CN104450529A - Schizochytrium limacinum strain as well as mutagenesis method and application thereof - Google Patents
Schizochytrium limacinum strain as well as mutagenesis method and application thereof Download PDFInfo
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- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
Abstract
The invention provides a schizochytrium limacinum strain as well as a mutagenesis method and an application thereof. In the prior art, although how to improve the content of DHA in a strain is reported, since the consideration factor is often single, how to significantly improve the content of DHA in the strain is not comprehensively explored which also is a key to achieve the industrialization of schizochytrium limacinum. Schizochytrium limacinum TC5-2 is obtained by virtue of an ion beam mutagenesis method, by combining a culture medium, optimizing a fermentation process and adding an exogenous factor clethodim, the content of DHA in the strain is significantly improved which is conductive to achieving the industrialization of production of DHA by virtue of schizochytrium limacinum.
Description
Technical field
The invention belongs to field of microbial fermentation, relate to a kind of schizochytrium limacinum bacterial strain and mutafacient system thereof and application.
Background technology
Docosahexenoic acid (DocosahexaenoicAcid, DHA) is the one of the necessary polyunsaturated fatty acid of mammalian growth, but owing to need absorb from the external world, is thus called as indispensable fatty acid.Research shows, DHA has very important physiological action to infant's brain and visual acuity, and has the physiological functions such as Prevention and Curation cardiovascular disorder, control senile dementia, suppression canceration, is thus widely used in food, medicine and feedstuff industry.
The traditional source of DHA is fish oil, but have that rate is low, resource-constrained and the defect such as purifying process is complicated.Marine microorganism is considered to the most promising commercial sources of DHA, wherein utilize micro-algae (comprising Crypthecodinium cohnii, fungi as schizochytrium limacinum and thraustochytriale etc.) to cultivate to produce DHA and there is larger advantage and produce potential, but the defect that ubiquity fermentation algae powder productive rate is low, DHA content is not high enough in lipid acid.In micro-algae, the content of schizochytrium limacinum fermentation production DHA is relatively high, is therefore also considered to the microorganism being hopeful most to realize DHA industrialization.
At present both at home and abroad about the research how improving DHA content mainly concentrates on following several respects: (1) finds the new strains of high yield DHA; (2) zymotechnique is improved; (3) Optimal Medium; (4) exogenous factor is added.The content that the DHA that marine fungus fission chytrid OUC 88 as cultivated is produced accounts for total fatty acids brings up to 23% ~ 44%; Produce in the research of the method for the micro-algae oil of DHA in another dry method, the method can be extracted and be up to 27.6g DHA in 1L fermented liquid; Improve in the research of DHA content at the third, by optimizing fragmentation cholerae strain fermention medium, the DHA that fragmentation vibrios is produced accounts for total fatty acid content and reaches 35%; Improve in the research of DHA content at the 4th kind, promoted the method for Microbe synthesis DHA by exterior addition factor, the method, by adding more than one acetic acid, citric acid and Simvastatin in the medium, makes DHA account for total fatty acid content and is up to 42.65%.Although more than research is all for the purpose of the DHA content improving lipid acid in bacterial strain, but often Consideration is single, how comprehensive discovery does not significantly improve DHA content in bacterial strain, make complex process, the cost increase of producing DHA, production efficiency is low, is unfavorable for realizing the industrialization that schizochytrium limacinum produces DHA.
Summary of the invention
The object of the embodiment of the present invention is that providing a kind of can grow and the schizochytrium limacinum TC5-2 of efficient synthesis DHA fast.
Another object of the embodiment of the present invention is to provide a kind of mutafacient system obtaining schizochytrium limacinum TC5-2.
The another object of the embodiment of the present invention is to provide the application that a kind of schizochytrium limacinum TC5-2 produces DHA.
In order to realize foregoing invention object, technical scheme of the present invention is as follows:
A kind of schizochytrium limacinum bacterial strain, its specific name is schizochytrium limacinum (Schizochytrium limacinum) TC5-2, and is deposited in China typical culture collection center on March 12nd, 2013, and its deposit number is CCTCC NO:M2013075.
