant hormone; The present invention has reached the purpose that promotes the schizochytrium limacinum growth and improve DHA content through when thalassiomycetes schizochytrium limacinum fermentative prodn, adding the plant hormone of proper concn.
For realizing the foregoing invention purpose, the present invention adopts following technical proposals to be achieved:
Add the substratum of plant hormone, it comprises following component:
With glucose is carbon source, and concentration is 10-100g/L;
With the yeast extract is nitrogenous source, and concentration is 10-80g/L;
Seawater accounts for the 20%-80% of substratum TV;
Add a kind of in 6-benzyladenine, furfuryl group aminopurine, Plant hormones regulators,gibberellins, indolebutyric acid, naphthylacetic acid and the indolylacetic acid respectively.
Further improvement to technique scheme: said 6-benzyladenine concentration is 1-10mg/L; Furfuryl group aminopurine concentration is 1-20mg/L; Plant hormones regulators,gibberellins concentration is 1-10mg/L; Said indolebutyric acid concentration is 1-10mg/L; NAA concentration is 1-10mg/L; Indolylacetic acid concentration is 1-10mg/L.
Further improvement to technique scheme: the optimization concentration of said glucose is 40-80g/L.
Further improvement to technique scheme: the optimization concentration of said yeast extract is 20-60g/L.
Further improvement to technique scheme: it is 30%-70% that said seawater accounts for the Optimum of culture medium ratio.
The present invention also provides the substratum that adds plant hormone in the schizochytrium limacinum fermentation, to use.
Further improvement to technique scheme: said schizochytrium limacinum is schizochytrium limacinum OUC88, culture presevation number: CGMCC NO.1240, preservation date: 2004.10.27.
Further improvement to technique scheme: said schizochytrium limacinum inserts in the said substratum with the inoculum size of 1-10%.
Further improvement to technique scheme: the fermentation culture temperature of said schizochytrium limacinum is 18-30 ℃, and the pH value is 4-8, and rotating speed is 100-250rpm, shaking culture 3-7 days.
Compared with prior art; Advantage of the present invention and positively effect are: the present invention is through adding the plant hormone of suitable concentration in the schizochytrium limacinum fermention medium; To detect the influence that plant hormone produces schizochytrium limacinum; Said plant hormone is respectively phytokinin 6-benzyladenine, furfuryl group aminopurine, Plant hormones regulators,gibberellins, plant hormone indolebutyric acid, naphthylacetic acid and indolylacetic acid, and their independent interpolations all can significantly improve the schizochytrium limacinum speed of growth and increase DHA content; And, adopt technical scheme according to the invention, can significantly reduce the fermentation costs of schizochytrium limacinum, improve fermentation efficiency, thereby promote industry development.
After advantages embodiment of the present invention, other characteristics of the present invention and advantage will become clearer.
Description of drawings
Fig. 1 shows that 6-BA is to the influence of schizochytrium limacinum growth and DHA content among the present invention.
Fig. 2 shows that KT is to the influence of schizochytrium limacinum growth and DHA content among the present invention.
Fig. 3 shows that GA is to the influence of schizochytrium limacinum growth and DHA content among the present invention.
Fig. 4 shows that IBA is to the influence of schizochytrium limacinum growth and DHA content among the present invention.
Fig. 5 shows that NAA is to the influence of schizochytrium limacinum growth and DHA content among the present invention.
Fig. 6 shows that IAA is to the influence of schizochytrium limacinum growth and DHA content among the present invention.
Embodiment
Below in conjunction with accompanying drawing and embodiment technical scheme of the present invention is done further detailed explanation.
In the research binding mode of plant hormone, mainly examine or check the content of DHA in living weight and the cell of schizochytrium limacinum, because both are two key factors judging that production efficiency improves to schizochytrium limacinum.
In order to improve the productivity effect of schizochytrium limacinum; The present invention is through adding the influence of 6 kinds of different these plant hormones of plant hormone checking to schizochytrium limacinum respectively in the fermention medium of schizochytrium limacinum; And determine the optimization addition of various plant hormones through further experiment; 6 kind of plant hormones according to the invention comprise: phytokinin 6-benzyladenine (BA), furfuryl group aminopurine (KT), Plant hormones regulators,gibberellins (GA), plant hormone indolebutyric acid (IBA), naphthylacetic acid (NAA) and indolylacetic acid (IAA); With the substratum that do not add plant hormone as contrast; The living weight of more different group schizochytrium limacinums and DHA content, experiment of the present invention mainly comprise two types:
1. more different plant hormones is to the living weight of schizochytrium limacinum and the effect of DHA accumulation;
2. the effect that more same kind of plant hormone accumulates schizochytrium limacinum growth and DHA under the various dose condition.
