CN102676393A - Saccharopolyspora spinosa for producing spinosad and culture and application of saccharopolyspora spinosa - Google Patents
Saccharopolyspora spinosa for producing spinosad and culture and application of saccharopolyspora spinosa Download PDFInfo
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- CN102676393A CN102676393A CN2011104295501A CN201110429550A CN102676393A CN 102676393 A CN102676393 A CN 102676393A CN 2011104295501 A CN2011104295501 A CN 2011104295501A CN 201110429550 A CN201110429550 A CN 201110429550A CN 102676393 A CN102676393 A CN 102676393A
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- pleocidin
- spinosad
- saccharopolyspora spinosa
- saccharopolyspora
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- 241000868102 Saccharopolyspora spinosa Species 0.000 title claims abstract description 16
- JFLRKDZMHNBDQS-UCQUSYKYSA-N CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C(=C[C@H]3[C@@H]2CC(=O)O1)C)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C.CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C=C[C@H]3C2CC(=O)O1)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C Chemical compound CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C(=C[C@H]3[C@@H]2CC(=O)O1)C)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C.CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C=C[C@H]3C2CC(=O)O1)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C JFLRKDZMHNBDQS-UCQUSYKYSA-N 0.000 title abstract description 9
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses saccharopolyspora spinosa for producing spinosad and belongs to the field of microorganisms. The strain number of the saccharopolyspora spinosa BCSsBFSg<5>-006 is CGMCC (China general microbiological culture collection center) No.5546, and a fermentation medium for the saccharopolyspora spinosa of the spinosad comprises glucose 40g/L, soluble starch 30g/L, corn steep liquor 15g/L, cottonseed concentration powder 20g/L, yeast powder 20g/L and calcium carbonate 1g/L, and is prepared by distilled water with the pH (potential of hydrogen) of 7.0-7.2 regulated by NaOH 1mol/L. Compared with conventional spinosad production strains, the mutant strain BCSsBFSg<5>-006 is higher in spinosad production capacity, and accordingly yield of the spinosad can be effectively increased, and production cost is reduced.
Description
Technical field
The invention belongs to the fermentation engineering field, be specifically related to thorn saccharopolyspora strain and cultural method thereof that a strain produces pleocidin in a large number.
Background technology
Pleocidin [spinosad] claims thorn sugared rhzomorph again, is through aerobic fermentation polyketone route of synthesis synthetic macrolide antibiotics by thorn saccharopolyspora strain (Saccharopolyspora spinosa).This kind microbiotic has the quick-acting of chemical pesticide and the security of biological pesticide concurrently, can not produce environment and pollute, and to fish, human security coefficient height, the development research of this environment friendly agricultural has in recent years obtained develop rapidly.Pleocidin; As a kind of biological pesticide; Have some unique physics, chemistry and biology characteristic; Wide like insecticidal spectrum, the mode of action is unique, insecticidal activity is high, the transformation period is short, residual low, no resistance, to person poultry harmless, characteristic such as low in the pollution of the environment, be a kind of have tag and the toxic novel microorganism botanical pesticide of ingesting, obtained U.S.'s " presidential green chemical challenge prize (Presidential Green Chemistry Challenge Award) " in 1999.These characteristics have determined pleocidin in husbandry is produced, to have broad application prospects, and this Products Development has good economic benefit and social benefit.