And the mutafacient system of a kind of schizochytrium limacinum TC5-2, comprises the following steps:
Schizochytrium limacinum strain expanded culture is obtained seed liquor, described seed liquor is seeded to schizochytrium limacinum and carries out fermentation culture without in bacteria fermentation culture medium, obtain logarithmic phase bacterium liquid;
Described logarithmic phase bacterium liquid is made mycoderm, and utilizes energy to be the N of 10 ~ 30keV
+ionic fluid adopts pulsed to carry out mutagenic treatment to described mycoderm, wherein, and the N in described mutagenic treatment process
+the implantation dosage of ionic fluid is 50 × 10
17~ 200 × 10
17ions/cm
2;
Mycoderm after mutagenic treatment is carried out cultivate, filter out the high schizochytrium limacinum TC5-2 of DHA output.
And above-mentioned schizochytrium limacinum strain TC5-2 or the mutafacient system according to above-mentioned schizochytrium limacinum TC5-2 obtain the application of schizochytrium limacinum TC5-2 for the production of DHA.
Above-mentioned novel schizochytrium limacinum TC5-2 fecundity is strong, and heritability is stablized, and is the desirable strain of high yield DHA.
In the mutafacient system of above-mentioned schizochytrium limacinum TC5-2, pass through N
+ion beam mutation mode obtains best mutagenic strain TC5-2, and this mutafacient system is simple and easy to implement, and mutagenesis obtained strains mutation rate is high, and heritability is stablized.
Above-mentioned schizochytrium limacinum strain TC5-2 is for the production of DHA, and its application art is simple, and condition is easily controlled, and efficiency is high, and can significantly improve the content of total fatty acids and DHA, is conducive to realizing the industrialization that schizochytrium limacinum produces DHA.
Accompanying drawing explanation
Fig. 1 is the mutafacient system process flow sheet of embodiment of the present invention schizochytrium limacinum TC5-2;
Fig. 2 is the thalli morphology figure of embodiment of the present invention schizochytrium limacinum TC5-2.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The embodiment of the present invention provides a kind of schizochytrium limacinum bacterial strain, its specific name is schizochytrium limacinum (Schizochytrium limacinum) TC5-2, and being deposited in China typical culture collection center on March 12nd, 2013, its deposit number is CCTCC NO:M2013075.The fecundity of this schizochytrium limacinum TC5-2 is strong, and heritability is stablized, and is the desirable strain of high yield DHA.
Correspondingly, the embodiment of the present invention also provides the mutafacient system of a kind of schizochytrium limacinum TC5-2, and as shown in Figure 1, the method comprises the following steps in its mutafacient system technical process:
S01. the cultivation of schizochytrium limacinum starting strain: with the schizochytrium limacinum before mutagenesis for starting strain, the strain expanded culture that will set out obtains seed liquor, described seed liquor is seeded to schizochytrium limacinum and carries out fermentation culture without in bacteria fermentation culture medium, obtains logarithmic phase bacterium liquid;
S02. adopt ion beam mutagenesis: aseptically, the logarithmic phase bacterium liquid in step S01 is made mycoderm, and utilize energy to be the N of 10 ~ 30keV
+ionic fluid adopts pulsed to carry out mutagenic treatment to described mycoderm, wherein, and N in mutagenic treatment process
+the implantation dosage of ionic fluid is 50 × 10
17~ 200 × 10
17ions/cm
2;
S03. the mycoderm after mutagenesis is carried out cultivating, Screening Treatment: the mycoderm after mutagenesis in step S02 is carried out cultivate, filter out the high schizochytrium limacinum TC5-2 of DHA output.
In above-mentioned steps S01, the starting strain of schizochytrium limacinum TC5-2 can select known schizochytrium limacinum, preferably derives from the schizochytrium limacinum bacterial strain of Beihai Fisheries Base Guangxi Province seabeach mangrove forest separation screening gained.
In this step S01, this schizochytrium limacinum can directly select existing schizochytrium limacinum without bacteria fermentation culture medium without bacteria fermentation culture medium, be preferably following utilize schizochytrium limacinum TC5-2 produce DHA method in use without bacteria fermentation culture medium.By improving existing fermention medium, schizochytrium limacinum of the present invention is nutritious without bacteria fermentation culture medium, can promote quick division and the growth and breeding of cell, significantly improve growth rate and the breeding potential of bacterial classification.