Embodiment 1:6-BA is to the influence of the living weight and the DHA content of schizochytrium limacinum
The schizochytrium limacinum substratum is carbon source with glucose, and concentration is 60g/L; With the yeast extract is nitrogenous source, and concentration is 20g/L; The ratio of seawater is 50% in the substratum.
In the schizochytrium limacinum substratum, add 6-BA then, add concentration and be respectively 0.5mg/L, 1mg/L, 3mg/L, 6mg/L and 10mg/.
Schizochytrium limacinum is inserted in the said substratum with 10% inoculum size.
The fermentation culture temperature is 23 ± 1 ℃, and the pH value is 6, and rotating speed is 200rpm, shaking culture 4 days.
As shown in Figure 1; The result shows: it is not obvious to the influence of the living weight of schizochytrium limacinum and DHA content when being 0.5mg/L that 6-BA adds concentration; Concentration is under 11mg/L and the 3mg/L condition, and 6-BA plays a driving role to schizochytrium limacinum growth and DHA content, and the schizochytrium limacinum living weight reaches peak during 3mg/L; Living weight is brought up to 9.28g/Ld than the 7.30g/Ld of control group, has improved 27.1%; DHA content brings up to 16.9% from 13.0%, has improved 30.0%.But when concentration was 6mg/L and 10mg/L, 6-BA produced restraining effect to schizochytrium limacinum growth and DHA content.
Embodiment 2:KT is to the influence of the living weight and the DHA content of schizochytrium limacinum
Schizochytrium limacinum substratum composition and cultural method such as embodiment 1 are said.
As shown in Figure 2, the result shows: KT when lower concentration (≤10mg/L) can improve the living weight and the DHA content of schizochytrium limacinum, when the KT addition was 10mg/L, the schizochytrium limacinum living weight reached 9.55g/Ld, than the high 2.25g/Ld of control group; DHA content 14.9% is higher by 1.9% than control group.When the KT addition is under the situation of 15mg/L or higher (20mg/L), to the growth and the effect of DHA accumulation generation obvious suppression of schizochytrium limacinum.
Embodiment 3:GA is to the influence of the living weight and the DHA content of schizochytrium limacinum
Schizochytrium limacinum substratum composition and cultural method such as embodiment 1 are said.
In substratum, add GA, add concentration and be respectively 0.5mg/L, 1.0mg/L, 3mg/L, 6mg/L and 10mg/L.
As shown in Figure 3, the result shows: when the GA addition was 1mg/L, schizochytrium limacinum living weight and DHA content all reached peak, were respectively 8.62g/Ld and 17.2%, respectively than the high 1.32g/Ld of control group and 4.2%.
Embodiment 4:IBA is to the influence of the living weight and the DHA content of schizochytrium limacinum
Schizochytrium limacinum substratum composition and cultural method such as embodiment 1 are said.
In the schizochytrium limacinum substratum, adding concentration is the IBA of 0.5mg/L, 1mg/L, 3mg/L, 6mg/L, 10mg/L.
As shown in Figure 4, the result shows: IBA is less to the influence of schizochytrium limacinum growth and DHA content, and along with the increase of IBA addition, schizochytrium limacinum living weight and DHA content all slowly rise.During at 3mg/L, the schizochytrium limacinum growth reaches peak with DHA content at the IBA addition, respectively than the high 1.36g/Ld of blank and 3.1%, plays restraining effect at addition above 3mg/L.
Embodiment 5: NAA (NAA) is to the influence of the living weight and the DHA content of schizochytrium limacinum
Schizochytrium limacinum substratum composition and cultural method such as embodiment 1 are said.
The NAA that in the schizochytrium limacinum substratum, adds 0.5mg/L, 1mg/L, 3mg/L, 6mg/L, 10mg/L respectively.
As shown in Figure 5; The result shows: when NAA addition during from 0.5mg/L to 6mg/L, along with the increase of NAA addition, schizochytrium limacinum living weight and DHA content increase gradually; But amplification is less; Reach maximum 8.40g/Ld at the NAA addition during for 6mg/L, 15.8%, respectively than the high 1.10g/Ld of control group and 2.8%; Addition is 10mg/L, and the obvious suppression effect is arranged.
Embodiment 6: indolylacetic acid (IAA) is to the influence of the living weight and the DHA content of schizochytrium limacinum
Schizochytrium limacinum substratum composition and cultural method such as embodiment 1 are said.
In the schizochytrium limacinum substratum, add IAA, concentration is 0.5mg/L, 1mg/L, 3mg/L, 6mg/L and 10mg/L.
