CN103031264A - Salinomycin high-yield strain - Google Patents
Salinomycin high-yield strain Download PDFInfo
- Publication number
- CN103031264A CN103031264A CN2012105892006A CN201210589200A CN103031264A CN 103031264 A CN103031264 A CN 103031264A CN 2012105892006 A CN2012105892006 A CN 2012105892006A CN 201210589200 A CN201210589200 A CN 201210589200A CN 103031264 A CN103031264 A CN 103031264A
- Authority
- CN
- China
- Prior art keywords
- salinomycin
- strain
- mutagenesis
- yield
- streptomyces
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a salinomycin high-yield strain which has a classification designation of streptomycesalbums S-610-11 and a preservation serial number of CCTCC (China Center for Type Culture Collection) No.M2012492 Streptomycesalbums S-610-11 is obtained by adopting plasma mutagenesis at normal pressure and temperature and combining with a high-throughput screening method, so that the content of salinomycin can be increased to a larger extent, the stability is good, and the titer of salinomycin after continuous passage of the strain for 5 times has no obvious variation. By employing shake flask fermentation, the yield of salinomycin reaches 56000u/ml, and increases by 70% compared with an original strain F2-1<#>. The salinomycin high-yield strain completely meets mutagenesis and screening demands of industrial strain production with salinomycin, can be applied to industrial fermentation production, and has a significant economic value.
Description
Technical field
The present invention relates to and belongs to industrial strain screening and Application Areas, is specifically related to a kind of High Yield Strains of Salinomycin.
Background technology
Salinomycin. is one of China's main antibiotic for animals of producing, use and exporting, can effectively kill the various coccidias in the poultry body, is widely used in fodder additives, is desirable anticoccidial drug, fodder additives.It is a class by streptomyces albus (
Streptomyces albums) the monocarboxylic acid polyethers that produces is the animal specific microbiotic, and most of gram-positive microorganisms (comprising mycobacterium and some filamentous funguss) and various coccidia are had stronger inhibition and killing action.Salinomycin. is by the anticoccidiosis medicine of drugs approved by FDA, by the countries in the world widespread use.
Salinomycin. is typical ion carrier antibiotic, and it is to the positively charged ion in the cell, especially K
+, Na
+, Rb
+Avidity strong especially, the necessary positively charged ion of cell activities is strengthened by the impregnability of lipid barrier on the film, the cell of can freely coming in and going out, hinder the inside and outside cationic normal delivery of cell, make the inside and outside ionic concn of cell occur sharply to change, break the normal equilibrium state of ion, thereby the osmotic pressure of cell is increased, finally make cell disruption, play germicidal action.
Strain improvement technology (strain improvement) commonly used can be divided into random seed selection (random screening) and rational seed selection (rationalized selection) two large classes according to its principle at present.Although the genetically engineered take molecular biology as the basis, metabolic engineering and learn the systems biology research that forms as directed constructing function cell strain provides bright prospects by various groups will really realize cultivating to reach the transformation target with these functioning cells extremely difficult.Because complicacy and many nodes property of microbe intracellular metabolite network, the metabolism of bacterial strain flows to toward the direction towards desired design not and shifts after the genetic manipulation.Especially in the complex system of the secondary meta-bolitess such as generation microbiotic, expect good industrial producing strain, mainly still adopt random breeding technique.For a long time for
Streptomyces albusMainly be that what to adopt is traditional breeding techniques such as ultraviolet mutagenesis and LiCl mutagenesis in the random breeding technique of strain improvement, and obtained certain effect, yet these mutagenic compound of life-time service are processed same bacterial classification, because the non-directiveness of sudden change and the accumulation of sudden change so that when obtaining high yield bacterial strain can obviously weaken at aspects such as viability, sporulation quantities, and repeatedly mutagenesis often causes higher negative mutation rate and resistance saturated, thus so that the insufficient space that bacterial strain improves.
Summary of the invention
The problem to be solved in the present invention is to provide a kind of High Yield Strains of Salinomycin.
In order to address the above problem, the technical solution used in the present invention is as follows:
A kind of Salinomycin. Producing Strain
,Classification And Nomenclature be streptomyces albus (
Streptomyces albums) S-610-11, described bacterial strain deposit number is CCTCC M 2012492.