In above-mentioned steps S02, adopt nitrogen ion beam injection mode mutagenic species, cause schizochytrium limacinum to morph, and with the stable increase of implantation dosage, lethality rate is also on the slow rise, and is shown in Table 1.Preferably, above-mentioned nitrogen ion beam implantation dosage can be 50 × 10
17, 100 × 10
17, 150 × 10
17, 200 × 10
17ions/cm
2.
In above-mentioned steps S03, the mutagenic species through the process of step S02 ion beam mutagenesis is numbered screening.Concrete screening method is that the mutagenic species of numbering is carried out enlarged culturing, and more various mutagenic strain produces the content of DHA, selects the mutagenic species that DHA productive rate is the highest.Through screening, find that the DHA productive rate of schizochytrium limacinum TC5-2 is the highest, up to 46.3%, therefore selected schizochytrium limacinum TC5-2 is optimum strain, and its thalli morphology as shown in Figure 2.
In this step S03, this mutagenic species can be carried out enlarged culturing without bacteria fermentation culture medium by the existing schizochytrium limacinum be directly seeded in above-mentioned S01 by the enlarged culturing of mutagenic species, be preferably directly seeded to following utilize schizochytrium limacinum TC5-2 produce DHA method in use carry out enlarged culturing without bacteria fermentation culture medium.
From the above mentioned, in the mutafacient system of above-mentioned schizochytrium limacinum TC5-2, N is passed through
+ion beam mutation mode obtains best mutagenic strain TC5-2, and this mutafacient system is simple and easy to implement, and mutagenesis obtained strains mutation rate is high, and heritability is stablized.
Correspondingly, the embodiment of the present invention also provides the application of schizochytrium limacinum TC5-2, specifically by schizochytrium limacinum TC5-2 for the production of DHA.
In a particular embodiment, the method utilizing schizochytrium limacinum TC5-2 to produce DHA comprises the steps:
Above-mentioned schizochytrium limacinum TC5-2 is seeded to schizochytrium limacinum and carries out enlarged culturing without in bacteria fermentation culture medium.
As preferred embodiment, above-mentioned schizochytrium limacinum comprises the component of following content without bacteria fermentation culture medium:
The above-mentioned carbon source without bacteria fermentation culture medium is glucose, and nitrogenous source is corn steep liquor, soy peptone and yeast extract paste.In addition, also add composite artificial seawater in substratum, except utilizing in seawater except naturally occurring inorganic salt, substratum is in addition again by adding synthetic sea water to provide sufficient P source, S source and Mn
2+, Zn
2+plasma, except ensureing that certain salt is outside one's consideration, also keeps the osmotic balance of cell, and provides required material for the growth and breeding of cell.
In further preferred embodiment, above-mentioned synthetic sea water comprises the component of following content:
Above-mentioned VITAMIN of also adding abundant species without bacteria fermentation culture medium.After VITAMIN different for various function is mixed, the common adjustment participating in organism metabolism, thus be applicable to more schizochytrium limacinum growth, promote the accumulation of DHA in schizochytrium limacinum further.If the present invention is by contrast experiment, when namely not adding vitamin complex (comparative example 2), its DHA content, compared with interpolation vitamin complex (embodiment 6), reduces 40.7%, shows that vitamin complex improves the DHA content of schizochytrium limacinum TC5-2.
In further preferred embodiment, above-mentioned Theravite comprises the component of following content:
In addition, 10 ~ 30 μm of ol/L exogenous factor clethodims are also added in above-mentioned substratum.
After adding the clethodim of trace in this substratum, effectively can improve the accumulation of lipid acid and DHA content in schizochytrium limacinum TC5-2.With do not add compared with clethodim (comparative example 3), the bacterial strain DHA content of adding clethodim (embodiment 6) increases by 48.33%, shows that clethodim has promoter action to schizochytrium limacinum DHA synthesis, can significantly improve the content of DHA in schizochytrium limacinum.
As preferred embodiment, the condition of above-mentioned enlarged culturing can carry out enlarged culturing according to the culture condition of other schizochytrium limacinum routines.But in order to the productive rate that improves DHA and efficiency, in a preferred embodiment, the condition of this enlarged culturing is: in first 40 hours, temperature is 21-25 DEG C, then temperature controls as 15-20 DEG C.