As shown in Figure 6; The result shows: the IAA of lower concentration (0.5mg/L, 1mg/L and 3mg/L) has certain promoter action to schizochytrium limacinum growth and DHA content; Living weight and DHA content all reached peak when addition was 3mg/L; Be respectively 8.29g/Ld and 16.0%, respectively than the high 0.99g/Ld of control group and 3.0%.
Embodiment 7:3 kind of plant hormone is to schizochytrium limacinum growth and DHA content influence
In order to test the various plants hormone to the growth of schizochytrium limacinum and the influence of DHA content; The present invention has selected to have 3 kinds of hormones (6-BA, KT and GA) of obvious promoter action in the effective concentration scope, to carry out three factors, three horizontal quadratures test (6-BA:0mg/L; 1.5mg/L, 3.0mg/L; KT:0mg/L, 5.0mg/L, 10.0mg/L; GA:0mg/L, 0.5mg/L, 1.0mg/L).
The schizochytrium limacinum substratum composition and the culture condition of orthogonal experiment are identical with embodiment 1-6.
Orthogonal experiment results is as shown in table 1, and living weight is the highest is the 6th group (6-BA 1.5mg/L KT10mg/L), reaches 8.88g/L.d, improves 28.9% than control group; DHA content is the highest is the 4th group (6-BA1.5mg/L GA0.5mg/L), is 14.2%, has improved 13.6%.With embodiment 1, embodiment 2 and embodiment 3 relatively, the compound use of 3 kind of plant hormones is starkly lower than 6-BA, KT and the GA of independent use to the promoter action of DHA accumulation, explain in some makes up even antagonistic effect occurred, DHA content decreases.Therefore an amount of plant hormone is added in suggestion separately in the schizochytrium limacinum fermentative prodn.
Table 1:3 kind of plant hormone is to the orthogonal experiment of schizochytrium limacinum growth with the DHA content influence
Embodiment 8, in the 100L fermentor tank 6-BA to schizochytrium limacinum growth influence with DHA content
Therefore take in the schizochytrium limacinum fermention medium, to add separately the mode of plant hormone, present embodiment is chosen the 6-BA that in the said 6 kind of plant hormones schizochytrium limacinum is had the greatest impact and is carried out the enlarged culturing experiment, and culture medium prescription is specific as follows.
The 100L fermentation tank culture medium:
Glucose 70g/L;
Yeast extract 40g/L;
Seawater 50%;
The plant hormone experimental group, 6-BA 3mg/L;
The plant hormone control group, 6-BA 0mg/L;
Other conditions:
Original ph, 7.0
Leavening temperature, 25 ℃
Inoculum size, 10%
Air flow, 1: 1
Fermentation time, 72h.
After fermentation was accomplished, centrifugal collection thalline after the lyophilize, was surveyed somatic cells dry weight and the content of measuring DHA in the thalline.
Table 2:6-BA is to the influence (100L fermentor tank) of schizochytrium limacinum growth with DHA content
| Living weight (g/l d) | DHA content (%) | |
| Experimental group | 13.8 | 17.3 |
| Control group | 12.0 | 14.8 |
Find out from table 2; When the 100L retort was fermented, the concentration of adding 6-BA was 3mg/L, the living weight of schizochytrium limacinum from 12.0g/l d to 13.8g/l d; Improved 15.0%; DHA content from 14.8% to 17.3% has improved 16.9%, proves that 6-BA has promoter action significantly to the growth and the DHA accumulation of schizochytrium limacinum.
Schizochytrium limacinum substratum of the present invention is carbon source with glucose, and concentration is 60g/L; With the yeast extract is nitrogenous source, and concentration is 20g/L; The ratio of seawater is 50% in the substratum.It is 10-100g/L that above-mentioned normal experiment operation by one of skill in the art can be prepared glucose concn, and yeast extract concentration is 10-80g/L, and seawater accounts for the schizochytrium limacinum substratum of the 20%-80% of substratum TV; And schizochytrium limacinum is inoculated in the substratum of said preparation, the influence that detected plant hormone produces is identical with the experimental result of embodiment 1-6.And drawing glucose through same experimental implementation, to optimize concentration be 40-80g/L, and it is 20-60g/L that yeast extract is optimized concentration, and it is 30%-70% that seawater accounts for the Optimum of culture medium ratio, and under the substratum of optimizing concentration formula, the schizochytrium limacinum growing state is best.