Salinomycin. Producing Strain, its Classification And Nomenclature be streptomyces albus (
Streptomyces albums) S-610-11, this bacterial strain is preserved in Chinese Typical Representative culture collection center, depositary institution address on December 2nd, 2012: Wuhan University's loujia hill belongs, deposit number are CCTCC M 2012492.
Salinomycin. Producing Strain of the present invention (
Streptomyces albums) preparation method of S-610-11 is: provided with industrial producing strain F by the applicant
2-1
#Be starting strain, pass through first normal pressure and temperature plasma body (ARTP) mutagenic treatment and obtain mutant strain, then obtain high productive mutant by High Throughput Screening Assay, obtain at last the Salinomycin. Producing Strain of genetic stability by shaking flask checking, the cultivation of going down to posterity.
The morphology of bacterial strain of the present invention, Physiology and biochemistry and utilization of carbon source feature are as follows:
The morphology of bacterial strain of the present invention and physiological characteristic
The selection of High Yield Strains of Salinomycin provided by the present invention, concrete steps are as follows:
(1) utilizes normal temperature and pressure plasma body (ARTP) mutagenesis starting strain
The spore suspension of preparation starting strain, and under sterile wind, dry up, then place ARTP mutagenesis system to carry out plasma irradiating 1～10min.Sample is washed with physiological saline, be coated on behind the gradient dilution on the plate culture medium, lucifuge is cultivated 5～8d.
(2) mutant strain high-throughput primary dcreening operation
Single colonial mutation strain of picking step (1) gained is inoculated in two 24,48 or 96 orifice plates that 1～6ml solid medium and 0.3 ~ 1.5ml liquid fermentation medium be housed respectively with aseptic toothpick point simultaneously, the former 25～30 ℃ leave standstill and cultivate 4～8d, 25～37 ℃ of the latter, humidity 50～90%, 200～300r/min cultivate 4～8d.After the cultivation, detect the Salinomycin. content of fermented liquid in each hole, choose and produce the mutant strain that plain level is higher than starting strain 30%, be the primary dcreening operation superior strain.
(3) the mutant strain high-throughput sieves again
The solid culture of the high productive mutant of step (2) gained is connected to aseptic toothpick point in 24, the 48 or 96 hole depth plates that 0.8 ~ 1.5ml seed culture medium is housed, about 25～37 ℃, humidity 50～90%, 200～300r/min shaking culture, 20～28h.Transfer in 24, the 48 or 96 hole depth plates that 0.8 ~ 1.5ml fermention medium is housed shaking culture 6～9d with 5～15% inoculum size.After the cultivation, detect the Salinomycin. content of fermented liquid in each hole, choose and produce the mutant strain that plain level is higher than starting strain 30%, be multiple sieve superior strain.
(4) mutant strain shaking flask checking
Step (3) gained superior strain is inoculated in the 250ml shaking flask that 20～50ml seed culture medium is housed, about 25～35 ℃, 200～300r/min vibration, 25～30h.Transfer in the 250ml shaking flask that 20～50ml seed culture medium is housed with 5～15% inoculum size, continue shaking culture 5～10d.
(5) mutant strain genetic stability checking
The superior strain that to verify through shaking flask is through 3～5 generations of 250ml shaking flask cultured continuously.
Remarkable advantage of the present invention:
The present invention adopts the mutagenesis of normal pressure and temperature plasma body and obtains the Salinomycin. Producing Strain in conjunction with high-throughput screening method
Streptomyces albusS-610-11 can improve the output of Salinomycin. by a relatively large margin, and good stability, and Salinomycin. is tired and all maintained same higher level after the bacterial strain continuous passage 5 times.Utilize shake flask fermentation, Salinomycin. output reaches 56000u/ml, than starting strain F
2-1
#Improved 70%.The present technique invention meets mutagenesis and the screening needs that the industrialization Salinomycin. is produced bacterial strain fully, and can be applicable to industrial fermentation production, has great economic worth.
Description of drawings
Fig. 1 is the colonial morphology that Salinomycin. produces bacterium, and as can be seen from the figure Salinomycin. produces bacterium 4 kinds of colonial morphologies: full type, the volcano shape of the mouth as one speaks, straw hat type and the type that subsides.The full type of G-1, the G-2 volcano shape of the mouth as one speaks, G-3 straw hat type, the G-4 type that subsides.