In further preferred embodiment, the air flow in above-mentioned enlarged culturing process is 0.5 ~ 2.0vvm, and this enlarged culturing is carried out in airlift reactor.
Like this, above-mentioned enlarged culturing preferably first improves cell concn under higher culture temperature, then reduces temperature to accumulate DHA; Meanwhile, the present invention preferably uses airlift reactor significantly to reduce the suppression of mechanical stirring to thalli growth, is convenient to the accumulation of DHA.In addition, pass into a certain amount of air and contribute to improving oxygen concn in substratum, be conducive to the Growth and reproduction of the schizochytrium limacinum of heterotrophism aerobic-type.
From the above mentioned, above-mentioned schizochytrium limacinum TC5-2 cultivating without bacteria fermentation culture medium not only through optimization process, add exogenous factor clethodim simultaneously, and combining the suitability for industrialized production that the zymotechnique optimized carries out DHA, culture condition considers that application art is simple comprehensively, condition is easily controlled, efficiency is high, and can significantly improve the content of total fatty acids and DHA, is conducive to wideling popularize of this bacterial strain and application art thereof.
Now for the mutagenesis of schizochytrium limacinum TC5-2, screening and the method for producing DHA, the embodiment of the present invention is further elaborated.
Embodiment 1
The mutagenesis of schizochytrium limacinum TC5-2, comprises the following steps:
S11. the cultivation of schizochytrium limacinum: with the schizochytrium limacinum (the schizochytrium limacinum strain of Beihai Fisheries Base Guangxi Province seabeach mangrove forest separation screening gained) before mutagenesis for starting strain, select single bacterium colony of starting strain, at 10mL liquid asepsis fermention medium, (composition comprised without bacteria fermentation culture medium is: synthetic sea water 600g/L; Soy peptone 30g/L; Yeast extract paste 40g/L; Glucose 90g/L; Corn steep liquor 15g/L; Theravite 5g/L; Clethodim 20 μm of ol/L.Wherein, synthetic sea water formula is: NaCl28g/L, MgCl
25g/L, KCl1g/L, CaCl
21.5g/L, NaHSO
46g/L, Na
2cO
38g/L, Na
2sO
410g/L, MgSO
43g/L, MnSO
45g/L, KH
2pO
44g/L, K
2hPO
47g/L, ZnSO
43g/L; Theravite formula is: vitamins B
13g/L, vitamins B
210g/L, vitamins B
58g/L, vitamins B
64g/L, vitamin PP 10g/L, vitamin H 12g/L, folic acid 45g/L, inositol 4g/L.) in test tube, under 25 DEG C and 150r/min condition, shaking culture is after 36 hours, is inoculated in the triangular flask that 50mL fermention medium (the same) is housed with 10% inoculum size, under 25 DEG C and 150r/min condition, shaking culture 36 hours, obtains logarithmic phase bacterium liquid;
S12. ion beam mutagenesis: aseptically, adds 1mL above-mentioned logarithmic phase bacterium liquid, smoothens and make mycoderm in the empty plate of each sterilizing, is the N of 20keV by energy
+ionic fluid injects the mycoderm be made up of logarithmic phase bacterium liquid with duoplasmatron source pulsed, wherein implantation dosage is the dosage of hereafter embodiment 1 in table 1.
Embodiment 2
The mutagenesis of schizochytrium limacinum TC5-2, the mutagenesis steps of this mutagenesis reference embodiment 1, wherein, in the ion beam mutagenesis step of S12, duoplasmatron source pulsed injects the mycoderm be made up of logarithmic phase bacterium liquid, and implantation dosage is the dosage of hereafter embodiment 2 in table 1.
Embodiment 3
The mutagenesis of schizochytrium limacinum TC5-2, the mutagenesis steps of this mutagenesis reference embodiment 1, wherein, in the ion beam mutagenesis step of S12, duoplasmatron source pulsed injects the mycoderm be made up of logarithmic phase bacterium liquid, and implantation dosage is the dosage of hereafter embodiment 3 in table 1.
Embodiment 4
The mutagenesis of schizochytrium limacinum TC5-2, the mutagenesis steps of this mutagenesis reference embodiment 1, wherein, in the ion beam mutagenesis step of S12, duoplasmatron source pulsed injects the mycoderm be made up of logarithmic phase bacterium liquid, and implantation dosage is the dosage of hereafter embodiment 4 in table 1.