The thorn saccharopolyspora strain belongs to saccharopolyspora strain and belongs to (Saccharopoly), is unique bacterial strain that can produce pleocidin through aerobic fermentation of finding so far.Nineteen eighty-two; Serve pedotheque from gathering near the depleted sugarcane system sweet wine factory in passing when chemist of EliLilly and Company (gift comes company) natural product research department spends a holiday in the Caribbean Sea, the personnel of this department screen, separate and ferment sample subsequently.People such as Boeck have isolated pleocidin component A, B, C, D, E, F, H, J in cluster needling saccharopolyspora strain QYLZ 88912 nutrient solutions first; Its main active ingredient is A component (spinosynA) and D component (spinosyn D); Wherein the A component accounts for 85-90%; The D component accounts for 10-15%, its mixture called after Spinosad, Chinese pleocidin by name.1986, pleocidin producing bacteria was determined and belongs to the Saccharopoly genus, and Full Name in English is Saccharopolyspora Mertz & Yao.1989, Dow Chemical and Li Lai Merger of Company their crop science department, become The Dow Agrosciences, LLC., after again then become Dow Agrosciences LLC (Dow AgroSciences Company) and come common R and D pleocidin.1994; The Dow Agrosciences, LLC. is submitted to U.S. Environmental Protection Administration (EPA) with this novel biological pesticide; In view of this medicament is not stranded in the environment, and quite low to Mammals and hydrobiological toxicity, and U.S. Environmental Protection Administration gave preferential registration in 1997.The Dow Agrosciences, LLC. is with this biological pesticide successful implementation commercialization immediately, and commodity are called Tracer (urge and killing), and registration is used to prevent and treat the lepidoptera pest in cotton field.On 24 countries surpass 100 various crop, registered in 1999, trade(brand)name also is useful on the Success (dish happiness) of greengrocery, Conserve and Spintor.At present, pleocidin has been registered to be used to prevent and treat on the 200 various crop insects in more than 60 country and has been used about 200,000,000 dollars of annual sales amount.Pleocidin in China's registration is mainly used in " urge and killing " (pleocidin 48% suspension agent) on the cotton and is used for " dish happiness " (pleocidin 2.5% suspension agent) on the vegetables.2007; The s-generation product of pleocidin (trade(brand)name DELEGATETM WG) through estimating, has obtained first whole world registration in nz, and this product consumption is few; It is faster in environment, to degrade, and is used to control carpocapsa pononella (a kind of thorny primary pest on the fruit tree).At present, it is higher that the research field of external pleocidin has extended to the exploitation insecticidal activity, and on the better pleocidin verivate of biological safety, and China does not still have industrialized report.Although domestic research to the fermentative prodn pleocidin is started late,, also has very big gap than the foreign study level though also obtained very big progress recent years.Therefore strengthen the research of pleocidin,, China's husbandry production is had important use be worth and environmental benefit for China's pesticide field is opened up a kind of novel green product.
The main method of at present synthetic pleocidin is mikrobe liquid fermentation method and microorganism solid fermentation method.The microorganism solid fermentation method is used for the synthetic of pleocidin, and the subject matter that faces is that zymotechnique difficultly control, yielded poorly, the extraction cost height, is difficult to large-scale production, and therefore, this method does not possess using value.It is synthetic that the preparation method of pleocidin mainly concentrates on the microbial liquid fermentation; The biosynthesizing pleocidin has outstanding advantage; Processing condition are more easy to control, constant product quality, extraction are comparatively convenient; But mikrobe liquid state fermentation production pleocidin output is very low, and the fermentation period of length fails to realize large-scale industrial production, so that has influenced the application of this material.
Summary of the invention:
The technical problem that the present invention solves provides thorn saccharopolyspora strain and the cultural method thereof that pleocidin is produced in a strain;
The present invention utilizes the complex mutation technology screening to obtain strain pleocidin superior strain thorn saccharopolyspora strain (Saccharopolyspora spinosa) BCSsBFSg
5-006, bacterium numbering CGMCC No.5546.
This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 7th, 2011, the classification name: the thorn saccharopolyspora strain; Depositary institution address: in the Institute of Microorganism, Academia Sinica of Datun Road, Chaoyang District, Beijing City.CGMCC No.5546 has improved the output of pleocidin, effectively reduces production cost, has realized the suitability for industrialized production of pleocidin.