Fig. 2 is that normal pressure and temperature plasma body (ARTP) mutation time is on the impact of streptomyces albus lethality rate.
Fig. 3 is mutant strain high flux screening process.1.～3.: primary dcreening operation; 4.～7.: multiple sieve; 8.: the shaking flask checking.
Embodiment
For the present invention there being clear and definite understanding, now in conjunction with example the present invention is done a step explanation.
(1) utilizes normal temperature and pressure plasma body (ARTP) mutagenesis starting strain
Preparation starting strain F
2-1
#Spore suspension, and under sterile wind, dry up, then place to utilize under the ARTP mutagenesis system and carry out plasma irradiating 10min.Sample is washed with physiological saline, be coated on behind the gradient dilution on the plate culture medium, lucifuge is cultivated 5d.
(2) mutant strain high-throughput primary dcreening operation
Single colonial mutation strain of picking step (1) gained is connected in two 96 orifice plates that 6ml solid medium and 0.3ml liquid fermentation medium be housed respectively with aseptic toothpick point simultaneously, the former 25 ℃ leave standstill and cultivate 8d, 25 ℃ of the latter, humidity 90%, 200r/min cultivate 8d.
(3) the mutant strain high-throughput sieves again
The solid culture of the high productive mutant of step (2) gained is connected to aseptic toothpick point in the 96 hole depth plates that the 0.8ml seed culture medium is housed, about 25 ℃, humidity 90%, 200r/min shaking culture 28h.Transfer in the 24 hole depth plates that the 1.5ml fermention medium is housed shaking culture 9d with 5% inoculum size.
(4) mutant strain shaking flask checking
Step (3) gained enhanced variant is inoculated in the 250ml shaking flask that the 20ml seed culture medium is housed, about 35 ℃, 200r/min vibration 30h.Transfer in the 250ml shaking flask that the 50ml seed culture medium is housed with 5% inoculum size, continue shaking culture 5d.
(5) mutant strain genetic stability checking
The superior strain that to verify through shaking flask is through 3 generations of 250ml shaking flask cultured continuously.
(1) utilizes normal temperature and pressure plasma body (ARTP) mutagenesis starting strain
Preparation starting strain F
2-1
#Spore suspension, and under sterile wind, dry up, then place under the ARTP mutagenesis system and carry out plasma irradiating 1min.Sample is washed with physiological saline, be coated on behind the gradient dilution on the plate culture medium, lucifuge is cultivated 8d.
(2) mutant strain high-throughput primary dcreening operation
Single colonial mutation strain of picking step (1) gained is connected in two 24 orifice plates that 1ml solid medium and 1.5ml liquid fermentation medium be housed respectively with aseptic toothpick point simultaneously, the former 30 ℃ leave standstill and cultivate 4d, 37 ℃ of the latter, humidity 50%, 300r/min cultivate 4d.
(3) the mutant strain high-throughput sieves again
The solid culture of the high productive mutant of step (2) gained is connected to aseptic toothpick point in the 24 hole depth plates that the 5ml seed culture medium is housed, about 37 ℃, humidity 50%, 300r/min shaking culture 20h.Transfer in the 96 hole depth plates that the 0.8ml fermention medium is housed shaking culture 6d with 15% inoculum size.
(4) mutant strain shaking flask checking
Step (3) obtained strains is inoculated in the 250ml shaking flask that the 50ml seed culture medium is housed, about 25 ℃, 300r/min vibration 25h.Transfer in the 250ml shaking flask that the 20ml seed culture medium is housed with 15% inoculum size, continue shaking culture 10d.
(5) mutant strain genetic stability checking
The superior strain that to verify through shaking flask is through 5 generations of 250ml shaking flask cultured continuously.
The checking of mutant strain genetic stability
With the superior strain through being sieved to again (
Streptomyces albums) the S-610-11 cultivation of going down to posterity, to detect its genetic stability, the result is as follows for the strain passage fermenting experiment:
The enhanced variant streptomyces albus (
Streptomyces albums) test of S-610-11 genetic stability
Can find out from experimental result, process goes down to posterity for 3-5 time, the mutant strain streptomyces albus (
Streptomyces albums) Salinomycin. of S-610-11 tires basicly stable, show and have preferably genetic stability, the target bacterial classification produces plain level and finally maintains about 56000u/ml, and (32000u/ml) improved about 70% with respect to starting strain, so the mutant strain streptomyces albus (
Streptomyces albums) S-610-11 can be used as the production bacterial strain of further research and development.