Mycoderm after embodiment 1-4 mutagenic treatment is carried out the mensuration of lethality rate, measurement result is as described in Table 1:
Table 1
As shown in Table 1, at implantation dosage respectively in 50,100,150,200 × 10
17ions/cm
2under the condition that gradient is gone forward one by one, the trend that the lethality rate of implantation dosage and bacterial classification keeps similar direct ratio to increase.Consider the positive and negative mutation rate of bacterial classification, N
+ion beam mutation dosage is chosen to be 100 × 10
17ions/cm
2.
Embodiment 5
Through N
+the screening of the mutagenic species after ion beam mutagenesis:
Wash out according to the mutagenesis mycoderm 9mL nutrient solution after the mutafacient system process of above-described embodiment 2, then dilution is coated with dull and stereotyped respectively.Cultivate 72 hours under 25 DEG C of conditions, after growing bacterium colony, choosing colony, and be numbered according in following table 2, shake-flask culture subsequently, detect lipid acid and the DHA of each bacterial strain of gained after 72 hours, determine the mutagenic strain that DHA output is the highest thus.Wherein, after 72 hours, each lipid acid of numbering bacterial strain and the detected result of DHA see the following form 2.
Table 2
As shown in Table 2, the mutagenic strain DHA output being numbered TC5-2 is the highest, and the output of total fatty acid content is also high, for producing the desirable strain of DHA.
Embodiment 6
The schizochytrium limacinum TC5-2 of screening in embodiment 5 is utilized to carry out enlarged culturing for extracting DHA.
By in embodiment 5 screening schizochytrium limacinum TC5-2 be seeded in embodiment 1 step S11 without in bacteria fermentation culture medium, and postvaccinal substratum is placed in 5L mechanically stirred reactor carry out enlarged culturing thus fermentation produce DHA, control temperature 20 DEG C, control stirring velocity 200r/min, air flow 0.5vvm.Detect after cultivating 60h, bacterial strain amount reaches 32.3g/L, and total fatty acid content reaches 56.3%, and DHA content reaches 46.3%.
Comparative example 1
Detect after the starting strain of embodiment 1 step S11 is directly carried out cultivation 60h according to the culture condition of embodiment 6 without mutagenic treatment, bacterial strain amount reaches 22.6g/L, and total fatty acid content reaches 45.4%, and DHA content reaches 38.2%.
Comparing embodiment 6 is known with comparative example 1, starting strain is through mutagenic treatment, the schizochytrium limacinum TC5-2 bacterial strain amount filtered out after mutagenesis improves 42.9%, total fatty acid content improves 24.01%, DHA content improves 21.2%, these data show under same fermentation condition, and the bacterial strain amount of the schizochytrium limacinum TC5-2 that nitrogen ion beam is mutagenic obtained, total fatty acid content and DHA content all significantly improve.
Comparative example 2
Enlarged culturing is carried out for extracting DHA according to embodiment 6 couples of schizochytrium limacinum TC5-2, difference from Example 6 is, without the Theravite of bacteria fermentation culture medium not containing 5g/L, the schizochytrium limacinum TC5-2 by screening in embodiment 5 is seeded to following without bacteria fermentation culture medium:
Synthetic sea water 600g/L; Soy peptone 30g/L; Yeast extract paste 40g/L; Glucose 90g/L; Corn steep liquor 15g/L; Clethodim 20 μm of ol/L.Wherein, synthetic sea water formula is: NaCl28g/L, MgCl
25g/L, KCl1g/L, CaCl
21.5g/L, NaHSO
46g/L, Na
2cO
38g/L, Na
2sO
410g/L, MgSO
43g/L, MnSO
45g/L, KH
2pO
44g/L, K
2hPO
47g/L, ZnSO
43g/L.
Detect after cultivating 60h, biomass reaches 15.3g/L, and total fatty acid content reaches 32.5%, and DHA content reaches 27.5%.Compared with embodiment 6, its biomass reduces 52.6%, and total fatty acids reduces 42.3%, and DHA content reduces 40.7%, shows that Theravite provides abundant nutrient environment for schizochytrium limacinum, is conducive to the raising of the DHA content of schizochytrium limacinum TC5-2 further.