Mutagenic strain thorn saccharopolyspora strain BCSsBFSg
5-006 (Saccharopolyspora spinosa) CGMCC No.5546) be to be that starting strain obtains behind ultraviolet mutagenesis with thorn saccharopolyspora strain (Saccharopolyspora spinosa) the bacterial strain QYLZ 88912 that filters out near the pedotheque China Guangxi depleted sugarcane system sweet wine factory.
Mutagenic strain thorn saccharopolyspora strain BCSsBFSg of the present invention
5-006 (Saccharopolyspora spinosa) CGMCC No.5546) mutagenic processes is following:
1. adopt many group complex mutation technology seed selection pleocidin superior strains
(1) selects well-grown thorn saccharopolyspora strain (Saccharopolyspora spinosa) bacterial strain QYLZ 88912 slant strains, wash spore, manufacture 10 with SPSS
6The spore suspension of individual/mL, under the 30W uv lamp, 5s, 10s, 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s are shone respectively in the 30cm place, and the spore suspension gradient dilution is coated with flat board, and lucifuge is cultivated, and calculates lethality rate;
(2) select the irradiation time of high, medium and low three various dose respectively the spore suspension of bacterial strain QYLZ 88912 to be carried out the uv irradiating processing according to lethality rate in (1);
(3) mix the bacteria suspension of three different irradiation times in (2) then, gradient dilution is coated with flat board, and lucifuge is cultivated;
(4) mutagenic strain in (3) is inoculated into cultivates in the seed culture medium that contains the 10mg/L Sulphaguanidine;
(5) seed liquor in (4) is inoculated into 10% inoculum size cultivates in the seed culture medium that contains the 10mg/L Streptomycin sulphate;
(6) seed liquor in (5) is inoculated into 10% inoculum size cultivates in the seed culture medium that contains 35mg/L Oxacyclotetradecane,erythromycin deriv;
(7) seed liquor in (6) is inoculated into 10% inoculum size cultivates in the seed culture medium that contains the 10g/L rhamnosyl;
(8) seed liquor in (7) is inoculated into 10% inoculum size cultivates in the seed culture medium that contains the 30g/L Sodium Propionate;
(9) get on the solid plate that seed liquor in (8) is inoculated into fermention medium and cultivate, bacterium colony is the plum blossom shape on the screening culture medium, and colony edge is a spination, positive white, drying, the tangible resistant strain of intermediate recess;
(10) get the multi-strain bacteria strain that obtains in (9) and be inoculated into respectively in the seed culture medium, under 29 ℃, 200r/m shaking culture 4d; Inoculum size with 10% inserts in the fermention medium, and 29 ℃, 240r/m bottom fermentation 7d shake lixiviate 2h with fermented liquid with equal-volume methyl alcohol room temperature; The centrifugal 10min of 5000r/m; Get supernatant,, sift out pleocidin superior strain BCSsBFSg again with the mass concentration that HPLC measures pleocidin in the supernatant
5-006.
2. sting saccharopolyspora strain BCSsBFSg
5-006 cultural method is:
(1) actication of culture: with slant strains---thorn saccharopolyspora strain BCSsBFSg
5-006 (Saccharopolyspora spinosa) slant medium of transferring, 29 ℃ of activation culture 5d;
(2) seed culture: choose well-grown inclined-plane as seed, add the 8mL sterilized water and wash slant pore, draw the 4mL spore suspension and insert in the seed culture medium, in 29 ℃, 180～200r/m cultivates 36～54h down, makes seed liquor;
(3) fermentation culture: the seed culture fluid in (2) is inoculated in the fermention medium with 8～10% (v/v) inoculum size, under 29 ℃, 240r/m shaking culture 7～8d;
3. thorn saccharopolyspora strain QYLZ 88912 used substratum are:
(1) preservation isolation medium: Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L, agar 20g/L, pH6.5～6.7,121 ℃ high pressure steam sterilization 30min;
(2) seed culture medium: Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L, pH6.5～6.7,121 ℃ high pressure steam sterilization 30min;
(3) fermention medium: 40g/L glucose, the 30g/L Zulkovsky starch, the immersion liquid of 15g/L corn, the 20g/L cottonseed concentrates powder, 20g/L yeast powder, 1g/L lime carbonate, pH7.0～7.2,121 ℃ high pressure steam sterilization 30min;
(4) select substratum:
1. Sulphaguanidine 10mg/L, Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L, pH6.5～6.7,121 ℃ high pressure steam sterilization is transferred 6.5～6.7,121 ℃ of sterilizations of pH 30min with 1mol/L NaOH.