The product detection method that arrives involved in the present invention
(1) preparation of mark product: take by weighing 1g Salinomycin. solid mark product, be mixed with the Salinomycin. mark product solution of 1000u/ml with dehydrated alcohol.
(2) preparation of developer: take by weighing 1.5g to dimethylbenzaldehyde, add an amount of dehydrated alcohol, add again the 1.5ml vitriol oil, be settled to 500ml with dehydrated alcohol after the dissolving, be stored in the brown bottle after shaking up.
The mensuration of (3) tiring:
The high throughput testing of orifice plate fermented liquid: after the orifice plate fermentation ends, directly in each hole, add the 4ml dehydrated alcohol, ultrasonic extraction 20min, the centrifugal 10min of 3000r/min, getting supernatant liquor dilutes in right amount, then with sample: dehydrated alcohol: behind the ratio mixing of developer=1: 9: 10,70 ℃ of water-bath 20min, after the cooling with placing 96 hole check-out consoles to detect OD with microplate reader
600 nm
The conventional sense of shake flask fermentation liquid: draw the 1ml fermented liquid in tool plug pipe, add in the 9ml dehydrated alcohol, leave standstill 30min, use filter paper filtering, getting filtrate dilutes in right amount, then with sample: dehydrated alcohol: behind the ratio mixing of developer=1: 9: 10,70 ℃ of water-bath 20min detect OD with spectrophotometer after the cooling
600 nmThe measuring method of mark product solution is the same.
The calculation formula that fermented liquid is tired is as follows:
Claims (2)
1. High Yield Strains of Salinomycin, Classification And Nomenclature be streptomyces albus (
Streptomyces albums) S-610-11, it is characterized in that described bacterial strain deposit number is CCTCC M 2012492.
2. the application of High Yield Strains of Salinomycin claimed in claim 1 in anticoccidial drug and fodder additives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210589200.6A CN103031264B (en) | 2012-12-31 | 2012-12-31 | Salinomycin high-yield strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210589200.6A CN103031264B (en) | 2012-12-31 | 2012-12-31 | Salinomycin high-yield strain |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410237201.3A Division CN104017756A (en) | 2012-12-31 | 2012-12-31 | Salinomycin strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103031264A true CN103031264A (en) | 2013-04-10 |
| CN103031264B CN103031264B (en) | 2014-12-03 |
Family
ID=48018666
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210589200.6A Active CN103031264B (en) | 2012-12-31 | 2012-12-31 | Salinomycin high-yield strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103031264B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505915A (en) * | 2014-09-26 | 2016-04-20 | 安徽工程大学 | Method for screening high-activity formaldehyde-tolerant formaldehyde-degrading bacterium mutant strains through atmospheric and room temperature plasma (ARTP) mutagenesis |
| CN110527630A (en) * | 2019-05-24 | 2019-12-03 | 浙江工业大学 | One plant of Gibberella fujikuroi mutant strain and application using the breeding of ARTP induced-mutation technique |
| CN111662831A (en) * | 2020-06-12 | 2020-09-15 | 浙江工业大学 | Aspergillus niger Rha-N1 and application thereof |
-
2012
- 2012-12-31 CN CN201210589200.6A patent/CN103031264B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| M.HUMAYOUN AKHTAR ET AL: "Microwave extraction of incurred salinomycin from chicken tussues", 《JOURNAL OF ENVIRNMENTAL SCIENCE AND HEALTH,PART B:PESTICIDES,CONTAMINANTS,AND AGRICULTURAL WASTES》 * |
| 储消和等: "盐霉素高产菌的推理选育", 《中国抗生素杂志》 * |
| 杨亚勇等: "提高白色链霉菌盐霉素产量的工艺研究", 《中国抗生素杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105505915A (en) * | 2014-09-26 | 2016-04-20 | 安徽工程大学 | Method for screening high-activity formaldehyde-tolerant formaldehyde-degrading bacterium mutant strains through atmospheric and room temperature plasma (ARTP) mutagenesis |
| CN110527630A (en) * | 2019-05-24 | 2019-12-03 | 浙江工业大学 | One plant of Gibberella fujikuroi mutant strain and application using the breeding of ARTP induced-mutation technique |
