CN104017756A - Salinomycin strain - Google Patents

Abstract

The invention discloses a salinomycin strain with the class name of streptomycesalbums S-610-11, and the preservation number is CCTCCM2012492. The salinomycin high-yielding strain streptomycesalbums S-610-11 is obtained by combination of plasma mutagenesis at normal temperature under normal pressure with high-throughput synergy screening method, the content of salinomycin can be greatly improved, and the stability is good, after 5 times of continuous passages of the strain, and the salinomycin titer has no obvious change. By use of shake flask fermentation, the yield of salinomycin reaches 56000u / ml, and is improved by 70% compared with that of original strain F2-1#. The salinomycin strain is fully consistent with mutagenesis and screening requirements of industrialization salinomycin production strains, and can be applied in industrial fermentation production, and has great economic value.

Description

A kind of Salinomycin. bacterial strain

Technical field

The present invention relates to and belongs to industrial strain screening and Application Areas, is specifically related to a kind of Salinomycin. bacterial strain.

Background technology

Salinomycin. is one of China's main antibiotic for animals of producing, use and exporting, can effectively kill the various coccidias in 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 animal specific microbiotic, and most of gram-positive microorganisms (comprising mycobacterium and some filamentous funguss) and various coccidia are had to stronger inhibition and killing action.The anticoccidiosis medicine that Salinomycin. is ratified by U.S. FDA, by countries in the world widespread use.

Salinomycin. is typical ion carrier antibiotic, and it is to the positively charged ion in 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 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.

Conventional strain improvement technology (strain improvement) can be divided into random seed selection (random screening) and the large class of rationality seed selection (rationalized selection) two according to its principle at present.Although the molecular biology of take is learned the systems biology research that forms as directed constructing function cell strain provides bright prospects as basic genetically engineered, metabolic engineering and by various groups, really realize and with these functioning cells, cultivate to reach that to transform target extremely difficult.Due to complicacy and many nodes of microbe intracellular metabolite network, after genetic manipulation, the metabolism of bacterial strain flows to toward not shifting towards the direction of desired design.Especially in producing the complex system of the secondary metabolites such as microbiotic, expect good industrial producing strain, mainly still adopt random breeding technique.For a long time for streptomyces albusin the random breeding technique of strain improvement, be mainly that what to adopt is traditional breeding techniques such as ultraviolet mutagenesis and LiCl mutagenesis, and obtained certain effect, yet these mutagenic compound of life-time service are processed same bacterial classification, because non-directiveness and the accumulation of sudden change of sudden change makes bacterial strain when obtaining high yield can obviously weaken at aspects such as viability, sporulation quantities, and repeatedly mutagenesis often causes higher negative mutation rate and resistance saturated, thus the insufficient space that bacterial strain is improved.

Summary of the invention

The problem to be solved in the present invention is to provide a kind of Salinomycin. bacterial strain.

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 has been preserved in Chinese Typical Representative culture collection center, depositary institution address on December 2nd, 2012: Wuhan University's loujia hill belongs, deposit number is CCTCC M 2012492.

Salinomycin. Producing Strain of the present invention ( streptomyces albums) preparation method of S-610-11 is: by applicant, provided with industrial producing strain F ₂-1 ^#for starting strain, first pass through normal pressure and temperature plasma body (ARTP) mutagenic treatment and obtain mutant strain, then by High Throughput Screening Assay, obtain high productive mutant, finally by shaking flask, verify, go down to posterity and cultivate the Salinomycin. Producing Strain of acquisition genetic stability.

The morphology of bacterial strain of the present invention, Physiology and biochemistry and utilization of carbon source feature are as follows:

The selection of High Yield Strains of Salinomycin provided by the present invention, concrete steps are as follows:

(1) utilize normal temperature and pressure plasma body (ARTP) mutagenesis starting strain

The spore suspension of preparing starting strain, and dry up under sterile wind, be then placed in ARTP mutagenesis system and carry out plasma irradiating 1～10min.Sample is washed down with physiological saline, be coated on plate culture medium after gradient dilution, 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 ℃ of standing cultivation 4～8d, 25～37 ℃ of the latter, humidity 50～90%, 200～300r/min cultivate 4～8d.After cultivation, detect the Salinomycin. content of fermented liquid in each hole, choose and produce plain level higher than the mutant strain of starting strain 30%, be primary dcreening operation superior strain.