The present invention has tested the influence to schizochytrium limacinum growth rate and DHA of phytokinin 6-benzyladenine (BA), furfuryl group aminopurine (KT), Plant hormones regulators,gibberellins (GA), plant hormone indolebutyric acid (IBA), naphthylacetic acid (NAA) and indolylacetic acid (IAA).The present invention adds micro-plant hormone through experiment showed, in the schizochytrium limacinum substratum, can significantly improve DHA content in the speed of growth and the cell of schizochytrium limacinum.And the present invention is simple and easy to do, and input-output ratio is high, for improving the schizochytrium limacinum fermentation production efficiency, reduces cost, and promotes industry development, and positive effect is arranged.
Above embodiment is only in order to explaining technical scheme of the present invention, but not limits it; Although the present invention has been carried out detailed explanation with reference to previous embodiment, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of relevant art scheme break away from the spirit and the scope of the present invention's technical scheme required for protection.
Claims (9)
1. add the substratum of plant hormone, it is characterized in that it comprises following component:
With glucose is carbon source, and concentration is 10-100g/L;
With the yeast extract is nitrogenous source, and concentration is 10-80g/L;
Seawater accounts for the 20%-80% of substratum TV;
Add a kind of in 6-benzyladenine, furfuryl group aminopurine, Plant hormones regulators,gibberellins, indolebutyric acid, naphthylacetic acid and the indolylacetic acid respectively.
2. the substratum of interpolation plant hormone according to claim 1 is characterized in that: said 6-benzyladenine concentration is 1-10mg/L; Furfuryl group aminopurine concentration is 1-20mg/L; Plant hormones regulators,gibberellins concentration is 1-10mg/L; Said indolebutyric acid concentration is 1-10mg/L; NAA concentration is 1-10mg/L; Indolylacetic acid concentration is 1-10mg/L.
3. the substratum of interpolation plant hormone according to claim 2 is characterized in that: the optimization concentration of said glucose is 40-80g/L.
4. the substratum of interpolation plant hormone according to claim 2 is characterized in that: the optimization concentration of said yeast extract is 20-60g/L.
5. the substratum of interpolation plant hormone according to claim 2 is characterized in that: it is 30%-70% that said seawater accounts for the Optimum of culture medium ratio.
6. add the application of substratum in the schizochytrium limacinum fermentation of plant hormone.
7. the application of the substratum of interpolation plant hormone according to claim 6 in schizochytrium limacinum fermentation, it is characterized in that: said schizochytrium limacinum is schizochytrium limacinum OUC88, culture presevation number: CGMCC NO.1240, preservation date: 2004.10.27.
8. the application of the substratum of interpolation plant hormone according to claim 6 in the schizochytrium limacinum fermentation, it is characterized in that: said schizochytrium limacinum inserts in the said substratum with the inoculum size of 1-10%.
9. the application of the substratum of interpolation plant hormone according to claim 6 in the schizochytrium limacinum fermentation, it is characterized in that: the fermentation culture temperature of said schizochytrium limacinum is 18-30 ℃, and the pH value is 4-8, and rotating speed is 100-250rpm, shaking culture 3-7 days.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104726508A (en) * | 2015-02-03 | 2015-06-24 | 昆明藻能生物科技有限公司 | Method for improving heterotrophic microbial fermentation via multiple chemical promoters to produce docosahexenoic acid |
| CN107699557A (en) * | 2017-11-10 | 2018-02-16 | 山东百龙创园生物科技股份有限公司 | A kind of preparation method of high-purity D psicoses |
| CN110558222A (en) * | 2019-09-05 | 2019-12-13 | 中国科学院南海海洋研究所 | Application of plant hormone in promoting growth rate, calcification and photosynthesis of large calcified seaweed |
| CN117947118A (en) * | 2024-03-25 | 2024-04-30 | 青岛农业大学 | Method for producing DHA by culturing schizochytrium limacinum by using kelp hydrolysate |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726508A (en) * | 2015-02-03 | 2015-06-24 | 昆明藻能生物科技有限公司 | Method for improving heterotrophic microbial fermentation via multiple chemical promoters to produce docosahexenoic acid |
| CN104726508B (en) * | 2015-02-03 | 2018-04-06 | 昆明藻能生物科技有限公司 | The method that a variety of chemical promoters improve microorganism heterotrophic fermentation production docosahexaenoic acid |
| CN107699557A (en) * | 2017-11-10 | 2018-02-16 | 山东百龙创园生物科技股份有限公司 | A kind of preparation method of high-purity D psicoses |
| CN110558222A (en) * | 2019-09-05 | 2019-12-13 | 中国科学院南海海洋研究所 | Application of plant hormone in promoting growth rate, calcification and photosynthesis of large calcified seaweed |
| CN117947118A (en) * | 2024-03-25 | 2024-04-30 | 青岛农业大学 | Method for producing DHA by culturing schizochytrium limacinum by using kelp hydrolysate |
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Application publication date: 20120104 |