Comparative example 3
Carry out enlarged culturing for extracting DHA according to embodiment 6 couples of schizochytrium limacinum TC5-2, difference from Example 6 is, without bacteria fermentation culture medium not containing clethodim component, the schizochytrium limacinum TC5-2 by screening in embodiment 5 is seeded to following without bacteria fermentation culture medium:
Synthetic sea water 600g/L; Soy peptone 30g/L; Yeast extract paste 40g/L; Glucose 90g/L; Corn steep liquor 15g/L; Theravite 5g/L.Wherein, synthetic sea water formula is: NaCl28g/L, MgCl
25g/L, KCl1g/L, CaCl
21.5g/L, NaHSO
46g/L, Na
2cO
38g/L, Na
2sO
410g/L, MgSO
43g/L, MnSO
45g/L, KH
2pO
44g/L, K
2hPO
47g/L, ZnSO
43g/L; Theravite formula is: vitamins B
13g/L, vitamins B
210g/L, vitamins B
58g/L, vitamins B
64g/L, vitamin PP 10g/L, vitamin H 12g/L, folic acid 45g/L, inositol 4g/L.
Detect after cultivating 60h, biomass reaches 30.1g/L, and total fatty acid content reaches 43.5%, and DHA content reaches 31.2%.Compared with embodiment 6, its biomass reduces 6.81%, and total fatty acids reduces 22.74%, and DHA content reduces 32.58%, shows that exogenous factor clethodim promotes that schizochytrium limacinum TC5-2 synthesizes DHA.
Embodiment 7
Enlarged culturing is carried out for extracting DHA: adopt mechanically stirred reactor fermentation to produce DHA according to the schizochytrium limacinum TC5-2 of screening in embodiment 5.
By in embodiment 5 screening schizochytrium limacinum TC5-2 be seeded in embodiment 1 step S11 without in bacteria fermentation culture medium, and postvaccinal substratum is placed in 5L mechanically stirred reactor carry out enlarged culturing thus fermentation produce DHA, control temperature 25 DEG C, controls dissolved oxygen 30%, air flow 1.5vvm.Cultivate 60h bacterial strain amount and reach 41.2g/L, total fatty acid content reaches 52.5%, and DHA content reaches 45.8%.
By this embodiment 7 compared with embodiment 6, the enlarged culturing of the present embodiment can make its biomass increase by 27.55%, total fatty acids reduces 6.75%, DHA content reduces 1.04%, show suitably to improve the Growth and reproduction that culture temperature and air flow can promote cell, but be unfavorable for the accumulation of total fatty acids and DHA, this may promote the breeding of cell with high temperature but suppress the synthesis of DHA relevant.
Embodiment 8
The schizochytrium limacinum TC5-2 of screening in embodiment 5 is utilized to carry out enlarged culturing for extracting DHA: to adopt airlift reactor fermentation to produce DHA.
By in embodiment 5 screening schizochytrium limacinum TC5-2 be seeded in embodiment 1 step S11 without in bacteria fermentation culture medium, and postvaccinal substratum is placed in 10L airlift reactor carry out enlarged culturing thus fermentation produce DHA, control temperature 25 DEG C, air flow 1.5vvm.Cultivate 60h bacterial strain amount and reach 40.1g/L, total fatty acid content reaches 55.2%, and DHA content reaches 49.2%.
Compared with this embodiment 8 being cultivated with the mechanically stirred reactor of embodiment 7, its biomass declines 2.67% slightly, but total fatty acids increases by 5.14%, and DHA content increases by 7.42%.Show that airlift reactor fermentation reduces churned mechanically shearing force, be conducive to the accumulation of DHA content.
Embodiment 9
Carry out enlarged culturing for extracting DHA according to embodiment 8 couples of schizochytrium limacinum TC5-2, difference from Example 8 is in enlarged culturing, and control temperature 25 DEG C, air flow is adjusted to 0.5vvm.Cultivate 60h bacterial strain amount and reach 35.2g/L, total fatty acid content reaches 48.4%, and DHA content reaches 46.2%.