(3) mutant strain high-throughput sieves again

The solid culture of the high productive mutant of step (2) gained is connected in 24, the 48 or 96 hole depth plates that 0.8 ~ 1.5ml seed culture medium is housed to 25～37 ℃, humidity 50～90%, 200～300r/min shaking culture, 20～28h left and right with aseptic toothpick point.With 5～15% inoculum size, transfer in 24,48 or 96 hole depth plates of 0.8 ~ 1.5ml fermention medium are housed, shaking culture 6～9d.After cultivation, detect the Salinomycin. content of fermented liquid in each hole, choose and produce plain level higher than the mutant strain of 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 to 25～35 ℃, 200～300r/min vibration, 25～30h left and right.With 5～15% inoculum size, transfer in the 250ml shaking flask of 20～50ml seed culture medium is housed, continue shaking culture 5～10d.

(5) mutant strain genetic stability checking

By the superior strain of verifying through shaking flask 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 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 after bacterial strain continuous passage 5 times, Salinomycin. is tired and all maintained same higher level.Utilize shake flask fermentation, Salinomycin. output reaches 56000u/ml, compared with starting strain F ₂-1 ^#improved 70%.This technological invention meets mutagenesis and the screening needs that industrialization Salinomycin. is produced bacterial strain completely, and can be applicable to industrial fermentation production, has great economic worth.

Accompanying drawing explanation

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 the impact of normal pressure and temperature plasma body (ARTP) mutation time on streptomyces albus lethality rate.

Fig. 3 is mutant strain high flux screening process.1.～and 3.: primary dcreening operation; 4.～and 7.: multiple sieve; 8.: shaking flask checking.

Embodiment

For the present invention being had to clear and definite understanding, now in conjunction with example, the present invention is further described.

Embodiment 1

(1) utilize normal temperature and pressure plasma body (ARTP) mutagenesis starting strain

Prepare starting strain F ₂-1 ^#spore suspension, and dry up under sterile wind, be then placed in to utilize under ARTP mutagenesis system and carry out plasma irradiating 10min.Sample is washed down with physiological saline, be coated on plate culture medium after gradient dilution, 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 ℃ of standing cultivation 8d, 25 ℃ of the latter, humidity 90%, 200r/min cultivate 8d.

(3) mutant strain high-throughput sieves again

The solid culture of the high productive mutant of step (2) gained is connected in the 96 hole depth plates that 0.8ml seed culture medium is housed to 25 ℃, humidity 90%, 200r/min shaking culture 28h left and right with aseptic toothpick point.With 5% inoculum size, transfer in 24 hole depth plates of 1.5ml fermention medium are housed, shaking culture 9d.

(4) mutant strain shaking flask checking

Step (3) gained enhanced variant is inoculated in the 250ml shaking flask that 20ml seed culture medium is housed to 35 ℃, 200r/min vibration 30h left and right.With 5% inoculum size, transfer in the 250ml shaking flask of 50ml seed culture medium is housed, continue shaking culture 5d.

(5) mutant strain genetic stability checking

By the superior strain of verifying through shaking flask through 3 generations of 250ml shaking flask cultured continuously.

Embodiment 2

(1) utilize normal temperature and pressure plasma body (ARTP) mutagenesis starting strain

Prepare starting strain F ₂-1 ^#spore suspension, and dry up under sterile wind, be then placed under ARTP mutagenesis system and carry out plasma irradiating 1min.Sample is washed down with physiological saline, be coated on plate culture medium after gradient dilution, 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 ℃ of standing cultivation 4d, 37 ℃ of the latter, humidity 50%, 300r/min cultivate 4d.

(3) mutant strain high-throughput sieves again

The solid culture of the high productive mutant of step (2) gained is connected in the 24 hole depth plates that 5ml seed culture medium is housed to 37 ℃, humidity 50%, 300r/min shaking culture 20h left and right with aseptic toothpick point.With 15% inoculum size, transfer in 96 hole depth plates of 0.8ml fermention medium are housed, shaking culture 6d.