2. Streptomycin sulphate 10mg/L (sterilization back add separately), Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L transfers 6.5～6.7,121 ℃ of sterilizations of pH 30min with 1mol/L NaOH.
3. Oxacyclotetradecane,erythromycin deriv 35mg/L (sterilization back add separately), Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L transfers 6.5～6.7,121 ℃ of sterilizations of pH 30min with 1mol/L NaOH.
4. rhamnosyl 10g/L (sterilization back add separately), Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L transfers 6.5～6.7,121 ℃ of sterilizations of pH 30min with 1mol/L NaOH.
5. Sodium Propionate 30g/L (sterilization back add separately), Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L transfers 6.5～6.7,121 ℃ of sterilizations of pH 30min with 1mol/L NaOH.
The present invention compared with prior art, beneficial effect is: mutagenic strain BCSsBFSg of the present invention
5-006 produces bacterium with conventional pleocidin compares, and its pleocidin throughput is more high, and can effectively improve the productive rate of pleocidin thus, reduces production costs.Output is 1200 ± 56.7mg/L, improves 783.5%. than original strain
Embodiment
Below in conjunction with specific embodiment the present invention is made further detailed description, following examples are descriptive, are not restrictive, can not limit protection scope of the present invention with this.
Embodiment 1 primary is produced the bacterial strain thorn saccharopolyspora strain QYLZ 88912 of pleocidin
1. thalli morphology: thalline is unicellular, and gramstaining is positive, on solid medium, breeds with conidial mode, forms mycelium pellet in the liquid medium within, mainly breeds new individuality through the substrate mycelium fragment.Thorn saccharopolyspora strain aerial hyphae is separated into the long spore chain of arranging with sickle shaped and open loop shape, also can observe short and incomplete spiral helicine mycelia.Spore is shaped as ellipse, and mean size is about 1.1 * 1.5 μ m.The color that gas is given birth to spore population is mainly light pink yellow, on some substratum, also produces white spore.
2. colonial morphology: on flat board, when substratum was moist, bacterium colony was the straw hat shape substantially, surface drying, and positive white, comparatively neat on every side, bacterium colony is opaque, intermediate projections; When substratum is dry, the coarse gauffer of bacterium colony, comparatively cave in the centre, and it is radial that the edge is more.
3. cultural characteristic: this bacterium is high oxygen consumption bacterium, and optimum growth temperature is 15～37 ℃, the righttest growth pH6-8; Responsive to N,O-Diacetylmuramidase; Can be under height oozes condition (11%NaCl) growth, can both well-grown on most of substratum (like ISP2, ATCC172), and form good aerial mycelium; At shaking speed 220～240r/min, cultivate 6～8d, pH 6.7～8.0, and fermented liquid has light bilgy odour, and when mostly microscopy was the mycelia fragment down, pleocidin output was the highest.Dissolved oxygen has remarkable effect to the generation of pleocidin in the fermenting process.
4. utilizable carbon source: glucose, SANMALT-S, ribose, wood sugar, fructose, semi-lactosi, seminose, N.F,USP MANNITOL, Zulkovsky starch, dextrin, Witconol 2301, oils etc., above-mentioned carbon source is generally mixed use, and single carbon source is of little use.