By this embodiment 9 compared with embodiment 8, its biomass reduces 12.22%, and total fatty acids reduces 12.32%, and DHA content reduces 6.10%.Show to reduce air flow separately, biomass, total fatty acids and DHA content can be made simultaneously to reduce.This is because schizochytrium limacinum belongs to heterotrophism aerobic-type microorganism, reduce the growth velocity that air flow suppresses its cell, especially directly reduce the content of biomass and total fatty acids.
Embodiment 10
The schizochytrium limacinum TC5-2 of screening in embodiment 5 is utilized to carry out enlarged culturing for extracting DHA: to adopt airlift reactor temperature inversion to control fermentation and produce DHA
By in embodiment 5 screening schizochytrium limacinum TC5-2 be seeded in embodiment 1 step S11 without in bacteria fermentation culture medium, and postvaccinal substratum is placed in 10L airlift reactor carry out enlarged culturing thus fermentation produce DHA, in culturing process, first 40 hours control temperature 25 DEG C, then control temperature 15 DEG C, air flow 2.0vvm.Cultivate 60h bacterial strain amount and reach 38.6g/L, total fatty acid content reaches 57.2%, and DHA content reaches 52.4%.
By this embodiment 10 compared with the constant temperature culture of embodiment 8, its biomass declines 2.67% slightly, but total fatty acids increases by 5.14%, and DHA content increases by 7.42%.Show that early stage cultivates under higher culture temperature and can improve cell concn, then reduce culture temperature and be conducive to accumulating DHA.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a schizochytrium limacinum bacterial strain, its specific name is schizochytrium limacinum (Schizochytrium limacinum) TC5-2, and being deposited in China typical culture collection center on March 12nd, 2013, its deposit number is CCTCC NO:M2013075.
2. the mutafacient system of schizochytrium limacinum TC5-2, comprises the following steps:
Schizochytrium limacinum strain expanded culture is obtained seed liquor, described seed liquor is seeded to schizochytrium limacinum and carries out fermentation culture without in bacteria fermentation culture medium, obtain logarithmic phase bacterium liquid;
Described logarithmic phase bacterium liquid is made mycoderm, and utilizes energy to be the N of 10 ~ 30keV
+ionic fluid adopts pulsed to carry out mutagenic treatment to described mycoderm, wherein, and the N in described mutagenic treatment process
+the implantation dosage of ionic fluid is 50 × 10
17~ 200 × 10
17ions/cm
2;
Mycoderm after mutagenic treatment is carried out cultivate, filter out the high schizochytrium limacinum TC5-2 of docosahexenoic acid output.
3. the mutafacient system of schizochytrium limacinum TC5-2 as claimed in claim 2, is characterized in that: describedly comprise following component without bacteria fermentation culture medium:
4. the mutafacient system of schizochytrium limacinum TC5-2 as claimed in claim 3, is characterized in that: described synthetic sea water formula comprises following component:
5. the mutafacient system of the schizochytrium limacinum TC5-2 as described in claim 3 or 4, is characterized in that, described Theravite formula comprises following component:
6. the mutafacient system of schizochytrium limacinum TC5-2 as claimed in claim 5, it is characterized in that, described schizochytrium limacinum is also added with exogenous factor clethodim without in bacteria fermentation culture medium, and content is 10 ~ 30 μm of ol/L.
7. the mutafacient system acquisition schizochytrium limacinum TC5-2 of schizochytrium limacinum strain TC5-2 as claimed in claim 1 or described schizochytrium limacinum TC5-2 as arbitrary in claim 2 ~ 6 is for the production of docosahexenoic acid.
8. the application of schizochytrium limacinum strain TC5-2 as claimed in claim 7, is characterized in that: described schizochytrium limacinum TC5-2 comprises the steps: for the production of the method for docosahexenoic acid
Described schizochytrium limacinum TC5-2 is seeded to as arbitrary in claim 2 ~ 6 as described in schizochytrium limacinum carry out enlarged culturing without in bacteria fermentation culture medium.
9. the application of schizochytrium limacinum strain TC5-2 as claimed in claim 8, is characterized in that: the condition of described enlarged culturing: in first 40 hours, temperature is 21 ~ 25 DEG C, and then temperature controls is 15 ~ 20 DEG C.