(4) mutant strain shaking flask checking

Step (3) obtained strains is inoculated in the 250ml shaking flask that 50ml seed culture medium is housed to 25 ℃, 300r/min vibration 25h left and right.With 15% inoculum size, transfer in the 250ml shaking flask of 20ml seed culture medium is housed, continue shaking culture 10d.

(5) mutant strain genetic stability checking

By the superior strain of verifying through shaking flask through 5 generations of 250ml shaking flask cultured continuously.

The checking of mutant strain genetic stability

By 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, strain passage fermenting experiment result is as follows:

Enhanced variant streptomyces albus ( streptomyces albums) test of S-610-11 genetic stability

From experimental result, can find out, through going down to posterity for 3-5 time, mutant strain streptomyces albus ( streptomyces albums) Salinomycin. of S-610-11 tires basicly stable, show to have good genetic stability, target bacterial classification produces plain level and finally maintains 56000u/ml left and right, with respect to starting strain (32000u/ml), has improved 70% left and right, so 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 arriving involved in the present invention

(1) preparation of mark product: take 1g Salinomycin. solid mark product, be mixed with the Salinomycin. mark product solution of 1000u/ml with dehydrated alcohol.

(2) preparation of developer: take 1.5g to dimethylbenzaldehyde, add appropriate dehydrated alcohol, then add the 1.5ml vitriol oil, be settled to 500ml with dehydrated alcohol after dissolving, be stored in brown bottle after shaking up.

(3) mensuration of tiring:

The high throughput testing of orifice plate fermented liquid: after orifice plate fermentation ends, directly in each hole, add 4ml dehydrated alcohol, ultrasonic extraction 20min, the centrifugal 10min of 3000r/min, getting supernatant liquor dilutes in right amount, then by sample: dehydrated alcohol: developer=1: after the ratio of 9: 10 mixes, 70 ℃ of water-bath 20min, cooling rear use is placed in 96 hole check-out consoles and detects OD by microplate reader _{600 nm}.

The conventional sense of shake flask fermentation liquid: draw 1ml fermented liquid in tool plug pipe, add in 9ml dehydrated alcohol, standing 30min, with filter paper filtering, getting filtrate dilutes in right amount, then by sample: dehydrated alcohol: developer=1: after the ratio of 9: 10 mixes, 70 ℃ of water-bath 20min, coolingly detect OD with spectrophotometer afterwards _{600 nm}.The measuring method of mark product solution is the same.

The calculation formula that fermented liquid is tired is as follows:

Claims (1)

1. a Salinomycin. bacterial strain, Classification And Nomenclature be streptomyces albus ( streptomyces albums) S-610-11, it is characterized in that the selection of High Yield Strains of Salinomycin, concrete steps are as follows:

(1) utilize normal temperature and pressure plasma body (ARTP) mutagenesis starting strain

The spore suspension of preparing starting strain, and dry up under sterile wind, be then placed in ARTP mutagenesis system and carry out plasma irradiating 1～10min; Sample is washed down with physiological saline, be coated on plate culture medium after gradient dilution, 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 ℃ of standing cultivation 4～8d, 25～37 ℃ of the latter, humidity 50～90%, 200～300r/min cultivate 4～8d;

After cultivation, detect the Salinomycin. content of fermented liquid in each hole, choose and produce plain level higher than the mutant strain of starting strain 30%, be primary dcreening operation superior strain;

(3) mutant strain high-throughput sieves again

The solid culture of the high productive mutant of step (2) gained is connected in 24, the 48 or 96 hole depth plates that 0.8 ~ 1.5ml seed culture medium is housed to 25～37 ℃, humidity 50～90%, 200～300r/min shaking culture, 20～28h left and right with aseptic toothpick point;

With 5～15% inoculum size, transfer in 24,48 or 96 hole depth plates of 0.8 ~ 1.5ml fermention medium are housed, shaking culture 6～9d; After cultivation, detect the Salinomycin. content of fermented liquid in each hole, choose and produce plain level higher than the mutant strain of 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 to 25～35 ℃, 200～300r/min vibration, 25～30h left and right;

With 5～15% inoculum size, transfer in the 250ml shaking flask of 20～50ml seed culture medium is housed, continue shaking culture 5～10d;

(5) mutant strain genetic stability checking

By the superior strain of verifying through shaking flask through 3～5 generations of 250ml shaking flask cultured continuously.