| CN110527630B (en) * | 2019-05-24 | 2021-04-20 | 浙江工业大学 | Aleurites lutescens mutant strain bred by ARTP mutagenesis technology and application thereof |
| CN111662831A (en) * | 2020-06-12 | 2020-09-15 | 浙江工业大学 | Aspergillus niger Rha-N1 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103031264B (en) | 2014-12-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103992978A (en) | Leuconostoc pseudomesenteroides and method for co-producing dextran and mannitol therefrom | |
| CN102286421A (en) | Liquid fermentation culture method for paecilomyces lilacinus | |
| CN104342390A (en) | Sinorhizobium meliloti strain and composition and application of sinorhizobium meliloti strain | |
| CN104087628A (en) | Method for reducing viscosity of gamma-polyglutamic acid fermentation liquid | |
| CN103031264B (en) | Salinomycin high-yield strain | |
| CN103074275B (en) | Lysinibacillus fusiformis for producing ethyl urethane hydrolase and application of lysinibacillus fusiformis | |
| CN103740606A (en) | Streptomyces phytohabitans, method for producing new antibiotics Novonestmycin from Streptomyces phytohabitans, and application of Novonestmycin | |
| CN103602596B (en) | Monochamus alternates hope beauveria bassiana space mutant B305 and application thereof | |
| CN104531535B (en) | One kind produces knob not Kangding B0Genetic recombination bacterial strain and selection and application | |
| CN103436465B (en) | Alcaligenes faecalis strain | |
| CN103627698A (en) | Breeding of acetoin high-tolerance bacterial strain and acetoin fermentation production with bacterial strain | |
| CN104531571A (en) | Pseudomonas fluorescens and biological preparation and application in preventing and controlling sugarcane smut | |
| CN104818236A (en) | Tea tree growth-promoting rhizobacterium Serratia marcescens and application thereof | |
| CN104830696B (en) | A kind of high yield CNNS Marasmius mutagenic strain and breeding method | |
| CN101748093A (en) | Streptomyces spectabilis SpLE-16 and application thereof to production of spectinomycin | |
| CN104017756A (en) | Salinomycin strain | |
| CN109797116A (en) | It is grown nonparasitically upon another plant altogether streptomycete HNS054 mutant strain by the sponge of the strong bacteriostatic activity of superposing type ARTP mutagenesis screening | |
| CN104531798A (en) | Method for improving output of tetraene macrolide antibiotics | |
| CN103602594B (en) | Monochamus alternates hope beauveria bassiana space mutant B252 and application thereof | |
| CN102080053A (en) | Novel yarrowia lipolytica and method for preparing gamma-decalactone with yarrowia lipolytica by castor oil biotransformation | |
| CN103451126B (en) | High-concentration ammonium ion tolerant escherichia coli capable of producing succinic acid and application thereof | |
| CN100529056C (en) | High toxicity bacillus thuringiensis mutant strain D1-23 of high producting Zwittermicin A and crystal protein and application thereof | |
| CN102533567B (en) | Paecilomyces tenuipes mutagenesis bacterial strain and culturing method thereof | |
| CN101948826B (en) | Method for screening methionine resistant deinsectization streptomyces avermitilis strain | |
| CN103667170A (en) | Method of preparing mature conidia of hirsutella sinensis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 313220 Huzhou City, Zhejiang Province, Deqing County Zhong Guan Town Industrial Zone Patentee after: Zhejiang Han Ye Limited by Share Ltd Address before: 313220 Huzhou City, Zhejiang Province, Deqing County Zhong Guan Town Industrial Zone Patentee before: Zhejiang Shenghua Biok Biology Co., Ltd. |
|
| CP01 | Change in the name or title of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20181123 Address after: 313220 Hengtang Bridge 81, Zhongguan Town, Deqing County, Huzhou City, Zhejiang Province Patentee after: Zhejiang biok Biology Technology Co. Ltd. Address before: 313220 Industrial Zone, Zhong Guan Town, Deqing County, Huzhou, Zhejiang Patentee before: Zhejiang Han Ye Limited by Share Ltd |
|
| TR01 | Transfer of patent right |