10. the application of schizochytrium limacinum strain TC5-2 as claimed in claim 8 or 9, it is characterized in that, described enlarged culturing is carried out in airlift reactor, and air flow is 0.5 ~ 2.0vvm.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105558305A (en) * | 2015-12-10 | 2016-05-11 | 新奥科技发展有限公司 | Schizochytrium limacinum and application thereof |
| CN106676041A (en) * | 2016-12-23 | 2017-05-17 | 广州富诺健康科技股份有限公司 | Fission vibrio strain and method for producing DHA (docosahexaenoic acid) by aid of fission vibrio strain |
| CN111235035A (en) * | 2019-12-30 | 2020-06-05 | 嘉必优生物技术（武汉）股份有限公司 | Schizochytrium limacinum mutant strain, and method and application thereof in preparation of docosahexaenoic acid grease |
| CN114456957A (en) * | 2021-12-22 | 2022-05-10 | 南京师范大学 | Schizochytrium limacinum genetic engineering strain, construction method and application thereof |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112481348A (en) * | 2019-09-11 | 2021-03-12 | 天津大学青岛海洋技术研究院 | Screening method of high-yield DHA Schizochytrium limacinum mutant strain |
| CN114672422A (en) * | 2022-03-08 | 2022-06-28 | 南京工业大学 | Cultivation method of thraustochytrid strain producing cis-11-hexadecenal |
| CN114437947B (en) * | 2022-04-08 | 2022-06-03 | 中国农业科学院北京畜牧兽医研究所 | High-protein high-DHA schizochytrium limacinum trap strain and application thereof as feed additive |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1264967C (en) * | 2004-12-08 | 2006-07-19 | 中国海洋大学 | Industrial use of marine fungus fission chytrid OUC88 |
| CN102839129A (en) * | 2011-06-23 | 2012-12-26 | 法国罗凯特兄弟公司 | Fragmentation chytrid mutagenesis method and variant produced by fragmentation chytrid mutagenesis method |
| CN102899254A (en) * | 2012-09-13 | 2013-01-30 | 温州大学 | Shizochytrium sp. and method of high density fermentation production of DHA by using same |
-
2013
- 2013-09-25 CN CN201710454236.6A patent/CN107164238B/en active Active
- 2013-09-25 CN CN201310443058.9A patent/CN104450529A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1264967C (en) * | 2004-12-08 | 2006-07-19 | 中国海洋大学 | Industrial use of marine fungus fission chytrid OUC88 |
| CN102839129A (en) * | 2011-06-23 | 2012-12-26 | 法国罗凯特兄弟公司 | Fragmentation chytrid mutagenesis method and variant produced by fragmentation chytrid mutagenesis method |
| CN102899254A (en) * | 2012-09-13 | 2013-01-30 | 温州大学 | Shizochytrium sp. and method of high density fermentation production of DHA by using same |
Non-Patent Citations (1)
| Title |
|---|
| 许永 等: "裂殖壶菌诱变筛选的研究", 《中国海洋大学学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105558305A (en) * | 2015-12-10 | 2016-05-11 | 新奥科技发展有限公司 | Schizochytrium limacinum and application thereof |
| CN105558305B (en) * | 2015-12-10 | 2019-12-03 | 新奥科技发展有限公司 | Schizochytrium limacinum mutant strain and its application |
| CN106676041A (en) * | 2016-12-23 | 2017-05-17 | 广州富诺健康科技股份有限公司 | Fission vibrio strain and method for producing DHA (docosahexaenoic acid) by aid of fission vibrio strain |
| CN106676041B (en) * | 2016-12-23 | 2020-01-14 | 广州富诺健康科技股份有限公司 | Fission arc strain and method for producing DHA (docosahexaenoic acid) by using same |
| CN111235035A (en) * | 2019-12-30 | 2020-06-05 | 嘉必优生物技术（武汉）股份有限公司 | Schizochytrium limacinum mutant strain, and method and application thereof in preparation of docosahexaenoic acid grease |
| CN114456957A (en) * | 2021-12-22 | 2022-05-10 | 南京师范大学 | Schizochytrium limacinum genetic engineering strain, construction method and application thereof |
| CN114456957B (en) * | 2021-12-22 | 2023-07-25 | 南京脂禾生物科技有限公司 | Schizochytrium limacinum genetically engineered strain, construction method and application thereof |
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| CN107164238A (en) | 2017-09-15 |
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