Embodiment 2 is that starting strain adopts many group complex mutation technology screenings to obtain bacterial strain BCSsBFSg with thorn saccharopolyspora strain QYLZ 88912
5-006.
1. the acquisition of mutagenic strain
(1) selects well-grown pleocidin to produce bacterial strain QYLZ 88912 slant strains, wash spore, manufacture 10 with SPSS
6The spore suspension of individual/mL, under the 30W uv lamp, 5s, 10s, 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s are shone respectively in the 30cm place, and the spore suspension gradient dilution is coated with flat board, cultivate in 29 ℃ of lucifuges, calculate lethality rate;
(2) select the irradiation time of 5s, 20s, three various dose of 33s respectively the spore suspension of bacterial strain QYLZ 88912 to be carried out the uv irradiating processing;
(3) mix the spore suspension of three different irradiation times then, carry out 10 times of serial dilutions 10
-1～10
-6, get 10
-4, 10
-5, 10
-6Three dilution spore suspension spread plates, 29 ℃ of lucifuges are cultivated 9～12d;
(4) mutagenic strain in (3) is inoculated into cultivates in the seed culture medium that contains the 10mg/L Sulphaguanidine; Seed liquor is placed 29 ℃ of constant temperature shaking tables, 200r/m, shaking culture 3～4d;
(5) seed liquor in (4) is inoculated into 10% inoculum size (v/v) cultivates in the seed culture medium that contains the 10mg/L Streptomycin sulphate;
(6) seed liquor in (5) is inoculated into 10% inoculum size (v/v) cultivates in the seed culture medium that contains 35mg/L Oxacyclotetradecane,erythromycin deriv;
(7) seed liquor in (6) is inoculated into 10% inoculum size (v/v) cultivates in the seed culture medium that contains the 10g/L rhamnosyl;
(8) seed liquor in (7) is inoculated into 10% inoculum size (v/v) cultivates in the seed culture medium that contains the 30g/L Sodium Propionate;
The seed liquor of (9) getting in (8) is carried out 10 times of serial dilutions, gets 10
-4, 10
-5, 10
-6Three dilution spore suspension coating slant mediums are dull and stereotyped, cultivate 9～12d for 29 ℃, positive white in the screening culture medium, and colony edge is a spination, surface drying, the tangible resistance marker bacterial strain of intermediate recess;
(10) get the multi-strain bacteria strain that obtains in (9) and be inoculated into respectively in the seed culture medium, under 29 ℃, 200r/m shaking culture 4d; Inoculum size with 10% inserts in the fermention medium, and 29 ℃, 240r/m bottom fermentation 7d shake lixiviate 2h with fermented liquid with equal-volume methyl alcohol room temperature; The centrifugal 10min of 5000rpm; Get supernatant, the mass concentration with HPLC measures pleocidin in the supernatant filters out pleocidin superior strain BCSsBFSg
5-006.
The fermentation culture based component is: 40g/L glucose, and the 30g/L Zulkovsky starch, the immersion liquid of 15g/L corn, the 20g/L cottonseed concentrates powder, 20g/L yeast powder, 1g/L lime carbonate, pH7.0～7.2,121 ℃ high pressure steam sterilization 30min.
Culture condition: 500mL shakes bottle, liquid amount 50mL, 29 ℃, 240r/m, shaking culture 7d.
2. mutagenic strain BCSsBFSg
5-006 characteristic
(1) thalli morphology: thalline is unicellular; Gramstaining is positive; On solid medium, breed with conidial mode; Forming mycelium pellet in the liquid medium within, mainly breed new individuality through the substrate mycelium fragment. thorn saccharopolyspora strain aerial hyphae is separated into the long spore chain of arranging with sickle shaped and open loop shape, also can observe short and incomplete spiral helicine mycelia.Spore is shaped as ellipse, and mean size is about 1.1 * 1.5 μ m.The color that gas is given birth to spore population is mainly light pink yellow, on some substratum, also produces white spore.
(2) colonial morphology: on flat board, bacterium colony was the plum blossom shape when substratum was moist, surface drying, and positive white, the edge is wavy and thicker, and is opaque, intermediate projections; When substratum is dry, the bacterium colony intermediate recess.
(3) cultural characteristic: 29 ℃, 220～240rpm down concussion cultivates 7d, and fermented liquid has bilgy odour denseer, under the microscopy mycelia dyeing weak, a small amount of phenomenon of rupture, pH6.7～7.5 arranged.
(4) utilizable carbon source: glucose, SANMALT-S, ribose, wood sugar, fructose, semi-lactosi, seminose, N.F,USP MANNITOL, Zulkovsky starch, dextrin, Witconol 2301, oils etc., above-mentioned carbon source is generally mixed use, and single carbon source is of little use.
(5) can be respectively on the resistant panel of the Sulphaguanidine of 10mg/L, 10mg/L Streptomycin sulphate, 35mg/L Oxacyclotetradecane,erythromycin deriv, 10g/L rhamnosyl, 30g/L Sodium Propionate, grow, and well-grown
1. the configuration of substratum
Preservation isolation medium: Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L, agar 15～20g/L, the zero(ppm) water configuration is with NaOH accent pH6.5～6.7, the 121 ℃ high pressure steam sterilization 30min of 1mol/L.
Seed culture medium: Zulkovsky starch 20g/L, glucose 5g/L, yeast extract 3g/L, steeping water 10g/L, MgSO
47H
2O 2g/L, the zero(ppm) water configuration is with NaOH accent pH6.5～6.7, the 121 ℃ high pressure steam sterilization 30min of 1mol/L, 29 ℃ of culture temperature.
Fermention medium: 40g/L glucose, the 30g/L Zulkovsky starch, the immersion liquid of 15g/L corn, the 20g/L cottonseed concentrates powder; The 20g/L yeast powder, 1g/L lime carbonate, zero(ppm) water configuration; NaOH with 1mol/L transfers pH7.0～7.2,121 ℃ high pressure steam sterilization 30min, 29 ℃ of culture temperature.
2. actication of culture
To sting saccharopolyspora strain switching BCSsBFSg
5-006 seed slant medium in constant incubator, is cultivated 4～5d for 29 ℃;
3. seed culture
Select well-grown activated inclined plane bacterial classification inoculation in the triangular flask that the seed culture medium that makes in above-mentioned 1 is housed (substratum of dress 50mL in the 500mL triangular flask), in 29 ℃, 200r/m cultivates 2～3d down;
4. fermentation culture
The seed culture fluid that makes in 3 is inoculated in (substratum of dress 50mL in the 500mL) in the fermention medium with 10% (v/v) inoculum size, in 29 ℃, 240r/m bottom fermentation 7d;
Detected result: thorn saccharopolyspora strain (Saccharopolyspora spinosa) BCSsBFSg
5-006 is 1200 ± 56.7mg/L in fermention medium fermentative prodn pleocidin output.
Claims (4)
1. the thorn saccharopolyspora strain BCSsBFSg of pleocidin is produced in a strain
5-006 (Saccharopolyspora spinosa), CGMCC No.5546.
2. the thorn saccharopolyspora strain BCSsBFSg of pleocidin
5-006 (Saccharopolyspora spinosa) CGMCC No.5546 forms with fermention medium: 40g/L glucose, 30g/L Zulkovsky starch, the immersion liquid of 15g/L corn; The 20g/L cottonseed concentrates powder, 20g/L yeast powder, 1g/L lime carbonate; The zero(ppm) water configuration, pH7.0～7.2.
3. as weighing the thorn saccharopolyspora strain BCSsBFSg of 2 said pleocidin
5-006 (Saccharopolyspora spinosa) CGMCC No.5546 fermentation culture method: comprise the steps: that seed culture fluid is inoculated in the fermention medium with 10% (v/v) inoculum size, in 29 ℃, 240r/m bottom fermentation 7d.
4. as weighing the application of 1 said CGMCC No.5546 in pleocidin is produced.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365813A (en) * | 2017-07-03 | 2017-11-21 | 齐鲁制药（内蒙古）有限公司 | A kind of pleocidin fermentation process for improving cytoactive |
| CN111849807A (en) * | 2020-06-30 | 2020-10-30 | 北大方正集团有限公司 | A kind of Saccharopolyspora spinosa DS190375 and its fermentation product, bacterial agent, fermentation and screening method and application |
| CN113337437A (en) * | 2021-06-17 | 2021-09-03 | 武汉臻智生物科技有限公司 | Method for preparing spinosad compounds by utilizing saccharopolyspora spinosa strain J1-DS1902 |
| CN113444659A (en) * | 2021-06-17 | 2021-09-28 | 武汉大学 | Saccharopolyspora spinosa for high yield of spinosad |
| WO2023116710A1 (en) * | 2021-12-21 | 2023-06-29 | 山东鲁抗医药股份有限公司 | Spinosad high-yield strain and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1252820A1 (en) * | 2001-04-25 | 2002-10-30 | JOHNSON & JOHNSON CONSUMER COMPANIES, INC. | Pediculicidal and ovacidal treatment compositions and methods for killing head lice and their eggs |
| CN101629203A (en) * | 2008-12-09 | 2010-01-20 | 湖南师范大学 | Process for preparing spinosad |
-
2011
- 2011-12-16 CN CN201110429550.1A patent/CN102676393B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1252820A1 (en) * | 2001-04-25 | 2002-10-30 | JOHNSON & JOHNSON CONSUMER COMPANIES, INC. | Pediculicidal and ovacidal treatment compositions and methods for killing head lice and their eggs |
| CN101629203A (en) * | 2008-12-09 | 2010-01-20 | 湖南师范大学 | Process for preparing spinosad |
Non-Patent Citations (2)
| Title |
|---|
| JIN ZH ET AL: "Enhanced production of spinosad in saccharopolyspora spinosa bu genome shuffling", 《APPL BIOCHEM BIOTECHNOL》 * |
| 蔡恒等: "刺糖多孢菌生产多杀菌素的研究进展", 《中国生物工程杂志》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107365813A (en) * | 2017-07-03 | 2017-11-21 | 齐鲁制药（内蒙古）有限公司 | A kind of pleocidin fermentation process for improving cytoactive |
| CN107365813B (en) * | 2017-07-03 | 2021-01-05 | 齐鲁制药（内蒙古）有限公司 | Spinosad fermentation method for improving cell activity |
| CN111849807A (en) * | 2020-06-30 | 2020-10-30 | 北大方正集团有限公司 | A kind of Saccharopolyspora spinosa DS190375 and its fermentation product, bacterial agent, fermentation and screening method and application |
| CN111849807B (en) * | 2020-06-30 | 2022-05-06 | 北大方正集团有限公司 | Saccharopolyspora spinosa DS190375 and fermentation product, microbial inoculum, fermentation and screening method and application thereof |
| CN113337437A (en) * | 2021-06-17 | 2021-09-03 | 武汉臻智生物科技有限公司 | Method for preparing spinosad compounds by utilizing saccharopolyspora spinosa strain J1-DS1902 |
| CN113444659A (en) * | 2021-06-17 | 2021-09-28 | 武汉大学 | Saccharopolyspora spinosa for high yield of spinosad |
| CN113444659B (en) * | 2021-06-17 | 2022-10-04 | 武汉大学 | Saccharopolyspora spinosa for high yield of spinosad |
| WO2023116710A1 (en) * | 2021-12-21 | 2023-06-29 | 山东鲁抗医药股份有限公司 | Spinosad high-yield strain and use thereof |
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